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Acute renal failure Morton, Kenneth Sherriffs 1953

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ACUTE RENAL FAILURE  by KENNETH SHERRIFFS MORTON  A Thesis Submitted i n Partial Fulfilment of the Requirements for the Degree of MASTER OF SCIENCE i n the Department of ANATOMY  We accept this thesis as conforming to the standard required from candidates for the degree ^ f MASTER OF SCIENCE  •Efembers <5f the Department of Anatomy  THE UNIVERSITY OF BRITISH COLUMBIA APRIL, 1953  - VI -  ABSTRACT  A brief review of the literature on traumatic anuria (acute tubular necrosis, lower nephron nephrosis) has been presented, including a complete bibliography.  Special attention  was paid to the pathology and pathogenesis of the syndrome and i t was concluded that Oliver's recent work (271) probably comes closest to presenting the true picture.  He describes tubular  necrotic lesions for which the chemical toxins (mercuric chloride, carbon tetrachloride) were responsible, and tubulorhectic lesions which were characteristic of the shock kidney.  These lesions  could appear at any level i n the renal tubule and were characterized by destruction of the basement membrane.  Pigment casts  were apparent i f intravascular pigment release was associated with the illness.  The work of Phillips, Van Slyke and associates  (291, 292, 355, 3 5 6 ) , of Oliver (271) and of Block et a l (41)  ;  lead one to conclude that renal ischemia i s the chief pathogenetic mechanism, though i t i s obvious that specific extrinsic renal toxins play a major role i n specific cases.  The role of hemo-  globin appears to be chiefly i n the production of obstructive casts later i n the course of the disease;  these pigments are  precipitated i n the lower nephron where urine i s concentrated; and acidified, and dehydration and oliguria contribute to their formation.  ..  ' •'  Three hundred rats were studied i n eighteen experiments concerning  crush syndrome*  i t "was concluded that the most  - VII important single factor tending to aggravate the renal effects of crushing injury Is the antecedent state of dehydration. Myoglobin i s not an essential factor i n the development of renal damage but tends to aggravate the existing uremia. failure was seen to be a late effect of shock;  Acute renal  animals developed  acute tubular necrosis only I f i n i t i a l shock was severe, but not severe enough to produce death from circulatory failure.  Devel-  opment of this delicate balance of factors was aided by reduction of renal reserve by unilateral nephrectomy.  A seldom described  but distinct and consistent phenomenon was observed i n the development of marked, immediate and persistent diuresis i n response to the trauma of limb ligation.  This polyuria was of a dilute  urine and was taken as an indication of i n i t i a l increased glomerular f i l t r a t i o n followed by decreased reabsorption of water because of tubular damage.  It was not an indication of a recov-  ery phase as i s recorded i n the c l i n i c a l syndrome. Testosterone propionate, desoxycorticosterone acetate, cortisone acetate and Compound F did not appear to be promising as therapeutic agents, although i n one experiment Compound F showed some promise.  Neither did combined therapy with testos-  terone and cortisone reduce the mortality rate or decrease uremia. Although there was no doubt that the syndrome of acute renal failure due to acute tubular necrosis could be produced i n large numbers of these relatively inexpensive laboratory animals by dehydration and limb ligation, production could not altogether be standardized and the syndrome ran such a short course that  - VIII -  serial observations were d i f f i c u l t to obtain and separation of shock deaths was occasionally impossible.  It i s f e l t that  future work might well make use of some other laboratory animal, perhaps the dog or cat, and that an i n i t i a l stress of controlled hypotension or renal artery occlusion could be used. It i s also our opinion that further investigation into the value of Compound F as a therapeutic agent i n this syndrome i s justified.  - I-  TABLE OF CONTENTS  Page REVIEW OF THE LITERATURE INTRODUCTION  1  HISTORY  3  AETIOLOGY  9  INCIDENCE  13  PATHOLOGY ..  15  PATHOGENESIS  20  Obstruction:  21  Myoglobin  25  Mechanism of Anuria  28  Nephrotoxin:  31  Renal Ischemia:  38  Trueta Shunt  51  Summary of Ischemia Theory  58  Summary of Pathogenesis  60  - II -  TABLE OF CONTENTS continued  EXPERIMENTAL AIM  Page 65  i  METHODS AND MATERIAL  66  REPORT OF EXPERIMENTS  75  DISCUSSION AND CONCLUSION  145  SUMMARY  168 171  BIBLIOGRAPHY  TABLES AND EXPERIMENTS LIST OF TABLES REPORT OF EXPERIMENTS  *  Ill V  -III-  LIST OF TABLES  TABLE  Page  IA  Dehydration i n Intact Rats  77  IB  Dehydration i n Right Nephrectomied Rats .......  78  2A  Myoglobin i n Intact Rats  82  2B  Myoglobin i n Intact Rats  83  2C  Myoglobin i n Intact Rats  84  3  Myoglobin and Dehydration in Intact Rats  85  4  Left Hind Limb Ligation i n Intact Rats  89  5A  Five Hours Ligation plus Dehydration  92  5B  Five and one-half Hours Ligation plus Dehydration  93  6  Ligation plus Myoglobin Injection i n Intact Rats  7  Dehydration, Ligation and Myoglobin in Intact Rats  7A  Statistical Analysis of Figures i n Table 7 . . . .  8  Dehydration and Bilateral Ligation i n Intact Rats Ligation and Dehydration i n Right Nephrectomied Rats Ligation and Dehydration i n Right Nephrectomied Rats  9A 9B 10  Mortality i n Dehydrated, Ligated Unlnephrectomied Rats  100 101 107 108 112 113 120  - IV -  LIST OF TABLES continued  TABLE  Page  11A  Testosterone i n Crush Syndrome  121  11B  Testosterone i n Crushed Female Rats  125  12  Testosterone i n Crushed Male Rats  126  13  Cortisone i n Crushed Female Rats  14  Cortisone i n Crushed Male Rats  15  Testosterone Plus Cortisone i n Crushed  .  Female Rats  130 131  133  16  Testosterone and Cortisone i n Crushed Male Rats ..  134  171  Compound F i n Crushed Male Rats  138  17B  Compound F i n Crushed Male Rats  139  18  Desoxycorticosterone i n Crushed Male Rats  143  - V-  REPORT OF EXPERIMENTS  OBSERVATIONS  Page  Experiment 1  75  Experiment 2  79  Experiment 3  86  Experiment 4  87  Experiment 5  •  Experiment 6 Experiment 7  99 •  102  Experiment 8 Experiment 9  91  105 •  110  Experiment 10  117  Experiment 11  118  Experiment 12  124  Experiment 13  127  Experiment 14  129  Experiment 15  ,  132  Experiment 16  136  Experiment 17  137  Experiment 18  142  ACUTE RENAL FAILURE  INTRODUCTION In the early years of the recent World War the heavy bombing of British cities resulted i n a great number of injuries to the population from falling masonry.  The subsequent course  run by many of these injured people was such that a "new" syndrome was described.  clinical  Because the injuries sustained were con-  sistently the result of prolonged exposure to the pressure of destroyed brick and concrete structures, this syndrome was f i r s t named the Crush Syndrome (69) and was typified by apparent early recovery from the crushing injury followed by a state of progressive, acute renal failure, with oliguria, anuria and uremia, frequently ending i n death.  In the ten years since that time  much work has been carried on to investigate the possible pathogeneses of the condition, with some progress being made.  In this  thesis, an attempt w i l l be made to synthesize the great multitude of papers published on the subject, to present principally the experimental aspect of i t s pathogenesis and to formulate a workable pathogenetic basis for treatment of the syndrome i n the light of more recent concepts.  It was soon realized that only a brief  summary of this voluminous literature was practical but an attempt  - 2-  has been made, nevertheless, to include as complete a bibliography as possible.  In addition, experiments designed to reproduce  consistently i n rats a syndrome resembling that seen i n human crush injuries are reported, as well as the results of using certain agents to lessen the effect of the presumably temporary cessation of renal function. Since Bywaters (69) f i r s t described the crush syndrome, a similar c l i n i c a l picture has been noted i n a great many other conditions and has been described under various t i t l e s .  "Traumatic  Anuria" i s perhaps a more general term, indicating that the anuria and i t s outcome i s a result of various forms of trauma.  The  pathological picture has been taken into consideration together with a slightly different etiological agent i n the description "Hemoglobinuria Nephrosis" (229) and i n 1946, Lucke (213) summarized the various conditions known to give rise to this syndrome and described the lesion i n the kidney as "Lower Nephron Nephrosis". Maegraith (226), emphasizing his opinion that the kidney damage Is a result of oxygen lack, has insisted that the "Renal Anoxia Syndrome" i s a better name, and more recently, other investigators (55) have tried to remain general i n their description of the pathological picture, at the same time avoiding the use of the undesirable term "nephrosis", by referring to i t as "Acute Tubular Necrosis".  A l l these descriptions, varying i n specificity and  point of view, are descriptions of forms of acute renal failure which are closely allied and must be discussed i n any consideration  - 3-  of traumatic anuria i t s e l f . If one must be restricted by the narrowness of definition then one could describe the c l i n i c a l syndrome and i t s experimental counterpart as a state of acute renal failure as exhibited by oliguria or anuria, retention of nitrogenous wastes within the body (i.e., uremia) and histological evidence of renal tubular damage which follows trauma.  It i s obvious that this statement  best defines "traumatic anuria", but to include a l l conditions likely to end i n this picture one needs merely to add the various other etiologies such as intravascular hemolysis, extrinsic chemical toxins and so on.  HISTORY  As i s the case with most "new" c l i n i c a l entities, the syndrome of traumatic anuria and i t s pathological picture are not new at a l l .  Renal deaths with hemoglobinuria after unmatched  blood transfusions were apparent as long ago as 1667 when Denys (12) cross-transfused blood from sheep to man.  Experimental  work with and c l i n i c a l t r i a l of blood transfusion continued through the subsequent years, notably i n the late nineteenth century, and in early editions of Osier's Principles and Practice of Medicine (277) reference is made to "acute parenchymatous nephritis" as a type of acute Bright's disease caused by various toxic agents such as turpentine, phenol and potassium chlorate, acting on the kidney.  The same pathological picture could be seen as a late  4 -  effect of burns and In toxemias of pregnancy;  i n later editions,  trauma and extensive surgery were added as causes of the subsequent renal damage.  Adami (3) i n 1909 added s a l i c y l i c acid, phosphorus,  bichloride of mercury and cholera as agents giving rise to the picture of "acute degenerative parenchymatous nephritis". However, in the reshuffling of classifications of kidney pathologies based on the work of Volhard and Fahr about thirty-five years ago, this particular entity was largely dropped or divided so that i t received less emphasis, at least in the English l i t e r a ture, until i t s rediscovery and description as "Crush Syndrome" by Bywaters and Beall (69) in 1941.  One must nevertheless be  careful not to malign the quite adequate powers of observation of the many clinicians and experimenters of those f i r s t forty years of the century, for cases which we would now classify as lower nephron nephrosis or traumatic anuria were noted and carefully Bell (27)  described.  considered under the description of  c l i n i c a l acute nephritis not only acute glomerulonephritis, but also tubular disease due to mercuric chloride and hemoglobin obstruction.  In a large measure, these entities which we are to  consider were included i n the term "extra-renal (pre-renal) uremia" (133 )•  This azotemia stands in contrast to that of  primary renal disease in which morphological kidney damage i s obvious.  Under extra-renal azotemia, Bell includes such causes  as diabetic coma, peritonitis, hypochloremia and external or internal haemorrhage, and specifies the absent or minimal kidney structural changes.  Fishberg (133)  anticipates to a large  - 5-  extent our present classifications by l i s t i n g prolonged vomiting, diarrhea (as i n cholera), hepato-renal syndrome, diabetic acidosis, Addisonian crisis and shock(traumatic, post-operative, peritonitis, burns, coronary thrombosis, etc.) as frequently giving rise to pre-renal uremia.  Also, focussing on a less  prominent feature of the pathological lesion i n the kidney, Kimmelstiel (192) described cases of "acute hematogenous inters t i t i a l nephritis" dying i n uremia as a result of septic abortion, burns and severe infections.  He recognized that his entity was  part of a picture of delayed renal tubular pathology i n infections and septicemia, conditions associated with hemolysis and the hepato-renal syndrome. In any case, Bywaters and Beall (69) i n 194-1 noted that casualties brought i n to hospital after being released from under fallen buildings soon developed signs of shock which went on to a picture of renal failure, uremia and death.  They report-  ed the f i r s t four such cases i n the British Medical Journal of March 21, 1941.  Bywaters soon discovered that the same entity  had been described adequately i n the German literature about the time of World War I, notably by Dr. Siego Minami (243) i n 1923, though von Colmers (245) had also encountered i t with the German r e l i e f expedition to the Messina earthquake of 1909.  Hackradt  (245) i n 1917 described a case of burial for nine hours with resultant renal damage i n which he emphasized tubular damage but Minami's description of the tubule damage with pigment casts  following "Verschuttung" ( b u r i a l ) , resulting i n death on about the  seventh day (Figure 1), was more complete both c l i n i c a l l y  and pathologically.  His cases t a l l i e d well with those of  d. wu«.  ^Uu*  ™ < «t*—)  •5.a B  sirs*.  7>*Mr. K.rlmrw " » » H « m » k »  < a * - 4 O flit (!•*.'•. F  I Hartuna in r"»nw nutUrl  \* ./A  •« ' " r "  ' V ,  iiMH t i l t ' mm MdlMI «nH *• •  XL* kAf 4 ™  (,lrr rVttn-lk-n) • • • I i •U*krw /x-rfall * • t W m brwntm: M i SwHim MnnrtarwW Mu.krlr. Aurh H^v»ku«Urr<arh«urni|ir ll.Kin.mtkw Art F « - m kuniirti »[»f k-n rim', wnn •ut'h 0riSkiaU|p ItuUc Mr h i n t t i i i * i « - h < i i Bi)<l Sitrm Htflk-n mwm ma Urn dar:  M  - i j K | * *} ? >•# *•  K% W  h r  Kr-h.Wrrt. K - l » ™ l . - n w h  * 1 mo *  liaAV'O / ' • M ^ m / W ^ i*«^™™t3r 'VB • •• / „  1 (MMN  •*  .IMIIH mil »inH vi* 1 • i' ilk »!)•! rahal - ui h * " \VitodrniOB Hi. .hi liW * V "Tt. * « « r r hlut^fullt l - r unH li.-bUhL NVI  S  ' • ' " ' ^ - - « - " ' - ! ' " l - ' * l . T .Nl.ih Vj-nrhO.ll 1111*  frinkm -hr -ninmluni:. I H • .-ni.-iii hm ^imnilimt:. U t M w l t i l i tnllw.Hi. uilM'liarf h> „  R I' I/I, 1 1 ) l U  , , „| „,lJ.., „AW t  i r  v  u - — • - -• T-r. k**- komim- M a w i-rw , . K|4ih.u*t, .kn <;i--i>b rulu-*|Mlktr •wnil.ii ntiritn-n O f a  Figure 1  Bywaters and he compared of horses.  them with p a r a l y t i c myohemoglobinuria  Although his description of pigment casts i n the  tubules of the renal pyramids and Bywaters  1  rediscovery of the  syndrome ani i d e n t i f i c a t i o n of the pigment as myoglobin (myohemoglobin, muscle hemoglobin) were separated by some twenty years, work had been going on, c l i n i c a l l y and experimentally before and during t h i s period, i n the f i e l d of intravascular hemolysis. Blackwater fever and incompatible transfusions i n p a r t i c u l a r were  - 7involved and an end result of renal failure with uremia and pigment cast formation i n the kidney tubules had been noted. The obvious similarity of these syndromes was soon realized and much of the older investigative work was applied to the new crush syndrome, with investigation in both fields receiving great impetus from the renewed importance of the c l i n i c a l entity.  In  1941, then, investigation into the possibility of a common pathogenesis for these various illnesses attracted new interest and since that time much experimental work has been carried out, many treatments tried and volumes of papers written.  Basing their  opinions on the work of Baker and Dodds (15) i n 1925,  Bywaters  and Beall (69) at f i r s t carried on the idea of obstruction of renal tubules by pigment casts into their theory of the pathogenesis, substituting myohemoglobin for hemoglobin.  Because  this concept did not satisfy a l l the observed facts, the idea, of renal anoxia was upheld by Maegraith (226)  i n his work on black-  water fever and he postulated some sort of short circuit of blood through the renal parenchyme (353)  i n 1947  (223).  When Trueta and associates  described just this phenomenon (which has come to  be known as the Trueta or Oxford Shunt) i t was felt that the answer, the common factor, had been found.  Trueta's classical  work, however, was soon followed by reports which east doubt on the importance —  and perhaps even the fact —  of this bypass  and at present the idea of pathogenesis appears to be in a state of flux, in which several modes of development appear to be acceptable, rather than one.  - 8-  It should be mentioned here, too, that a third line of investigation has been carried on i n the ten year period from 1941 to 1951? based on early observations of the toxic action of such chemical agents as mercury, uranium and phosphate on the renal tubules.  Nephrotoxins, acting directly on the renal  tubules, have been said to be released from ischemic muscle, and such workers as Eggleton  (125)?  Bywaters  (67, 75)  and Bielchowsky  and Green (30) have named breakdown products of muscle protein, myoglobin derivatives and released intracellular components as being responsible for the renal damage.  Two of the pathogenetic  theories are drawn together i n work on shock which produces hypotension and thus renal anoxia.  Corcoran and Page (85),  among many others, have identified a vaso-depressor substance released from tissues i n trauma whieh causes a prolonged lowering of blood pressure, which i n terms of Maegraith's concept (226) of renal anoxia, would damage the kidney i n such a way as to produce the acute renal failure seen c l i n i c a l l y . It can be seen, then, that the syndrome described by Bywaters (69) i n 1941 was not new, but had been encountered i n similar circumstances earlier i n the same century and described adequately by Minami (243) i n 1923•  In addition, the same end  result had been recognized and investigated i n conditions of release of hemoglobin into the bloodstream, notably i n incompatible blood transfusion and Blackwater fever.  The pathological  picture was described, at the turn of the century, as acute tubular nephritis, but with the identification of etiologies  - 9 -  responsible  i n recent y e a r s ,  s p e c i f i c d e s c r i p t i o n s such as  kidney, hemoglobinuria nephrosis were suggested.  and  lower nephron  As might be expected, w i t h the  that the kidney damage was f a c t o r s , the pendulum has  the common end returned,  genesis,  to be d i s c u s s e d  realization  of m u l t i p l e  Lowe (55)»  etiological  acute  time  tubular  Three main t h e o r i e s o f patho-  l a t e r , remain but  being viewed i n t h e i r proper p e r s p e c t i v e v a r y i n g degrees to the end  nephrosis  so that at the present  the term suggested by B u l l , Joekes and n e c r o s i s , seems more s a t i s f a c t o r y .  crush  these are perhaps  as each c o n t r i b u t i n g i n  r e s u l t of acute r e n a l  failure.  AETIOLOGY In the years s i n c e the crush syndrome was the concept has  broadened to i n c l u d e many more e t i o l o g i e s produc-  i n g the same end  result.  As mentioned p r e v i o u s l y , the  between t h i s syndrome and water f e v e r and  similarity  the r e n a l deaths encountered i n Black-  incompatible  t r a n s f u s i o n was  These e t i o l o g i e s are so numerous and seems a d v i s a b l e  rediscovered  soon r e a l i z e d .  appear so d i v e r s e that i t  to name the syndrome on the b a s i s o f a common  pathological picture.  For t h i s reason, the term acute  n e c r o s i s seems s a t i s f a c t o r y .  tubular  In Table A, an attempt has  been  made to group the causes of acute t u b u l a r n e c r o s i s under e i g h t headings.  I n d i c a t i v e o f the  confusion  as to the pathogenesis  the c o n d i t i o n i s the r a t h e r l a r g e column under and  "Miscellaneous",  i t w i l l be noted t h a t s e v e r a l e t i o l o g i e s appear under more  than one  heading,  a f a c t which i n d i c a t e s that more  of  - 10 -  TABLE A INTRAVASCULAR  HEMOLYSIS  1  Transurethral prostatectomy ... 2 1 9 , 2 0 7 , 9 4 , 3 6 7 .  2  Blackwater fever ...  3  Incompatible transfusion ... 1 2 ,  371,137,223. 229,96,45,365,16,106,109,  128,129,10,343,341,121,151,14,105,246,310,107,135,127.  4  Quinine ... 349, 2 7 8 .  5  Burns ... 2 3 6 , 5 3 , 3 2 8 , 1 5 3 , 2 7 2 , 1 2 2 .  6  Malaria ... 3 0 7  7  March hemoglobinuria ... 2 9 3 , 3 1 0 .  8  Paroxysmal cold hemoglobinuria ... 3 4 5 , 3 1 0 , 3 3 3 , 109-i  9  Paroxysmal nocturnal hemoglobinuria ...  10  Paralytic myohemoglobinuria  11  Toxins:  333*  ... 7 2 , 1 9 9 .  Favism ... 3 3 3 , 3 1 0 , 1 3 7 Snake venoms Mushroom poisoning ... 2 1 3 •  12  Myanesin ... 1 7 6 .  TRAUMA and SHOCK 1  Hemorrhagic shock ... § 4 , 3 2 2 .  2  Traumatic shock ... 8 7 , 8 8 , 1 7 5 , 2 2 9 , 1 0 0 , 3 5 5 , 2 8 0 , 2 4 9 , 2 5 0 , 2 5 1 , 252; 102 28'3,317,l64: 26,59,60,61,93,208,305; 116,162, 163,347,78,329,297,348,291,111,319. 2  3  Burn shock ... See "Burns".  4  Crush injury ... 6 3 , 6 4 , 6 5 , 6 6 , 6 7 , 6 9 , 7 0 , 7 1 , 7 2 , 7 4 , 7 5 , 2 5 , 1 7 3 , 212,227,228,239,245,247,351.  5  Peritonitis ...  244,177,193,221,315,364,187.  - 11 TABLE A continued  INFECTION 1  Typhus ... 150  2  Cholera ... 352  3  Malaria ... 307  4  Weil's disease ... 362  5  Welch i n f e c t i o n ... 178  6  Septic abortion  ...51  ELECTROLYTE IMBALANCE 1  P y l o r i c obstruction ... 54, 82, 133,187,28,333,214,221.  2  A l k a l o s i s ... 214,193,221,339,9,224.  3  Acidosis ... 160, 161.  4  Hyponatremia ... 315  5  Hypochloremia ... 177  6  Hypokalemia ... 136  CHEMICAL TOXINS 1  Mercuric chloride ... 77, 123, 303, 174, 357, 23.  2  Carbon tetrachloride ... 368,284,90,331,333,271,23-  3  Diethylene glycol ... 271,23.  EXPERIMENTAL TOXINS 1  Uranium ... 304,43,174,264,363.  2  Oxalates and' Urates ... 118, 119, 120  3  Phosphates ... 222, 218, 30.  - 12. -  TABLE A continued  4- Potassium chlorate ... 2 7 1 5  Sodium tetrathionate ... 3 3 2  7 Potassium dichromate ... 174 MASSIVE DESTRUCTION OF TISSUE 1  Burns ... See "Intravascular hemolysis" and "Shock".  2  Prolonged labor ... 3 7 3 , 3 7 2  3  Toxemia of pregnancy ... 8 3 , 3 6 0  4  Concealed, retroplacental hemorrhage ... 3 7 2 , 2 8 5 , 1 1 2 .  5  Welch infection ... I 7 8  6  Abortion ... 1 7 8 , 2 7 8 , 2 6 8 , 2 6 9 .  MISCELLANEOUS 1  Pulmonary infarction ... 1 9 8  2  Electroshock ... 1 5 2  3  Gastrointestinal hemorrhage ... 340, 1 8 8 , 3 5 6 , 34.  4  High altitude anoxemia ... 2 5 2 , 7 6 , 1 9 7  5 Hepatorenal syndrome ... 4 8 , 1 7 2 , 2 7 5 , 3 3 8 . 6  Heat stroke ... 2 5 2 , 2 1 3 , 146.  7  Intravenous soap... 3 5 9  . 8 Sulphonamides 9  ... 2 , 104, 2 9 0 , 142, 1 3 2 .  Allergy ... 2 1 1 , 142, 1 3 2 .  10  Myelomatosis ... 2 5 4  11  Lymphosarcoma ... 2 8 9  12  Volkmann's ischemic contracture ... 1 6 5 .  - 13 -  than one pathogenetic  factor i s involved.  Also, i n attempting  to find a common factor i n these many causes, i t i s often impossible to decide just what factor contributes to the renal failure, so that such headings as "Massive destruction of tissue", "Electrolyte imbalance" and "Infection", though unsatisfactory are, in the present state of our knowledge, unfortunately necessary. It i s impossible to review i n detail the many interesting intricacies of individual entities leading to the end picture of acute tubular necroses.  However, i t was f e l t that the many  references to these etiologies encountered i n the literature might well be included i n the Table for future reference.  INCIDENCE A brief review of the incidence of the syndrome as presented i n the literature i s advisable i n order to place i t i n i t s true perspective as a c l i n i c a l entity.  Bywaters (64) stated the  incidence of ischemic muscle necrosis (crush syndrome) as one to five per cent i n air-raid casualties. cases requiring hospital care.  Presumably these were  Douglas (114), i n a very complete  work, considered a random group of casualties, 764 i n a l l , admitted to hospital following an air raid.  Of these 764, .  77 (10.1 %) were buried for two or more hours, and six of these 77 (7.8$) developed crush syndrome.  That i s , six (0.79%) of the  total of 764 casualties were cases of crush syndrome and one of the six (16.6%) died.  The works of Lauson et a l (208), of  - 14 -  Cournand et a l (93) and of Burnett et a l (59, 60) present verycomplete renal function studies i n cases of trauma with or without shock, but these records are mainly i n the acute phase of trauma. Darmady (99) also found that blood urea nitrogens done i n 79 battle casualties were elevated i n 35$ of cases.  In 10,GOG casualties  reviewed by him there were 44 deaths, twelve of them due to renal failure;  a l l of these suffered shock from blood loss.  et a l (335)  Snyder  considered 1411 battle casualty deaths and found 68  deaths from lower nephron nephrosis, with 31 other deaths i n which lower nephron nephrosis played a part.  Of these 99 fatal cases,  56 had blood pressures below 100 mm. of mercury and only five had "no evidence of shock".  Moyer (256)  examined renal function  following major surgical procedures but again considered only the immediate post-operative period i n random cases i n which impairment of renal function was not c l i n i c a l l y apparent.  Gaberman et  al (146) searched widely for cases of the "renal anoxia syndrome" and found few  22 cases i n two years of admissions to two large  Chicago hospitals. Although these incidence figures are not high and the frequency of the syndrome i n hospital practice w i l l not be great, i t is apparent that i n times of violence cases of traumatic shock w i l l increase this incidence to perhaps 1% of cases treated.  An  understanding of i t s pathogenesis and an adequate regime of treatment therefore become of some importance.  - 15 -  PATHOLOGY With the great number of etiologies recorded, i t i s apparent that many minor variations of essential kidney pathology would be expected.  There must be, however, a basic common  factor i n the pathologies i n order that the syndrome be described as an entity in i t s e l f .  This essential factor i s , of course, by  definition renal tubular degeneration to necrosis. Although Adami's (3) description of the pathology of "acute degenerative parenchymatous nephritis" i n 190?  cannot  essentially be improved upon today, this pathologic entity received less and less emphasis i n the early 1900's, principally because of the reclassification of kidney pathologies based on the work of Volhard and Fahr.  Nevertheless, such men as Bell  (27) continued to describe i t , partly as "acute haemorrhagic glomerulonephritis" with i t s tubular obstruction by blood and hemoglobin casts, and also as "acute i n t e r s t i t i a l nephritis" and "pure tubular degeneration" as in mercury poisoning. Kimmelstiel (192)  Again,  described a number of cases i n which he empha-  sized the focal i n t e r s t i t i a l edema and i n f i l t r a t i o n by naming the renal pathology "acute hematogenous i n t e r s t i t i a l nephritis". In 1942, Bywaters (71) followed his report of the new crush syndrome with a f u l l description of i t s pathology.  The  most outstanding gross feature of the kidneys was the almost constant appearance of cortical pallor contrasting with a congested,  - 16 reddish-purple medulla.  Microscopically, the glomeruli were  noted to be essentially normal, except for the frequent appearance of intracapsular granular eosinophilic debris and occasionally cubical metaplasia of the capsular epithelium. essential lesion was i n the renal tubules;  Again, the  Bywaters describes  a catarrh of the proximal tubule and descending loop of Henle, while i n the ascending limb and distal tubule, degeneration and necrosis of tubules and herniation and rupture of casts through tubular walls were seen.  Outstanding were pigment casts formed  of myoglobin derivatives, i n the distal convolution and collecting tubule.  Bywaters' f i r s t report (69) emphasized the severe  degeneration of proximal tubules, but i n his more extensive consideration (71) he localized the severe changes to the distal convolution.  It w i l l be seen later that, i n Oliver's  (271)  opinion, the renal lesions i n crush syndrome were perhaps most accurately noted by Dunn, Gillespie and Niven  (117)'  Bywaters, of-course, noted the similarity of this pathology to that described i n transfusion reactions and Blackwater fever as hemoglobinuric nephrosis, as did Dunn et a l (117) lesions described by Dunn and Poison (120) r i t i s , and McFarlane (218) (229)  with uric acid neph-  with phosphate nephritis.  carefully listed the characteristics of the  kidney.  to the  Mallory  hemoglobinuric  Grossly, the kidney is enlarged with a pale cortex and  purplish pyramids, but may be normal. glomeruli are again largely normal.  Microscopically, the The f i r s t change noted is  - 17 fatty vacuolization of the ascending loop of Henle, becoming severe degeneration by three days, with herniation and rupture. Little change i s seen i n the proximal segments. the casts and these are of two types:  Prominent are  pigment casts, staining  red-orange i n Hematoxylin-Eosin sections, are seen i n the lower nephron, while hyalin casts are encountered higher up.  A  controversial feature described i s dilatation of the proximal tubules, which appears to be more prominent i n formalin fixed material.  It w i l l be seen later i n the discussion of patho-  genesis that the problems of frequency of casts and presence or absence of tubule dilatation are key points i n the argument. In addition, a focal and diffuse inflammatory i n f i l t r a t i o n of the i n t e r s t i t i a l tissue i s seen with a granulomatous reaction around herniated casts. A report which has dominated the literature on this syndrome since i t s publication i n 194-6 i s that of Lucke  (213),  who recognized the similarity i n c l i n i c a l picture and renal pathology i n these various c l i n i c a l entities.  He described  the common pathological picture and because he believed i t was essentially a lesion of the distal convoluted tubule, he named i t lower nephron nephrosis.  This name has persisted even  though opinions as to the location of the lesion have changed. Lucke's description (213) of the pathology as lower nephron nephrosis remains in common usage.  - 18 -  By means of an extremely meticulous technique, Oliver (270) has been able to study the nephron as a unit i n continuity. By his microdissection technique, complete individual nephrons are dissected out and stained. tion (271) of human material —  Ten years of careful investiga54 kidneys of crush injuries,  burns, transfusion reactions, Blackwater fever, obstetrical deaths, surgical shock, paroxysmal cold hemoglobinuria, sulfonamides, mercuric chloride, diethylene glycol, carbon tetrachloride, potassium chlorate and mushroom poisoning — and of animals subjected to induced shock or toxins, led Oliver and his coworkers (271) to conclude that there are two essential tubular lesions i n the kidney of acute tubular necrosis.  These lesions  are: (1) Nephrotoxic tubular necrosis — here the epithelium disintegrates between intact basement membranes, the lesions are seen only i n the proximal convolution and are evenly distributed i n a l l nephrons of a damaged kidney.  Such agents as mercuric  chloride, potassium chlorate, diethylene glycol and carbon tetrachloride produce the typical nephrotoxic lesion, but i t must be remembered that the second essential tubular lesion may also be observed i n these cases, presumably because the toxins also induce shock.  (2) Tubulorhexis, i n which there i s a localized destruc-  tion of the entire tubular wall.  The basement membrane disinte-  grates and there may be intralumenal material such as pigment casts i n a fortuitous distribution.  There may be an i n t e r s t i t i a l  granulation tissue reaction associated, and regeneration, though  - 19 i t may begin, i s impossible without the support of a basement membrane.  As to the localization of this tubulorhectic lesion,  Oliver states that i t has been found anywhere from the proximal convolution at the glomerulus to the lower nephron at i t s junction with the collecting tubule.  Maximum development i s usually in  the terminal portion of the proximal tubule which i s i n the outer stripe of the outer zone of the medulla.  The distribution of  the lesion i n any one kidney i s irregular —  irregular among  nephrons and within a nephron. This view that there are two characteristic lesions, tubulorhexis and nephrotoxic tubular necrosis, in the entity acute tubular necrosis would seem to be most acceptable because i t satisfies a l l the known facts —  the actual cytological disrup-  tion and the observed distribution of both types of lesion —  and  because i t i s as well based on a pathogenetic concept which i s becoming more widely accepted.  These ideas are hardly new.  Almost a l l the observations had been made previously.  But  Oliver's work organizes and classifies these observed facts and places them on a sound basis by persistent, meticulous and patient techniques. Though most reports have dealt with glomerular changes as being absent or consisting of, at most, ischemia, intracapsular eosinophilic debris and swelling of the capsular epithelium, i t i s true that some observers have searched for more significant changes i n the renal corpuscles of these kidneys. (155»  157,  158),  Goormaghtigh  in particular, has examined the glomerular  - 20 -  structures and found minute changes which he feels are important functionally.  French (143) believes that tubular changes are  not sufficient to explain the oliguria of lower nephron nephrosis, and uses Fahr's term "glomerulo-nephrosis" to emphasize his view that the glomerular changes of decreased blood, thickened capillary wall, thickened capsular epithelium and granular precipitate i n the capsular space are functionally important. These glomerular changes may well be present, but the large majority of investigators are agreed that most of the important faults i n function of the kidney of crush or trauma can be explained by the tubular lesions.  In any case, the domi-  nant tubular changes provide a suitable contrast to the many glomerular pathologies i n classifications of renal diseases.  PATHOGENESIS That an understanding of the pathogenesis i s the key to successful treatment of a disease entity i s an obvious truth which is no less a fact i n the case of acute tubular necrosis.  For  this reason, much of the experimental work done and the articles written on this subject are concerned with the pathogenetic mechanism.  Presentation of this material necessitates retracing  steps to consider early opinions on the matter, then following them to their logical end i n the most acceptable theories of today.  In doing this i t i s advantageous to group ideas into  three categories: (1) simple mechanical obstruction of tubules;  - 21 -  (2) toxic action of agents on renal tubules; ischemia.  and (3) renal  It w i l l be seen that these three theories of patho-  genesis overlap a good deal and are i n no way mutually exclusive. Obstruction: The earliest theory put forth, chiefly because f i r s t observations were made on the pigment nephropathies, was perhaps the simplest and most obvious, that of simple, mechanical obstruction of renal tubules by the prominently seen pigment casts.  Yorke and Nauss (37D and Foy, Altmann et a l (137)  trace the origin of this theory to the German literature of as long ago as 1883 and Yorke and Nauss themselves (370, 371), after observing rabbits injected with homologous hemoglobin solutions, believed that the renal tubules secreted the hemoglobin into the lumena where i t was precipitated, forming casts which plugged the tubules chiefly i n the thin loop of Henle.  They observed  dilatation of tubules, presumably as a result of the plugging, and believed that this i n turn impinged on adjacent patent tubules thereby increasing the obstruction.  They recognized  the fact that a lowered blood volume (shock) i n Blackwater fever aids production of anuria and precipitation of hemoglobin casts. A paper which appears to be the basis of many presentday opinions was published i n 1925 by Baker and Dodds (15). They examined kidneys from two fatal cases of transfusion reaction and were struck by the widely dilated tubules and capsules, together with casts, seen i n one.  Experimentally, they concluded  -  22  -  that the hemoglobin released intravascularly was excreted by the kidney as oxyhemoglobin, but i n the presence of acid urine (pH less than 6) this was converted to methemoglobin, which was precipitated by a concentration of inorganic sodium salts of at least 1%.  The precipitate was thought to be hematin  and this process was aided by the normal concentration of tubular fluid as i t proceeded down the renal tubules.  The hematin  casts then obstructed the tubules and accounted for the observed dilatation.  Baker and Dodds • conclusions were logical  and attractive, but nevertheless were based on experiments carried out on only a small number of rabbits.  The work does  not merit the devoted attention i t has been given over the last 25 years. Baker and Dodds* work was at f i r s t supported by De Gowin et a l (106)  (108)  in  1937* but  i n the following year, De Gowin  observed that i n five deaths in renal failure following  transfusion reactions, there was no microscopic anatomic basis for renal insufficiency —  few casts and l i t t l e  and dilatation of tubules.  De Navasquez (109)  degeneration followed this  work with the opinion that pigment cast obstruction was not the cause of oliguria because too few casts were seen and dilatation of tubules was rarely seen. did no harm with pH at 5.5  He believed that hemoglobinuria  to 6.3,  and that i f the glomerular  flow was sufficient, urine flow would wash out any casts formed. This opinion, i t w i l l be seen later, is more or less returned to by Jean Oliver in his classical report of December,  1951 (271).  - 23 In the past 10 years, the controversy of whether or not pigment casts can account for anuria by simple obstruction has continued and the role of aciduria i n the precipitation of hemoglobin casts has also been thoroughly discussed (96, 10, 137, 32,  62, 117, 202-206).  One of the chief objections to the obstruction  theory has been suggested by Bywaters and Dible (7D*  that not  enough casts are seen to account for obstruction and urinalyses indicate that there i s abnormal tubule function.  They point out  that, i f obstruction alone accounted for oliguria, that urine which was excreted would be from normal tubules and would be of normal makeup;  this, of course, i s not the case.  Ayer and Gould (10) concluded that necrosis of the dist a l convolution was the only progressive change seen i n the renal pathology and that casts do not produce the structural and functional changes.  They point out that kidneys of jaundiced infants  may show frequent casts without any evidence of renal dysfunction in l i f e and quote Huber as stating that slight dilatation of the tubules i s a normal variation. A number of investigators  (137»32, 377, 202-206) record  their belief that precipitation of pigment i s a sequel to, not the cause of renal failure, implying that renal damage and dysfunction is present before pigment casts appear and therefore the casts, i f they do obstruct the tubules, merely add to renal damage already present.  They add that oliguria appears within hours of i n i t i a l  injury and that the earliest structural change seen i n the kidney is lipid vacuolization i n the ascending loop of Henle  (229).  - 24 -  Maegraith and Findley (223)  i n a preamble to their  conclusion that a redistribution of renal blood flow i s responsible for anuria, l i s t four objections to the simple pigment cast obstruction theory: (1) Casts are not extensive enough in d i s t r i bution to discount the high renal reserve;  (2) dilatation of  tubules and capsular spaces is not always present;  (3) reaction  of urine played no role in production of anuria (in an analysis of 35 cases of Blackwater fever); and (4) the anuric state i s reversible. In contrast to these opinions, there are several arguments for the obstruction theory.  Corcoran and Page (85)  conclude  that renal damage in pigment nephropathy i s due to three factors  —  obstruction by casts, ingestion of pigment by cells and a cytotoxic action of hematin on d i s t a l tubules. (270)  They refer to Oliver's work  of the same year, i n which he points out that a renal lobule  which i n histological section appears only partially obstructed by pigment casts may be i n fact completely occluded since the casts form at different levels, as seen by microdissection. tally, Flink (135)  Experimen-  on the basis of studies i n dogs injected with  hemoglobin and examined by needle biopsies of an explanted kidney, concluded that the most severe renal insufficiency developed in those animals with most casts, and the amount of tubular epithelial injury correlated with the number of casts.  He believed that  hemoglobin casts and tubular epithelial damage were equal factors in the production of anuria and insufficiency.  - 25 -  Harrison and co-workers (168)  also believe that renal  impairment is partly explained by obstruction to flow of urine in tubules and Maluf (232) obstruction theory:  comes out strongly in favor of the  "The mechanism of renal failure from the  intravascular introduction of a moderate quantity of lysed red cells is primarily due to tubular obstruction from casts of hemochromogen combined with a low rate of glomerular f i l t r a t i o n . " The answer to these strongly held opinions i s probably the compromise stated so convincingly by Oliver i n his monograph of  1951 (271).  He points out that, as a pathologist, he cannot  ignore the fact that, in micro-dissected  kidneys from fatal  cases of pigment nephropathy, renal tubules are plugged with heterogeneous casts, often massive in extent and obviously contributing to the anuria by obstructing the tubules which normally conduct f l u i d .  He points out that pigment casts are found in  a l l cases where myoglobin or hemoglobin is liberated into the blood, but that there i s no correlation between pigment casts and tubular damage.  He concludes that simple mechanical obstruc-  tion of renal tubules by pigment casts is certainly a factor contributing to the oliguria and anuria seen in the pigment nephropathies. Myoglobin: Though most of the above work has been based on c l i n i cal observations on cases of intravascular hemolysis and experimental injection of hemoglobin solutions, i t is obvious that the  - 26 theory bin  a p p l i e s e q u a l l y w e l l t o those c o n d i t i o n s i n which myoglo-  i s the r e l e a s e d  pigment.  Muscle pigment entered the  d i s c u s s i o n w i t h the r e p o r t i n g o f the crush syndrome by Bywaters and B e a l l (69), who noted the l o s s o f t h i s pigment from p a l e , edematous, crushed s k e l e t a l muscle and were able  (70) t o  i d e n t i f y i t i n the u r i n e o f these i n j u r e d p a t i e n t s . M i l l i k a n (242) reviewed the p r o p e r t i e s o f muscle hemog l o b i n thoroughly and r e p o r t e d  t h a t i t was f i r s t  c r y s t a l l i z e d by T h e o r e l l i n 1932.  i s o l a t e d and  Myoglobin, w i t h a molecular  weight o f  17,500 (as compared  threshold  o n e - f i f t h that o f hemoglobin, i s v e r y s o l u b l e and i s  e a s i l y o x i d i z e d to the 'met' form. it  68,000)  t o hemoglobin's  According  has a r e n a l  t o Morgan  i s extremely s o l u b l e i n phosphate b u f f e r s at pH 6.6.  occurs i n red muscle and has a c h a r a c t e r i s t i c spectrum.  (253) It I t has  the t y p i c a l hemoglobin oxygen-carrying c a p a c i t y but p r o b a b l y a c t s c h i e f l y i n s t o r i n g oxygen r a t h e r than t r a n s p o r t i n g i t . I t s i s o - e l e c t r i c p o i n t has been reported Whipple (265) tubule  as 6.78  (7).  Newman and  stated t h a t t h i s pigment was not taken up by r e n a l  c e l l s , a f a c t w i t h which Y u i l e and C l a r k e  (375)  agree.  These workers found t h a t the pigment was r a p i d l y c l e a r e d from the blood old  —  25 times more r a p i d l y than hemoglobin —  and i t s t h r e s h -  v a l u e was 20 mg. p e r cent. When Bywaters and B e a l l (69) f i r s t  reported  the crush  syndrome they suspected t h a t the c i r c u l a t i n g muscle pigment might be r e s p o n s i b l e  f o r the kidney damage.  They soon i d e n t i f i e d the  - 27 myoglobin (70),  s p e c t r o g r a p h i c a l l y i n the u r i n e o f a i r - r a i d  casualties  though they could not i d e n t i f y i t i n the plasma because o f  i t s rapid clearance.  Bywaters and D i b l e (72) reviewed  seven  reported cases o f acute p a r a l y t i c myohemoglobinuria i n man, and added an e i g h t h case i n which the kidney pathology was the same as that seen i n the crush syndrome. (199) fied  K r e u t z e r , S t r a i t and K e r r  r e p o r t e d a n i n t h case i n which the pigment was a g a i n i d e n t i s p e c t r o g r a p h i c a l l y i n the u r i n e . I t was n a t u r a l t h a t experimental work i n v o l v i n g the  i n j e c t i o n o f myoglobin Bywaters and Stead  s o l u t i o n s would f o l l o w these o b s e r v a t i o n s .  (75) prepared  such s o l u t i o n s o f human myo-  g l o b i n by T h e o r e l l ' s method (242) and i n j e c t e d amounts c a l c u l a t e d to approximate per Kg.). produced  that r e l e a s e d i n a t y p i c a l crush i n j u r y  Using r a b b i t s , they found that myoglobin no kidney damage i n e i g h t animals;  f o l l o w i n g l e g compression  produced  alone  myoglobin  injections  o l i g u r i a , uremia and r e n a l dys-  f u n c t i o n w i t h c a s t s i n s i x o f s i x animals; injected  (150-200 mg.  and the pigment  i n t o . a n i m a l s w i t h ammonium c h l o r i d e a c i d i f i e d u r i n e  r e s u l t e d i n f o u r deaths i n 27 animals, w i t h a r i s e i n urea n i t r o g e n o f 100  t o 860  mg. % i n 15 r a b b i t s .  Corcoran and Page (87»  88) were a l s o a b l e t o produce  kidney damage i n r a t s , comparable to t h a t seen i n human c r u s h syndrome, by i n j e c t i n g myoglobin hours.  f o l l o w i n g limb l i g a t i o n f o r f i v e  T h e i r dose was 75 t o 180  mg. per Kg. and i n experiments  on dogs (85) w i t h u r i n e a c i d i f i e d by d i e t and sodium a c i d phosphate they produced  p a r t i a l l y r e c o v e r a b l e r e n a l i n j u r y which they  - 28 -  attributed to obstruction by casts and a cytotoxic action of hematin, a split-product of myoglobin.  They also injected  hematin i t s e l f and observed efferent, then afferent, arteriolar constriction and toxic cellular changes with resultant depression of renal function.  Kidney damage as a result of hematin  injection had been reported previously by Anderson et a l (6). These workers believed that the resultant renal failure was produced by a vascular effect rather than a nephrotoxic  or  obstructive one. On the basis of these few reports, no definite conclusions can be drawn as to the role of myoglobin i n the production of renal damage.  It seems probable, however, that the muscle  pigment w i l l contribute i n much the same way as hemoglobin Itself does, be i t obstructive or toxic.  As Oliver has stated  (271), i t is d i f f i c u l t to say that pigment cast obstruction does not contribute to renal dysfunction when microdissected specimens show the tubes to be plugged.  This effect may well be a  later phenomenon, but appears to be a definite one. not the pigments have a cytotoxic effect i s best  Whether or  considered  under the discussion of the nephrotoxic theory of renal dysfunction. Mechanism of Anuria: The problem of pathogenesis in acute tubular necrosis can be viewed to advantage as a question of the mechanism of anuria.  Four possible mechanisms have been suggested.  - 29 F i r s t , mechanical obstruction by casts, as discussed above, is an obvious cause of anuria.  Oliver (271) points out  that when a plumber sees a plugged pipe he concludes that fluid w i l l not flow.  So i t i s with the renal tubules obstructed with  pigment casts.  This plugging contributes to the anuria only  later, however, and may well be only a minor factor. Second, increased plasma osmotic pressure due to hemolysis has been considered by Foy et a l (137) to be a possible cause of oliguria i n Blackwater fever.  With 50% of the blood  hemolyzed, plasma protein would be increased by 8%, thus increasing the plasma osmotic pressure to counteract the hydrostatic pressure, reduce f i l t r a t i o n and result in oliguria. They concluded, however, that this change made no contribution to oliguria because both albumin and globulin levels i n plasma dropped in proportion to the hemoglobin rise so that the total osmotic pressure remained unchanged. Third, a decreased hydrostatic pressure could also conceivably result i n reduced urine output.  This i s an obvious  cause of anuria i n the i n i t i a l stage of shock, where renal blood flow may be interrupted completely.  With a prolonged low blood  pressure, below 60 to 100 systolic (29D» renal blood flow continues to be n i l , so that obviously no urine can form.  This  mechanism, then, involves the renal ischemia theory of pathogenesis and w i l l be discussed under that heading.  - 30 -  A fourth mechanism has been discussed as early as by Dunn and Jones (119) ritis.  1925  i n their experiments with oxalate neph-  This i s the "back diffusion theory".  They believed that  the urea retention and oliguria seen i n "experimental tubular nephritis" could be explained on the theory that the damaged tubule cells are unable to prevent indiscriminate reabsorption of glomerular f i l t r a t e with urea from the tubules into the connective tissue and vessels of the kidney.  They carried this idea  into their work on uric acid nephritis (120)  and recalled i t i n  a consideration of two cases of crush syndrome i n 1941 In  1929»  A. N. Richards  (303)  (117).  had reported that i n frogs made  anuric with mercuric chloride, glomeruli were more active and remarked, "The only explanation which I can reach i s that under these abnormal conditions the osmotic pressure of the blood proteins i s unobstructed by the normal qualities of the tubular epithelium and i s able to draw a l l or nearly a l l of the glomerular f i l t r a t e back into the blood stream." also to Nicholson et a l (266)  The idea appealed  i n their work with sodium tartrate  nephrosis, as i t did as well to Hayman et a l (17D nephrosis and to Bywaters and Dible (71).  i n uranium  In more recent  functional studies, Redish et a l (299) determined clearances and tubular maximums i n a case of sulphonamide anuria and found, in the third week, that these values were negative. positive at six weeks.  They became  They concluded that the i n i t i a l decreased  clearances (mannitol) were not indicative of the true glomerular  - 31 f i l t r a t i o n rate and that some back diffusion had occurred. Similarly, a negative Tm  pAH  indicated back diffusion of PAH  (para-amino hippuric acid) through the tubules.  This same  conclusion was reached by Govaerts (159) i n comparing urea and creatinine clearances i n mercury and bismuth poisoning cases and uranium and oxalate poisoning i n dogs. It would seem reasonable to conclude that anuria i s f i r s t the result of reduced or absent renal blood flow i n the i n i t i a l stages of shock.  If glomerular flow i s restored but  tubules have been damaged, anuria and oliguria may be continued by back diffusion of tubular fluid through the dead membrane of necrotic tubules into the more osmotically active plasma. When intravascular hemolysis and pigment cast formation i s involved, tubular obstruction i s undoubtedly a factor.  Nephrotoxin: The nephrotoxic theory, of renal damage i n the syndrome has, like the obstruction theory, been held for many years. Implied i n this theory i s that a substance toxic to the kidney is released into the circulation i n trauma, shock, crush, intravascular hemolysis or destruction of tissue, resulting i n the c l i n i c a l picture of acute tubular necrosis. modes of action of the toxin are considered:  Two possible  f i r s t , a direct  cytotoxic action whereby tubular cells are poisoned and put out of action;  and second, a vasospastic action i n which case the  renal ischemia theory of pathogenesis i s embraced to some extent.  - 32 -  In specific instances, the validity of the nephrotoxic theory cannot be doubted.  For many years, specific inorganic  compounds such as mercury bichloride (123» 174), uranium (174) and carbon tetrachloride (331» 368) have been known to damage renal tubular cells directly even to the point where various levels of action,, with reference to the nephrons, have been noted  (271). The more controversial aspect of the nephrotoxic theory  is encountered i n the belief that various intracellular components are released from damaged or shocked tissues, which, i n effect, have the same cytotoxic action as the chemical agents referred to above.  Reviews of recent years (213, 88) have  always included this explanation but of late i t has received less attention.  The observation of MacKay and Oliver (222) i n  1935 that rats fed an excess of inorganic phosphate developed lesions i n the terminal portions of the proximal convolution were confirmed by McFarlane (218) i n 1941, though the affected portion according to this observer was lower i n the ascending limb of Henle. These observations were a prelude to the work of Green (162, 163, 164) and of Bollman and Flock (44) and others, who attempted to isolate a shock-producing muscle.  factor from striated  Green (162) isolated an adenylic acid derivative from  crushed muscle, which Bielchowsky and Green (30) identified as adenosine triphosphate.  This compound, when injected into  0  -  33  -  animals, could produce hemoconcentration,  f a l l i n temperature  and anuria with casts and elevated NPN, results which compared favorably with results of experimental limb ischemia.  Stoner  and Green (342) followed this work with analyses of blood phosphate and adenosine levels following limb ischemia and shock and found these to be increased anywhere from 25 to 129$.  They  concluded that with a diminished blood supply to a large muscle mass, the blood returning from the area has increased adenosinelike action, and believed that adenosine triphosphate may play a role i n the renal failure death in rabbits.  Bollman and  Flock (44), however, i n careful experiments based on Green's work, were unable to demonstrate a toxic product released during exercise after limb isehemia, saw no.renal impairment and concluded that adenosine triphosphate was not released from ischemic muscle;  the adenosine triphosphate was hydrolyzed and only non-  toxic products were liberated. however, reiterating i n 1945  Green continued in his opinion,  (163) that adenosine triphosphate  is toxic to the kidney and in crush syndrome i t s effect i s magnified by that of myoglobin.  But later (164), i n discussing a  case of traumatic uremia, he concludes that renal anoxia (vasospasm) and toxic metabolites (myoglobin) are responsible for the renal damage. A second approach considered the toxicity of thoracic due.t lymph i n crush injury......trauma and tourniquet shock. Blalock (38) concluded that lymph from dogs suffering five hours  - 34 -  o f crush to one limb  contained  some f a c t o r lowering  sure and producing c a s t s i n u r i n e . a l s o found that blood  pressure  Katzenstein  The  was lowered f o l l o w i n g i n j e c t i o n  first  to t o u r n i -  The e f f e c t was, however, v a r i a b l e . work o f E g g l e t o n (124, 125?  126)  on crush i n j u r y  i n the c a t i s i n t r i g u i n g and has not been a l t o g e t h e r She  pres-  et a l (190)  of t h o r a c i c duct lymph taken from animals subjected quet shock.  blood  reported  discounted.  i n 1942 that crush syndrome could be r e p r o -  duced by a sudden r e l e a s e o f compression whereas gradual prevented development o f impaired  renal function.  release  An i n t a c t  l i v e r appeared to be e s s e n t i a l f o r the d e t o x i f i c a t i o n o f an agent g r a d u a l l y r e l e a s e d able to show (125)  muscle.  that an e x t r a c t o f ischemic  the c r e a t i n i n e clearance not;  from the ischemic  She was  muscle depressed  while an e x t r a c t o f f r e s h muscle d i d  t h a t e x t r a c t s of muscle dead f o r from four t o t e n hours  proved to be t o x i c ;  and that the e x t r a c t appeared t o be a break-  down product o f a l a r g e p r o t e i n molecule formed  anaerobically.  A l s o i n 1944, M l r s k y and F r e i s (244) i n j e c t e d t r y p s i n i n t r a p e r i t o n e a l l y i n t o r a t s and r a b b i t s and produced r e n a l damage. They concluded that t h e i r f i n d i n g s supported the theory extensive  that  t i s s u e damage r e l e a s e d p r o t e o l y t i c enzymes which i n  turn released  "catabolic factors" responsible  f o r r e n a l and hep-  atic injury. Most searches f o r these b i o c h e m i c a l  f a c t o r s have been  - 35 -  concerned with the etiology of shock and many agents have been described since Chambers, Zweifach et a l (78) demonstrated a vaso-excitor material (VEM) i n early shock and a vasodilator material (VDM) i n later shock. to present times  (329)? but  This work has been carried on  both Reinhard et a l  (302) and  Frank  and his co-workers (139) have discounted the importance of this VEM-VDM mechanism i n shock.  They do, however, substantiate the  importance of an intact, functioning liver i n the prevention of irreversible shock.  Page  (297> 302, 34-8) has  also been active  in this f i e l d , having described a serum vasoconstrictor, serotonin, which he believed was the ultimate effector mechanism i n renal ischemia.  He believed the primary stimulus to renal vaso-  constriction was neurogenic. Frank et a l (138,  139) found also that i n dogs suffering  haemorrhagic shock and treated by peritoneal irrigation or a r t i f i c i a l kidney, the blood chemistry picture improved but survival was not prolonged.  They concluded from their work that plasma  electrolyte disturbance, azotemia and hypoglycemia were not responsible for i r r e v e r s i b i l i t y of shock, and that there is no circulating depressor substance i n irreversible shock. To attempt to untangle the voluminous literature on the subject of humoral agents i n shock i s beyond the scope of this thesis, but one other lead i s of interest.  Moyer and Handley  (260) injected dogs with norepinephrine and epinephrine and found that both drugs produce a diminution of the number of active nephrons as indicated by functional tests.  It i s evident that  - 36 the newer investigations i n the field of nephrotoxins have been directed to the discovery of an agent which i s responsible f i r s t for the abnormal vascular responses of the body i n shock and also for the apparent lasting renal ischemia seen as a late complication of shock.  The search for a specific cellu-  lar nephrotoxic agent has therefore been overshadowed by this new concept, which is obviously an outgrowth of a strengthened faith i n what w i l l be described below as the renal ischemia theory of pathogenesis. The role of hemoglobin (and by implication, myoglobin) in the production of renal failure has always involved the question of a direct cytotoxic action of the pigment, as well as a vasospastic action.  Early opinion (21, 22) was that red  blood c e l l stroma was responsible for the symptoms observed i n hemoglobinemia.  Conflicting opinions were soon recorded, how-  ever, and Sellards and Minot (323)  injected small amounts of  laked red cells and found no symptoms and no renal damage. Bayliss (24) considered the problem "Is hemolyzed blood toxic?" and concluded that results of rabbit experiments were not valid because of sensitivities i n the animal and that incompatible transfusion damage was not due to hemolysis as such but was "rather an aspect of the action of foreign serum protein analogous to that responsible for anaphylactic shock".  Reid  (301)  was the f i r s t to suggest a vasospastic action of hemoglobin when he noted a marked but transient decrease i n kidney volume  - 37 -  following injection of d i s t i l l e d water or laked red cells. Mason and Mann (237) continued this work and found that associated with decreased kidney volume there were fewer blood f i l l e d glomeruli and more narrowed arterioles.  It was about this time  (45) that the idea of hemoglobin as a toxin began to be accepted but as Mason and Mann pointed out, most reports did not guarantee the purity of the injected pigment.  Another variable, that of  dosage, was emphasized i n the work of 0'Shaughnessy et a l (276) who found that a 5% solution of hemoglobin in Ringer's was  toler-  ated well as a blood substitute i n doses up to 50 gm. of hemoglobin.  In the years following the re-description of the crush  syndrome in 1941, experimental work was profuse and contradictory, chiefly because l i t t l e attention was paid to the amount of circulating hemoglobin involved (5)»  Other products of hemolysis  (histamine, potassium) were implicated (147), as were related' pigments (31» 32) and conflicting opinions as to the toxicity or vasospastic action of hemoglobin were frequent.  One i s forced  to the conclusion that whatever the action of hemoglobin and related pigments i s , i t is at least not very dramatic and at most only contributory.  That i t does play a role is indicated  in the work of Badenoch and Darmady (12) who  concluded on the  basis of rabbit experiments that hemoglobin per se i s not toxic, but with renal ischemia produced by renal artery constriction, added hemoglobin plays a significant part i n the severity and mortality of the i l l n e s s .  Yuile et a l (377) believed that a  specific renal vasoconstrictive action of hemoglobin was not an  - 38 important factor i n the development of renal insufficiency, hut more recent work by Miller and MacDonald (241) disagrees. These investigators injected homologous hemoglobin solutions into 25 normal males,and on the basis of PAH and inulin clearances postulated again a vasoconstrictor effect of hemoglobin. The cytotoxic action of hemoglobin has also received recent support i n the work of Rosoff and Walter (309) who suggest that "heme" competes with cytochromes i n the oxidative processes of the tubular cells, resulting i n damage, degeneration and necrosis of these cells. In summarizing present opinion on the nephrotoxic theory, i t i s apparent that the original idea, the release of specific cytotoxic agents, has been somewhat neglected and replaced by the search for agents which are responsible for the early phenomena of shock.  In this way, discussions of the nephro-  toxic and ischemic theories fuse to some extent.  The role of  hemoglobin and related pigments also enters the field i n that these agents have been reported to be both cytotoxic and vasospastic.  These aspects of the theory remain controversial and  only the very definite action of such chemical toxins as mercury, uranium and carbon tetrachloride, can be stated with certainty. Renal Ischemia: This third theory of the pathogenesis of acute tubular necrosis puts forth the idea that the renal disease i s primarily due to a diminished or absent renal blood flow.  As a result of  - 39 this ischemia the renal tubular cells undergo degeneration and necrosis, kidney function i s disrupted and the c l i n i c a l picture can progress from one of i n i t i a l shock to acute renal failure with i t s olguria, uremia and death.  Proponents of this theory  believe that shock i s the i n i t i a l event i n a l l cases, whether i t be from trauma, crush, haemorrhage, dehydration, burns or u  anaphylactic reactions.  With the i n i t i a l lowering of blood pres-  sure renal blood flow may cease altogether (probably at blood pressures of 60 to 100 mm. systolic (291) ), so that glomerular f i l t r a t i o n ceases, accounting for the immediate oliguria to anuria.  The tubule cells are also deprived of their blood supply  and, sensitive because of their comparatively high rate of metabolism, suffer varying degrees of anoxic damage.  When the hypo-  tensive or anoxic period i s prolonged, this damage i s severe and even though renal blood flow may be restored, degeneration continues to necrosis and the kidney recovers neither structurally nor functionally.  Death i n acute renal failure ensues.  The disput-  able point i n this theory i s , what i s responsible for the prolongation of renal ischemia beyond the period of i n i t i a l low blood pressure?  There appear i n the literature cases of acute tubular  necrosis, especially i n the field of gall bladder surgery (hepatorenal syndrome), i n which either no or only a very short period of hypotension was recorded and yet renal tubular degeneration occurred.  It w i l l be seen that nervous, humoral and hormonal  agents have been described as producing renal arteriolar constriction to account for the prolonged ischemia, together with  4G  -  the complicated Arterio-venous shunt suggested by Trueta (353). Fishberg (133)  i n 1937  believed that a decreased renal  blood flow, the result of decreased blood volume and cardiac output was the primary pathogenetic factor i n the development of what he then called pre-renal azotemia.  Tomb (351)  also  believed that the renal characteristics of the crush syndrome were due to anoxia and i n 194-5, Maegraith et a l (226) strongly for the renal anoxia theory.  came out  These workers point out  that pigments are not always present and therefore are not essential;  that the nephrotoxic theory demands a wide variety of  toxins;  that circulatory collapse alone i s common to a l l cases.  They leave open the question of what causes the circulatory collapse, but foresee the work of Trueta by suggesting the possibili t y of a redistribution of renal blood flow or glomerular bypass  (225). In his classical review of lower nephron nephrosis i n 1946,  Lucke (213)  devotes some time to the disturbed renal blood  flow theory, pointing out that as Haldane said, "Anoxia not only stops the machinery but wrecks the machine."  He appears, however,  to feel that the heme pigments and nephrotoxins are of f i r s t importance.  It was about this time that Trueta (353)  published  his work on renal vascular shunts and the shift of emphasis to the renal ischemia theory gained impetus.  There were some objec-  tions to the idea, however, notably by Bywaters (68),  who believed  that the characteristic lesion of renal ischemia i s cortical  - 41 -  necrosis and that renal ischemia short of this necrosis produces patehy degeneration of the proximal convolution.  He adds that  in crush and incompatible transfusion, the lesions are i n the distal tubule. In spite of these objections, much of the recent work on acute tubular necrosis has been concerned with i t s shock aspect. Page (280) believed that the entire cardiovascular musculature was altered i n shock and pointed out that experimentally i t i s always necessary to produce shock i n rats before injecting myoglobin solutions, i f lower nephron nephrosis i s to develop. Marshall and Hoffman (233) i n the same year analyzed six cases of lower nephron nephrosis on the basis of mannitol and PAH clearances and concluded that the renal lesion was diminished renal blood flow and loss of function of the lower nephron. They defined lower nephron nephrosis as "a syndrome of oliguria with progressive renal insufficiency following a shockrlike state produced by a variety of acute insults to the body, and i n many cases associated with the deposition i n the renal tubules of various derivatives of hemoglobin and, myoglobin."  This defini-  tion would.appear to be a most satisfactory one i n the light of present knowledge.  Because of the swing towards ischemia as  the chief pathogenetic factor, the description "Renal Anoxia Syndrome" suggested by Maegraith (226) has been emphasized by Gaberman, Atlas, et a l (146) who review the problem, report 22 cases and suggest an etiological classification.  42  C l i n i c a l and pathologic evidence supports this ischemic theory well.  The lesion, bilateral cortical necrosis has for  some years been described as an ischemic lesion (115) and cases of concealed placental haemorrhage  (112),  surgical shock  (286),  burns (53) and so on have been described i n which the renal lesion was anything from slight tubular degeneration to bilateral cortical necrosis.  Functional and pathologic studies indicating  a reduced renal plasma flow have been reported i n alimentary haemorrhage (31) and particularly i n traumatic shock (102, 99, 93, 208, 303, 322, 355, 356) and Herbut (175) l i s t s twelve cases of "severe degeneration to complete necrosis" of renal tubules, a l l of different etiology, in which he emphasizes the common factor of shock.  An interesting case of cardiac arrest for  thirty minutes was reported by Bailey and Rubenstein (13), i n which anuria and uremia developed but recovery occurred. It i s to be expected that, with the idea that a period of hypotension was the prime factor i n initiating the renal dysfunction, much experimental work with circulatory shock i n animals and functional studies i n patients suffering shock were reported.  Corcoran and Page (84), working with haemorrhagic  hypotension i n dogs, showed that the renal blood flow f e l l and f i l t r a t i o n decreased; unequally;  that renal blood flow was distributed  that renal denervation showed restoration of the  renal blood flow;  and that a humoral vasoconstrictor was res-  ponsible for the failure of the kidney to respond to transfusion. Olson et a l (273) used dogs subjected to haemorrhage, burns or  -43  -  crush and were able to produce renal damage which they believe was a result of low blood pressure, low blood volume, hemoglobinemia and an unknown substance, myoglobin or a toxin from ischemic Keele and Slome ( 1 9 D j using cats, found that the renal  muscle.  blood flow reduction was greater, in proportion, than the lowering of blood pressure by crushing the limb.  They therefore  believed that reduction of renal blood flow was not due only to reduction i n blood pressure.  Selkurt (322) confirmed this  finding i n dogs i n haemorrhagic shock, measuring renal blood flow both directly and by plasma extraction of PAH and diodrast. Although the kidney changes (described by Goldblatt) were minimal, i t seems l i k e l y that a true? acute tubular necrosis was obtained, i n view of Oliver's recent work (271).  Selkurt  concluded that i n shock the kidney received proportionally less of the cardiac output (5% instead of the usual 20%) due to intrarenal vascular resistance which may be of humoral or nervous origin.  The gradual onset suggested a humoral agent;  the  restoration suggested a nervous mechanism. The work of Van Slyke and his group (291» 355» 356) has perhaps been the most complete along these lines.  They pro-  duced shock i n dogs by haemorrhage or by blows on the thigh with a mallet to hold blood pressures below 70 to 80 mm.  of mercury.  They concluded that with a sudden massive haemorrhage there was an immediate drop i n blood pressure with renal arteriolar constriction.  If blood pressure dropped below 60 to 100  renal blood flow and function ceased.  mm.,  If the i n i t i a l loss of  - 44 -  blood was not too great, the blood pressure was restored byperipheral vasoconstriction, which i s slower than the renal response and kidney function was restored to less than the prehaemorrhagic level.  This cycle could be repeated to the toler-  ance of the animal and even then partial renal function could be restored by transfusion.  But i f the depletion of blood was too  great, transfusion became useless because peripheral constriction was replaced by dilatation and function failed.  Eventually  also efferent arteriolar construction, which maintained glomerular f i l t r a t i o n , failed, and complete failure ensued even though blood pressure was maintained at 60 to 1GG mm.  They found that  trauma produced a similar series of events and concluded that, while i n man i t appeared possible to restore the circulation by transfusion to prevent death by shock without restoring enough kidney function to maintain l i f e and so allow death i n uremia, i n dogs deaths appeared to be almost consistently from shock. Though they believed i t almost impossible to get uremia i n these animals, i t w i l l be seen that, i n Oliver's examination of kidneys from these dogs (27D characteristic tubular lesions were indeed seen. Functional studies i n c l i n i c a l cases tend to show the same reduction of renal blood flow in shock.  The work of  Cournand and Lauson (93, 208) i s again classical in the c l i n i c a l field.  Determinations of glomerular f i l t r a t i o n rates and renal  plasma flows i n cases of trauma, haemorrhage, peritonitis, burns and head injuries with and without shock showed that "the rate  - 45 -  of glomerular f i l t r a t i o n and effective renal plasma flow are significantly reduced i n nearly every patient suffering from shock, the degree of reduction being roughly proportional to the severity of shock".  Again, because renal blood flow  decreased more than did arterial pressure, they concluded that "a considerable degree of renal vasoconstriction must have been present",  and because glomerular f i l t r a t i o n rate f e l l  more than did the arterial pressure, they concluded that there must have been increased afferent arteriolar constriction. They noted that tubular damage apparently persisted for longer than impaired renal blood flow.  In later work Van Slyke  (355)  summarized results of this work by describing an ischemic phase of shock kidney i n which there is renal vasoconstriction as compensation for the low blood pressure, and a renal damage phase of shock kidney i n which reversible to irreversible renal failure i s seen.  That i s , there must be a degree of shock  sufficient to result i n renal failure, but not enough to cause death from shock.  These workers (111) also point out that nor-  mally the kidney extracts less oxygen than do other tissues, and the increased renal extraction of oxygen i n shock i s much less than the increased extraction i n other tissues. A second experimental approach to the problem has naturally been the production of definite renal ischemia by occlusion of the kidney blood supply.  As long ago as 1923  Marshall and Crane (234) noted that temporary closure of the  - 46 -  r e n a l a r t e r y r e s u l t e d i n a n u r i a f o r a p e r i o d l o n g e r than the c l o s u r e , presumably because the t u b u l e s were more s e n s i t i v e t o anoxia and f u n c t i o n was i n t e r f e r e d w i t h .  S t a r r (337)  produced  a l b u m i n u r i a i n animals and man by r e n a l a r t e r y c o n s t r i c t i o n and by a d r e n a l i n e and ephedrine was unable  i n j e c t i o n and emotional u p s e t .  to recognize r e n a l s t r u c t u r a l damage i n the animal McEnery et a l (217)  experiments.  r e p o r t e d e l e v a t e d blood  l e v e l s , o l i g u r i a and a n u r i a and c o r t i c a l p a l l o r w i t h c o n g e s t i o n i n kidneys blood  He  f o l l o w i n g temporary clamping  urea  medullary  o f the r e n a l  supply. The more recent e r a o f r e n a l a r t e r y o c c l u s i o n began soon  a f t e r the c r u s h syndrome r e c e i v e d so much a t t e n t i o n . Keele  (312)  crush kidney  d e s c r i b e uremia and r e n a l pathology  s i m i l a r t o the  (but w i t h proximal tubule degeneration)  i n g the r e n a l p e d i c l e f o r up to two hours.  S c a r f f and  a f t e r clamp-  They conclude,  "Thus  t h e r e i s a p o s s i b i l i t y t h a t the kidney l e s i o n i n cases o f c r u s h i n j u r y might be due to r e n a l ischemia  ... ".  S e l k u r t (320),  w i t h s h o r t e r p e r i o d s o f ischemia, records reduced  i n u l i n , diodrast  and PAH c l e a r a n c e s and tubule damage " s i m i l a r t o mild uranium poisoning";  he l a t e r confirmed  these f i n d i n g s by d i r e c t  t i o n s o f r e n a l blood flow and b e l i e v e d t h a t a f f e r e n t c o n s t r i c t i o n decreased  the glomerular  Badenoch and Darmady ( 1 1 ) , t e m p o r a r i l y , obtained  determina-  arteriolar  f i l t r a t i o n r a t e (321).  i n o c c l u d i n g the r e n a l a r t e r y o f r a b b i t s  e l e v a t e d blood urea l e v e l s and r e n a l t u b u l a r  damage which appeared s i m i l a r to t h a t seen i n human traumatic uremia.  K o l e t s k y and Gustafson  (195)  p o i n t e d out t h a t the r e n a l  - 47 -  lesion obtained by clamping the renal pedicle l£ to 2 hours i n rats was not the same as seen i n human or experimental crush syndrome or i n rats with tourniquet shock;  here the lesion  was proximal tubule degeneration whereas i n crush i t was lower nephron nephrosis.  Later work (194) demonstrated that healing  of the necrotic epithelium was possible.  Scheibe et a l ( 3 1 3 ) ,  in clamping the renal pedicle or vein alone, concluded also that the proximal convolution was more sensitive to anoxia. Again, the work of Phillips et a l (291, 292) i s outstanding.  In noting that renal artery compression depressed  urea clearance, they suggested that three possibilities were apparent:  decreased renal blood flow, decreased plasma f i l t r a t i o n or  increased reabsorption of urea by devitalized tubules.  They  clamped the l e f t renal artery of right nephrectomied dogs for two hours, then determined PAH and creatinine clearances.  They  observed that blood flow was soon re-established after two hours occlusion, but that PAH and creatinine extraction decreased progressively, indicating progressive tubular damage, and concluded that resultant urea retention was probably due to back diffusion. They emphasized i n this work the fact that PAH and diodrast clearances can be used as an indication of plasma flow only when the tubules are undamaged.  More recent work ( 3 0 8 , 41) has con-  firmed and elaborated on these conclusions based on renal artery occlusion i n dogs. These investigations leave l i t t l e doubt that renal  - 48 -  anoxia plays a prominent r o l e i n the p r o d u c t i o n  of r e n a l  damage i n many of the c l i n i c a l e n t i t i e s p r e s e n t i n g acute r e n a l f a i l u r e .  tubular  a picture of  They a l s o i n d i c a t e that a c t i v e r e n a l  a r t e r i o l a r c o n s t r i c t i o n i s a prominent f a c t o r i n the development of the anoxia, d e s p i t e that there causing  the statement of Schroeder and  i s l i t t l e r e n a l v a s o c o n s t r i c t i o n , i n shock.  50)  t h a t as long ago  of p e r i p h e r a l and Results  pigments).  splanchnic  nerves produced r e n a l  vasoconstric-  l e v e l s and  i n various  to conclude that p a i n impulses t r a v e l l i n g i n the  of shock.  t r a c t s may  Wolff (366)  anuria, believed  be r e s p o n s i b l e  a l s o was  S i r o t a (52)  (20)  and  resulted i n  led to conclude t h a t there  published  resumption o f kidney  I t was  i n f u l l i n 1946  that emphasized the importance of neurogenic r e n a l t i o n o r i g i n a t i n g i n damaged limbs. agents" stimulated  was  remarked on a r e f l e x r e n a l  ischemia from manipulating e x c i t a b l e r a b b i t s . work, begun i n 1942  of cystoscopy pro-  flow and  some f a c t o r which allowed r a p i d c e s s a t i o n and Brod and  dorsal  f o r the v a r i o u s phenomena  that the p a i n or d i s c o m f o r t  Donnelly (113)  f u n c t i o n , and  et a l  i n d i s c u s s i n g the mechanism of r e f l e x  duced s t i m u l i which reduced r e n a l blood anuria.  re-  stimulation  ways, p r i o r to muscle trauma shock, l e d Swingle, K l e i n b e r g  spinothalamic  the  of c o n t r o l l e d experiments i n dogs i n which the  nerve t r a c t s were i n t e r r u p t e d at v a r i o u s  (346)  agent  Darmady (99)  as 1859» Bernard observed t h a t  (316)  Two  have been suggested, the nervous and  humoral ( i n c l u d i n g endocrines and  tion.  The  t h a t v a s o c o n s t r i c t i o n has not been i d e n t i f i e d .  mechanisms (146,  ported  Steele  Trueta's (353)»  vasoconstric-  They b e l i e v e d that  "noxious  nerves p e r i p h e r a l l y or c e n t r a l l y to d i v e r t  - 49 -  renal blood flow to save the cortex from the t o x i n .  That i s ,  these workers suggested that there was neurogenic i n i t i a t i o n of a corticomedullary "shunt" of renal blood. w i l l be discussed f u l l y below. 361)  This concept  Several other reports ( 2 0 0 , 2 1 0 ,  appeared to support the idea that chronic stimulation of  the renal nerves was and Cort ( 9 2 ,  91)  responsible for renal vasoconstriction,  stated that trauma produced a s c i a t i c - s p l a n c h -  nic r e f l e x which could be interrupted by sympathetic block to allow d i u r e s i s .  Smith (333)? however, summarized opinion on  nervous control of renal hemodynamics by stating 'categorically, "the physiological control of the renal c i r c u l a t i o n remains almost a complete mystery," and points out that, i n animals under l o c a l anesthesia, spinal anesthesia or denervation of the kidney does not produce renal hyperemia and that renal blood flow i s normally determined by "autonomous i n t r i n s i c a c t i v i t y of the renal a r t e r i o l e s and i s not dependent upon tonic a c t i v i t y i n sympathetic pathways."  Nevertheless  i t would appear probable  that the immediate response of the kidneys to d r a s t i c blood loss i s neurogenic i n nature  (329).  Several humoral agents are known to constrict renal a r t e r i o l e s but whether they are responsible for this phenomenon i n the traumatic anuria syndrome i s not known. considers a number of these (adrenalin (316, angiotonin (321,  Smith  260),  (333)  renin or  42) and histamine) and notes that f r i g h t , exer-  cise and pain a l l decrease the renal plasma flow.  Corcoran and  Page (84) consider that humoral vasoconstriction i s responsible,  -  50  -  and eventually Rapport, Green and Page ( 2 9 7 ) isolated from beef serum this agent, serotonin, which had a vasoconstrictive power twice that of epinephrine. (34-8)  In later work, Taylor and Page  concluded that the primary stimulus to renal vasoconstric-  tion in tourniquet shock was neurogenic, but because the denervated kidney also showed vasoconstriction, believed that the ultimate effector mechanism was humoral.  Adenosine t r i -  phosphate, released from crushed muscle, was thought by Stoner and Green (342) to play a possible role in the production of renal ischemia, while Moyer and Handley ( 2 6 0 ) showed that norepinephrine had the same action as adrenaline, increasing renal resistance by efferent arteriolar constriction.  Finally,  as was mentioned i n the discussion of the role of pigments i n the production of anuria, i t has been suggested that hematin (85)  and hemoglobin i t s e l f ( 6 2 , 2 1 6 , 241) have a vasoconstric-  tive effect and may even produce "ischemic alteration of the glomerular capillaries". Whatever the cause, neurogenic or humoral, renal vasoconstriction appears to be a phenomenon, accepted by most observ ers, which is part of a general response to shock.  The  immediacy of this response would suggest that neural pathways are at least early responsible, while prolongation of vasoconstriction may be due to a humoral agent.  Investigations into  the pathogenesis of shock i t s e l f , such as carried out by Shorr et a l ( 3 2 9 ) and Frank et a l (140, 1 3 9 ) may produce progress i n  -  this  51  -  direction. Trueta's corticomedullary shunt of renal blood flow: It was i n 1947 that Trueta et a l (353)  f u l l results of experiments  begun i n 1942  published i n  (20, 141) to study  the problem of a r t e r i a l spasm i n response to trauma.  A com-  plete review of this very thorough work i s impossible at this point, but i n essence the theory i s based on morphological differences between c o r t i c a l and juxtamedullary glomeruli (Figure 2), which, under stimulus of trauma, shock and so on,  Figure 2  allow the renal blood flow to be shunted through the  juxtamedul-  l a r y glomeruli, bypassing the c o r t i c a l ones and the main mass of tubules.  Trueta et a l believe that a reflex neurovascular  - 52 -  mechanism i s responsible for these profound alterations of intrarenal circulation.  They observed this change by angiographs, by  direct observation of cortical pallor and "stream-lines" and by injection masses (neoprene latex and india ink) i n response to tourniquets, sciatic nerve stimulation, haemorrhage and various drugs (adrenalin, p i t u i t r i n , pitressin, ephedrine and staphylococcus toxin);  they made observations on dogs, cats, rats,  guinea pigs but chiefly i n rabbits.  Because of this shunt,  cortical glomeruli (which constitute 85% of renal glomeruli) are rendered ischemic which i n turn renders at least an equal percentage of renal tubules ischemic (see Figure 2 ) , accounting for the renal dysfunction seen i n traumatic uremia, crush syndrome, b i lateral cortical necrosis and other renal diseases. Trueta's shunt theory implies the following: (1) that so called juxta-medullary glomerular differences are present i n species other than rabbits.  Trueta states that these differences are  seen most often i n rabbits, but less so i n the dog, cat, guinea pig and rat, and that the differences are less evident i n man; (2) that these juxta-medullary glomeruli function differently from the cortical glomeruli.  Trueta suggested that they constitute a  virtual arterio-venuous anastomosis and pointed out that the close relationship of the vasa recta and loop of Henle may have some significance in water reabsorption;  (3) that the shunt, when i n  operation, allows blood to bypass tubules whose function i s essent i a l for normal kidney function with the result that less oxygen is utilized by the kidney.  Trueta observed streams of red,  - 53 oxygenated blood i n the renal vein when the shunt was said to be i n operation and therefore implied that the arterio-venous oxygen difference was decreased i n this syndrome;  (4) that,  essentially, the total renal blood flow was not reduced but that this total flow was merely short-circuited through the "lesser circulation of the kidney".  In his early work, how-  ever, Trueta himself contradicted this implication by his observation that the renal blood supply was reduced by neurogenic renal vasoconstriction.  It i s on these four points that  Trueta's shunt theory has been most severely c r i t i c i z e d . It i s interesting that, many years before Trueta suggested his corticomedullary shunt, observations were made on kidney pathologies which support his ideas.  Renal pathology i n fatal  blood transfusions was frequently described as an enlarged kidney with pale cortex and congested, red-brown medulla;  micro-  scopically the glomeruli were bloodless and interlobular capillaries (vasa recta) were engorged  (365)•  And Trueta him-  self pointed to a strong support of his ideas in the kidneys described as bilateral cortical necrosis (115> 336) i n which the entire cortex becomes necrotic because of interference with its blood supply.  This would appear to be the extreme instance  of Trueta's shunt, as suggested by Heggie (179)-  It i s interest-  ing, too, that i n 1944, Maegraith and Findley (223) predicted Trueta's theory when they described the kidneys of Blackwater fever as having an anemic cortex but congested medulla and  - 54 -  suggested that the renal glomerular flow was short-circuited. Again i n 1946 (225) they pointed out that the renal blood flow must be redistributed i n the kidney and from the histological appearance, suggested a corticomedullary diversion. The shunt theory received some early support from the work of Simken et a l (330) who injected into the renal artery glass spheres of such a size that, when they were recovered, these workers were forced to conclude that arterio-venous bypasses existed i n the kidney or i t s capsule.  Experiments were  carried out i n rabbits, dogs and human kidneys.  The controversy  of arterio-venous anastomoses has been apparent for many years and i s reviewed adequately i n Smith's book  (333)•  Arcadi and  Farman ( 8 ) were able to duplicate Trueta's work by India ink injections i n rabbits, as ware Goodwin et a l (154), using tourniquets or sciatic nerve stimulation i n rabbits, dogs, eats and monkeys.  They observed kidneys directly, used india ink and  evans blue injections and visualized the renal vasculature with thorotrast, and believed they demonstrated a neurovascular cont r o l of the renal circulation i n which renal ischemia started i n the cortex and spread to the medulla.  They saw the possibility  of a true ischemia here, however, and questioned whether the phenomenon was a shunt or rather a progressive peripheral vasoconstriction.  Black and Saunders (35)  also supported Trueta's  observations with the reservation that, before the shunt i s accepted, three criteria must be satisfied:  (1) low inulin and  PAH clearances with increased C i to Cp^g ratio, n  ( 2 ) PAH  - 55 extraction less than 80% and (3) absence of gross changes i n general circulation, since efferent arteriolar constriction, rise i n renal vein pressure or f a l l i n systemic pressure could produce a picture simulating"the shunt. Evidence against the shunt also appeared early and has continued to accumulate since Barclay et a l (17) expressed their unhappiness that clearance methods had not been used by Trueta. Most criticism has been from this point of view.  Trueta's obser-  vations imply that total renal blood flow i s not reduced i n the syndrome, the change being merely a short-circuiting of blood rather than a reduction i n flow; blood flow, however.  he did not measure the renal  Based on many reports of functional studies  carried out in animals (257» 302a, 258, 183» 259, 260) and i n man (347, 302a, 331> 18) i t appears conclusive that i n the kidneys of sciatic stimulation (347» 257> 258, 259), adrenalin injection (302a, 183, 258, 259, 260), carbon tetrachloride poisoning (33D and incompatible transfusion (18), there is. i n fact a true reduction of renal blood flow.  Moyer et a l (258) substantiated this  claim by direct measurement of renal blood flow.  Further inroads  into Trueta's theory have been made i n attempts to demonstrate the shunt by injection methods.  Maluf (232) got no shunting of blood  from cortex to medulla as shown by India ink injections i n dogs dehydrated and receiving hemoglobin injections.  Kahn, Sheggs  and Shumway (189) injected India ink under pressure into kidneys of rabbits treated with epinephrine, pitressin, amyl nitrate, haemorrhage, central sciatic stimulation and renin and saw no  - 56 evidence of a bypass.  Schlegal and Moses (314) formed the  same conclusions using a fluorescent dye to visualize renal blood vessels of rabbits in tourniquet shock, and Block et a l (40), using neoprene, state that "the only consistently existing vessels which directly communicate between the renal arteries and veins ... are situated at the hilum of the kidney." Trueta's implication that oxygen utilization by the kidney was reduced has also been discounted.  Repeated determin-  ations of arterio-venous oxygen differences by various workers (258, 259? 260, 257, 302A, 331, 18, 183) have shown that this difference, normally very small, has remained the same or has increased, rather than decreased as implied.  Trueta's observa-  tion of arterialization of renal venous blood is not substantiated by direct measurement of oxygen levels. A fourth interesting contradiction of Trueta's work is reported by Mukherjee ( 2 6 3 ) , who states that in dogs subjected to tourniquet shock, radioactive isotopes indicate that renal anoxia i s diffuse, not localized as Trueta suggests.  He notes  that the proximal nephron i s affected as well as d i s t a l . No criticism of the work of Trueta et a l can be complete without a consideration of the conclusions reached by Maxwell, Breed and Smith (238) on.the significance of the renal juxtamedullary circulation i n man.  These men point out (1)  that,  since the proximal segment i s responsible for excretion, when blood i s perfusing the vasa recta which are in contact only with  - 57 -  d i s t a l and thin segments PAH clearance should be low;  (2) that  with the bypass, f i l t e r i n g surface i s reduced and therefore inulin extraction should be reduced;  (3) that renal arterio-  venous oxygen difference should be decreased with shunt and (4) that reduction in PAH and inulin extractions alone would not indicate finally a shunt, because proximal convolution damage dould do i t ;  but reduction of these with a normal renal blood  flow would be good evidence of a shunt.  They found that none  of these criteria were satisfied i n cases of old age, pitressin or adrenalin injection, hypertension, congestive heart failure and shock anuria.  They concluded as follows:  (1) juxtamedul-  lary glomerular function does not differ from cortical;  there  is the essential relationship between tubules and vasa recta which allows usual kidney function;  (2) i f diversion did occur,  to produce cortical ischemia, renal function would continue by way of juxta-medullary glomeruli;  (3) evidence i s against  diversion of blood through uncleared channels;  (4) juxtamedul-  lary circulation in man has no unique functional significance. It i s seen i n the rabbit only as a species difference. a •  On the basis of evidence cited above, the conclusions of Maxwell et a l are justified.  Observations made on renal  blood flow and arterio-venous oxygen differences are not compatible with Trueta's concept of a corticomedullary shunt of renal blood i n the traumatic anuria syndrome.  The thoroughness of  Trueta's experiments, however, convince one that the phenomenon is of frequent occurrence at least i n rabbits, and the occurrence  -  58 -  of c l i n i c a l cases of bilateral cortical necrosis adds to one's conviction that the Trueta shunt may indeed have a place i n the scheme of things as an extreme i n a series which includes anything from undisturbed renal blood flow to complete cessation of that flow. Such a survey of the pertinent literature leads on inevitably to the conclusion that renal ischemia i s of prime pathogenetic importance i n the development of acute renal failur in shock, burns and crush.  The work of Cournand's group (93»  208) showed conclusively that i n shock from trauma with or without haemorrhage, peritonitis, abdominal injury and burns, "the rates of glomerular f i l t r a t i o n and effective renal plasma flow are significantly reduced ... the degree of reduction being roughly proportional to the severity of shock."  The work, too,  of Van Slyke's group (355» 356, 291, 111) has substantiated thes observations in dog experiments i n which shock was induced by haemorrhage and by trauma.  Phillips and Hamilton (292) and  others (312, 320, 11, 195, 194, 313) completed the cycle of i n formation when, by clamping the renal artery to produce ischemia they produced renal dysfunction as measured by clearance techniques and tubular lesions described by Oliver (271) as being identical to those seen i n shock or crush kidneys.  Both the  functional and structural changes seen i n the kidney i n human cases of shock from any cause, therefore, have been observed i n animals subjected to experimental shock and in animal kidneys with obvious ischemia induced by clamping of the renal artery.  - 59 -  The  p a t h o g e n e t i c s i g n i f i c a n c e was apparent t o B l o c k et a l (41),  when they undertook t o c a r r y out t h i s complete c y c l e o f e x p e r i ments i n dogs.  They subjected  28 dogs to p e r i o d s  of 70 mm. o f mercury f o r p e r i o d s studied  o f hypotension  o f from s i x t o 26 hours and  kidney f u n c t i o n and l a t e r h i s t o l o g i c a l changes i n f i f t e e n  animals, the e a r l y h i s t o l o g y i n t h i r t e e n .  They a l s o  the e f f e c t o f r e n a l a r t e r y o c c l u s i o n from three  studied  t o s i x hours on  24 dogs and 23 r a t s (one t o three hours o c c l u s i o n ) , as w e l l as the e f f e c t o f epinephrine on e i g h t dogs. observed changes i n r e n a l tubules  In a l l c a s e s , they  from d e g e n e r a t i o n t o complete  c o r t i c a l n e c r o s i s , but found that death i n r e n a l f a i l u r e was rare;  i t required  kidney.  almost complete i s c h e m i c d e s t r u c t i o n o f the  They s t a t e t h a t these r e s u l t s were independent o f the  nerve supply and that there was no evidence f o r T r u e t a ' s shunt. I t i s a l s o o f importance to c o n s i d e r r e n a l ischemia i s produced. that an i n i t i a l p e r i o d  the means by which  In cases o f shock i t i s obvious  o f hypotension i s r e s p o n s i b l e  c e s s a t i o n o f r e n a l blood flow ( 2 9 1 ) .  Renal v a s o c o n s t r i c t i o n  f o l l o w s and appears to be p r i m a r i l y neurogenic, humoral  f o r the  secondarily  (348). A t h i r d p o s s i b l e source o f anoxia i s a decreased oxygen  e x t r a c t i o n by the kidney; the c o n t r a r y ,  arterio-venous  most i n v e s t i g a t o r s p o i n t out t h a t , on oxygen d i f f e r e n c e s a r e increased  that i s , that oxygen e x t r a c t i o n by the kidney i n shock i s increased  r a t h e r than decreased  ( 2 5 8 , 259> 3 0 2 a , 1 8 3 ,  111).  —  - 60 -  Normal r e n a l oxygen e x t r a c t i o n , however, i s s i n g u l a r l y i n e f f i c i e n t and  arterio-venous  oxygen d i f f e r e n c e s are so s l i g h t  that  they may  be w i t h i n the e r r o r o f methods o f d e t e r m i n a t i o n  (39,  238).  Summary of Pathogenesis In c r y s t a l l i z i n g an o p i n i o n on the pathogenesis o f acute r e n a l f a i l u r e due  to acute t u b u l a r n e c r o s i s , one  impressed by the c o n c l u s i o n s 1951  and  reached by O l i v e r et a l (271)  by the work of P h i l l i p s , Van  to a l a r g e extent, O l i v e r ' s opinions  Slyke,  afforded  and  the c o n f i r m a t i o n  in  et a l , on which  were based.  made c l i n i c a l l y by Lauson, Cournand et a l (208, convincing  is  Observations 93)  of experimental  are  also  conclusions  by the work of B l o c k , et a l , i n September 1952  (41/)  appears t o complete the p i c t u r e of p a t h o g e n e s i s . I t i s at f i r s t of two  e s s e n t i a l to emphasize the  existence  pathogenetic mechanisms of prime importance.  there appears l i t t l e doubt that i n c e r t a i n c l i n i c a l s p e c i f i c e x t r i n s i c t o x i c agents are r e s p o n s i b l e of tubular  1  Firstly, entities,  for disruption  c e l l metabolism, producing the acute t u b u l a r  necrosis.  Common among these agents are b i c h l o r i d e of mercury, carbon t e t r a c h l o r i d e and  uranium s a l t s .  O l i v e r (271)  has named the  they produce "nephrotoxic t u b u l a r n e c r o s i s " and v i n c i n g l y the p o i s o n i n g s and damage.  described  lesions con-  c h a r a c t e r i s t i c p a t h o l o g i c a l l e s i o n seen i n these the d i f f e r e n c e s from the  second type o f  tubule  - 61 -  Secondly, there i s a l a r g e group of c l i n i c a l c o n d i t i o n s i n which shock i s a common f a c t o r and which f r e q u e n t l y (146, 335)  g i v e r i s e to the syndrome which we  r e n a l f a i l u r e due  p r e f e r to c a l l  to acute t u b u l a r n e c r o s i s " .  (252):  "acute  Such c o n d i t i o n s  have been d e s c r i b e d e a r l i e r i n t h i s t h e s i s but can be briefly  114,  trauma, haemorrhage, c r u s h , burns,  listed  peritonitis,  h e p a t o r e n a l syndrome, r e t r o p l a c e n t a l haemorrhage and o t h e r s . There i s no doubt that t h i s second p a t h o g e n e t i c mechanism, r e n a l ischemia, i s of prime importance  here.  p a t h o p h y s i o l o g i c a l phenomena may  w e l l be as f o l l o w s :  induced from any cause the i n s u l t may  A resume o f the probable i n shock  be so severe o r the  r e s i s t a n c e t o i t so low t h a t the i n d i v i d u a l progresses to the socalled  i r r e v e r s i b l e stage and d i e s i n p e r i p h e r a l  failure;  or the i n d i v i d u a l may  circulatory  respond w e l l to the s t r e s s  recover u n e v e n t f u l l y from the hypotensive shock p e r i o d ; may  and  or he  make an apparent r e c o v e r y from the acute shock p e r i o d o n l y to  pass i n t o what might be c a l l e d a l a t e e f f e c t o f shock, a s t a t e of  acute r e n a l f a i l u r e w i t h o l i g u r i a , a n u r i a and, once a g a i n ,  e i t h e r r e c o v e r y or death i n uremia. acute r e n a l f a i l u r e , i s t y p i f i e d  This third  possibility,  by cases i n which massive haem-  orrhage i n i t i a t e s the r e n a l f a i l u r e .  With the haemorrhage there  i s an immediate f a l l i n systemic blood p r e s s u r e which, i f s l i g h t , may  still  a l l o w glomerular f i l t r a t i o n due  renal efferent a r t e r i o l a r constriction. blood p r e s s u r e f a l l s below 60 to 1G0  mm.  to the compensatory But i f the systemic of mercury (291)  renal  blood flow and f u n c t i o n cease. ' Accompanying t h i s e a r l y hypo-  - 62 t e n s i v e p e r i o d there i s r e n a l v a s o c o n s t r i c t i o n which i s p a r t o f a g e n e r a l i z e d compensatory v a s o c o n s t r i c t i o n which may the i n d i v i d u a l compensating  enough to s u r v i v e the acute shock  p e r i o d f o l l o w i n g the haemorrhage. e i t h e r by t h i s compensation  Blood p r e s s u r e can be r e s t o r e d  or by t r a n s f u s i o n and the kidney  c i r c u l a t i o n returned to something levels.  result i n  l e s s than  pre-haemorrhagic  In s p i t e of t h i s apparent r e t u r n to normal,  some cases  go on to t u b u l a r d e g e n e r a t i o n and n e c r o s i s w i t h c l i n i c a l renal f a i l u r e .  Because i n both c l i n i c a l and  acute  experimental cases  c l e a r a n c e techniques and d i r e c t measurements show r e n a l blood flow to be d i m i n i s h e d , and because experimental o c c l u s i o n of the r e n a l a r t e r y i n animals can produce  lesions i d e n t i c a l with  those seen i n c l i n i c a l acute t u b u l a r n e c r o s i s , the damage has been blamed p r i m a r i l y on i s c h e m i a .  Whether the i n i t i a l p e r i o d  of hypotension, when prolonged, i s s u f f i c i e n t i n i t s e l f to produce the damage, or whether the e a r l y (probably neurogenic) r e n a l v a s o c o n s t r i c t i o n i s prolonged e i t h e r by nerve impulses by humoral agents t o p r o l o n g the anoxemia cannot be  or  stated  definitely.  The r o l e of i n t r i n s i c n e p h r o t o x i c agents, presumably released  from damaged or ischemic t i s s u e s , i n the  pathogenesis  of t h i s syndrome can be l e s s c o n v i n c i n g l y s t a t e d .  The s t a t u s  of the v a r i o u s e x t r i n s i c chemical agents has been mentioned previously.  However, the presence of a p r o t e i n breakdown  product of ischemic muscle (125)  o r a t o x i n from m a s s i v e l y des-  troyed t i s s u e s , to d i s r u p t the metabolism  of t u b u l a r c e l l s would  - 63 -  appear to be unnecessary to explain the renal damage since i n most of these cases, shock and reduced renal blood flow are accompaniments.  Other humoral agents (renin, VDM, serotonin,  adenosine triphosphate), i f they prove to be of some importance, probably are so by virtue of their shock-producing or renal vasoconstrictive properties.  Hemoglobin pigments as well play at  least only a minor nephrotoxic  role either by an unproven cyto-  toxic action ( 6 , 309) or by a renal vasospastic action. It i s therefore implied that cellular anoxia i s responsible for the tubular damage, which i n turn accounts for the renal dysfunction.  The mechanism of this dysfunction is prob-  ably "back diffusion" (303, 1 1 7 ) , in which the dead tubule cells act as a membrane through which tubular f l u i d , urea and other wastes diffuse back to the i n t e r s t i t i a l fluid and thence into the circulation. The place of pigment cast obstruction i n the development of renal dysfunction, so evident i n the intravascular hemolyses, has been given i t s proper place by Oliver et a l ( 2 7 D and Block et a l (41).  Pigment cast obstruction i s at least unnecessary.  Clinical lower nephron nephrosis i s seen i n cases in which no pigment release i s involved and acute tubular necrosis can be produced experimentally without appearance of hemoglobin or related substances.  Again, c l i n i c a l l y , intravascular release  of pigments usually occurs i n cases in which there i s associated shock —  transfusions, Blackwater fever, post-operative  1  - 64 -  transurethral prostatectomy, crush, burns —  so that ischemia  is probably contributing more to the renal failure than i s tubule obstruction.  But, as Oliver has pointed out, i n cases  i n which pigment casts are prominent, one cannot ignore the fact that the involved tubules are plugged and w i l l not carry urine.  If there i s back pressure i n these plugged tubules  and tubule dilatation occurs, or i f tubular fluid escapes into the interstitium, then occlusion of adjacent, otherwise patent tubules might well occur, adding to the obstruction or damage by increased intrarenal pressure ( 2 8 9 , 364).  Although i t  appears possible to produce renal shutdown from induced hemoglobinemia ( 2 3 2 , 1 6 8 , 241), i t i s always easier to induce the renal damage when dehydration (202-206) or renal anoxia (309, 41) are present.  It would therefore appear that the presence  of circulating pigments merely adds to renal damage induced by renal ischemia originating i n shock or severe dehydration. Associated with pigment cast obstruction i s the problem of precipitation of hemoglobin, myoglobin or related pigments. It i s probable that a variety of factors (15, 376, 240), urine pH, glomerular f i l t r a t i o n , urine salt content, tubular reabsorption, combine i n the lower nephron and collecting tubules to produce conditions favorable for heme pigment precipitation.  EXPERIMENTAL  AIM As was s t a t e d e a r l i e r , i t was thought  advisable to  repeat i n a systematic way some o f the work done by other i n v e s t i g a t o r s i n order to determine the r o l e o f c e r t a i n f a c t o r s i n the p r o d u c t i o n o f traumatic a n u r i a .  Preliminary  then, were c a r r i e d out w i t h the aim o f producing  experiments,-,  "lower  nephron  n e p h r o s i s " i n a standard way i n a s u b s t a n t i a l p r o p o r t i o n o f t e s t rats.  Once t h i s s t a n d a r d i z a t i o n was accomplished,  experiments were designed  further  t o t e s t the e f f i c a c y o f c e r t a i n  hor-  monal agents i n the a l l e v i a t i o n o f the kidney damage. A sampling  o f the experimental  l i t e r a t u r e on t h i s  sub-  j e c t has shown that three f a c t o r s p l a y a prominent r o l e i n the pathogenesis  o f acute t u b u l a r damage.  The pigment c a s t o b s t r u c -  t i o n theory can be a s s o c i a t e d c l o s e l y w i t h the n e p h r o t o x i c of pathogenesis  and so the f i r s t v a r i a b l e f a c t o r chosen was the  r o l e o f myoglobin (muscle damage.  theory,  hemoglobin) i n the p r o d u c t i o n o f kidney  I t was not considered c r u c i a l t o t h i s work whether the  pigment produced i t s e f f e c t by a t o x i c a c t i o n o r by o b s t r u c t i o n , though o b s e r v a t i o n s on t h i s problem w i l l be made. f a c t o r considered was what may be c a l l e d  The second  c l i n i c a l shock, and here  - 66 -  a c r u s h i n j u r y to the limb of the t e s t animal was production.  the means of  Again, whether the p a t h o g e n e t i c mechanism was  one  of r e l e a s e of nephrotoxic m a t e r i a l s from damaged t i s s u e s or simply one o f p r o d u c t i o n o f prolonged hypotension was ered, though some c o n c l u s i o n s w i l l be drawn. controlled  i n . t h e f o l l o w i n g experiments  animals, s i n c e d e h y d r a t i o n (202, emphasize, i n some way,  203,  was  232)  not c o n s i d -  The t h i r d  factor  the h y d r a t i o n o f the has been shoira to  the t u b u l a r damage to the kidney.  I t can be seen that the aim of the experiments  reported  below has not been p r i m a r i l y to i n v e s t i g a t e the pathogenesis  of  acute t u b u l a r n e c r o s i s , but r a t h e r to s t a n d a r d i z e the p r o d u c t i o n of the syndrome i n the white r a t , and to i n v e s t i g a t e the t h e r a p e u t i c p o s s i b i l i t i e s of v a r i o u s hormones. mechanism i n v o l v e d , based  Statements  as to the  on these experiments, w i l l t h e r e f o r e  be impressions r a t h e r than c o n c l u s i o n s drawn from c o n t r o l l e d  ex-  periments.  METHODS AND  MATERIALS  Bothemale and female white r a t s of the Sprague-Dawley and W i s t a r s t r a i n s , weighing  from 200 to 350  i n o r d e r t o b r i n g out any sex d i f f e r e n c e .  grams, were used These animals  are  convenient g e n e r a l l y because of t h e i r s i z e , r e l a t i v e economy, a v a i l a b i l i t y and h a r d i n e s s ;  s p e c i f i c a l l y , they are of use  because the s k e l e t a l muscle c o n t a i n s l i t t l e ,  i f any,  myoglobin  so that i n experimental work designed to t e s t the r o l e of  - 67 -  myoglobin r e l e a s e and o f crush i n j u r y , these two f a c t o r s can be c o n v e n i e n t l y  separated.  Dehydration  was accomplished merely by withdrawing  water f o r p e r i o d s ranging  from 24 to 72 hours.  The e f f e c t i v e -  ness o f t h i s method was e a s i l y a s c e r t a i n e d by the massive weight l o s s recorded  (up to 20% o f body weight i n 48 to 72 hours) and  by the p r o d u c t i o n o f u r i n e which was low i n volume and h i g h i n c o n c e n t r a t i o n , o f feces which were s m a l l , hard as reduced i n amount.  and d r y as w e l l  U r i n e s p e c i f i c g r a v i t i e s were not d e t e r -  mined, but gross o b s e r v a t i o n o f c o l o r , and i n some  cases  v i s c o s i t y , gave a rough index o f that f a c t o r . Myoglobin was obtained  commercially  c r y s t a l l i n e form and was administered  i n a purified  i n t r a v e n o u s l y i n t o the  femoral v e i n a f t e r d i s s o l v i n g i t i n a phosphate b u f f e r o f pH = 7*35 ( 1 7 0 ) .  D i f f i c u l t y was encountered i n d i s s o l v i n g the  p r o t e i n which had been dehydrated so completely lized  form.  to t h i s  crystal-  I t was not thought a d v i s a b l e t o use s o l u t i o n s o f  pH too f a r removed from i t s i s o e l e c t r i c p o i n t o f 6 . 7 8 (7)> i n j e c t i n g a s o l u t i o n o f v e r y a c i d o r a l k a l i n e pH would a complicating  factor.  since  introduce  The p o s s i b i l i t y o f d i s s o l v i n g the  pigment i n r a t serum was considered  but a g a i n i t was thought  a d v i s a b l e to avoid the added q u e s t i o n o f s e n s i t i v i t y r e a c t i o n s to n e c e s s a r i l y heterologous  serum as w e l l as the d i f f i c u l t y i n  keeping such a s o l u t i o n s t e r i l e .  I t was found f a i r l y  satisfac-  t o r y to make up a s o l u t i o n o f 25 mg. p e r c c . o f b u f f e r which was  - 68 -  kept 24 t o 48 hours i n an oven at 65% C, w i t h frequent  shaking.  Complete d i s s o l u t i o n was not o b t a i n e d , but i t was estimated t h a t 60 to 70 per cent o f the p r o t e i n d i s s o l v e d and the remainder could be suspended by v i g o r o u s shaking p r i o r t o i n j e c t i o n . Dosage administered ranged gram o f body weight,  a f i g u r e based  Bywaters ( 7 5 ) , and used  from 0 . 1 to 0 . 1 5 mg. per on t h a t c a l c u l a t e d by  a l s o by Corcoran and Page ( 8 5 ) .  amount n e c e s s i t a t e d the i n j e c t i o n o f a volume up t o 1.6 a 250 gm. r a t , but i f g i v e n s l o w l y , no i l l  This cc. i n  e f f e c t s were observed.  I n j e c t i o n s were made w i t h a #25 hypodermic needle w i t h the r a t under e t h e r a n e s t h e s i a , a t times considered to simulate as c l o s e l y as p o s s i b l e the time r e l a t i o n s h i p s encountered "crush syndrome" —  i . e . , immediately  i n human  on r e l e a s e o f the c r u s h i n g  ligature. L i g a t i o n was c a r r i e d out on the hind limb o f the animal, either l e f t  o r both, i n the manner i l l u s t r a t e d  Figure 3  i n F i g u r e 3*  - 69 •i  Under e t h e r a n e s t h e s i a , the limb was  c l i p p e d of h a i r from ankle  to g r o i n i n order that the l i g a t u r e would not s l i p . heavy twine, the limb was  Using  then wrapped t i g h t l y from ankle to as  h i g h on the limb as p o s s i b l e without i n t e r f e r i n g w i t h the u r e t h r a l o u t l e t or i n c u r r i n g the r i s k o f l o o s e n i n g . l i g a t u r e was level.  usually tied  The l i g a t u r e was  at what was  T h i s meant that the  e s s e n t i a l l y the "mid-thigh"  l e f t i n p l a c e f o r i n t e r v a l s of f o u r to  f i v e and one h a l f hours, being p r o t e c t e d by adhesive tape i n case the animal attempted t h i s time the r a t was gm.  of body weight  needed.  to b.ite l o o s e the s t r i n g .  sedated w i t h p e n t o b a r b i t a l 2.5  (5  mg.  wrapping During  mg.  per  100  per cc.) g i v e n i n t r a p e r i t o n e a l l y as  The f i v e hour p e r i o d was  spent i n l a r g e g l a s s funnels  which allowed f o r more easy o b s e r v a t i o n and handling as w e l l as f o r convenient c o l l e c t i n g of u r i n e . completed,  With the p e r i o d o f c r u s h  animals were removed from f u n n e l s , the l i g a t u r e  was  removed under the remaining nembutal s e d a t i o n or ether a n e s t h e s i a , the i n j u r e d limb was tency and  massaged u n t i l i t l o s t i t s "doughy" c o n s i s -  the foot appeared  placed i n a metabolism not emptied  was  cage f o r o b s e r v a t i o n .  then  The b l a d d e r  by compression at the completion of the f i v e  crush because recorded.  b r i g h t r e d , and the animal was  o n l y the t o t a l 24 hour u r i n e volume was  was  hours  to be  In animals which were normally hydrated, d i f f i c u l t y  encountered  i n that they would b i t e the i n j u r e d l i m b , r e s u l t -  i n g i n haemorrhage and an at times severe anemia. made to p r o t e c t the limb i n some way not r e s t r i c t the s w e l l i n g .  Attempts  were  to prevent the b i t i n g but  L o o s e l y a p p l i e d adhesive tape, which  could be added as necessary, was  found to be most s a t i s f a c t o r y i n  - 70 -  this regard. crushed  Dehydration animals were observed  to not b i t e the  limb and f r e q u e n t l y these were l e f t unwrapped.  The  e f f e c t i v e n e s s o f the l i g a t u r e i n producing a t y p i c a l c r u s h i n j u r y was observed  i n the almost immediate s w e l l i n g o f the limb  ( F i g u r e 4 ) and at autopsy by the appearance o f subcutaneous and muscle edema as w e l l as d i s c o l o r a t i o n o f the muscle ( F i g u r e 5 ) .  Figure 5 Because p r o d u c t i o n o f the syndrome was not completely s a t i s f a c t o r y i n the i n t a c t animal, and because the r a t i s known  - 71 -  to  have a formidable r e n a l r e s e r v e , i t was  t h i s r e s e r v e i n as p h y s i o l o g i c a l a way nephrectomy was  decided to reduce  as p o s s i b l e .  t h e r e f o r e c a r r i e d out i n l a t e r  Under e t h e r a n e s t h e s i a , the r i g h t kidney was i o r l y and decapsulated  suture and  approached p o s t e r -  the p e d i c l e was  gms.  silk  given p o s t - o p e r a t i v e l y .  weight were g i v e n f o u r days i n which to  recover b e f o r e the s t r e s s of experiment began; were allowed o n l y three days.  r e c o v e r y allowed  the  A s i n g l e subcutaneous i n j e c t i o n o f  9,000 u n i t s o f p e n i c i l l i n i n o i l was  gms.  tied,  the wound c l o s e d w i t h a s i n g l e b l a c k  skin c l i p s .  Animals under 200  experiments.  i n order to assure t h a t the a s s o c i a t e d  s u p r a r e n a l gland remained i n s i t u ; kidney removed and  Right  Too  200  long a p e r i o d o f  f o r compensatory hypertrophy  kidney, while the p e r i o d a l l o t t e d  those over  them allowed  of the  remaining  f o r complete  r e c o v e r y as s i g n i f i e d by g a i n i n weight. Test hormones used were a c r y s t a l l i n e p r e p a r a t i o n o f t e s t o s t e r o n e p r o p i o n a t e , a. s a l i n e suspension o f c o r t i s o n e a c e t a t e a s a l i n e suspension of compound F  (17 h y d r o x y c o r t i c o s t e r o n e  —  21 - a c e t a t e ) and a watery suspension o f d e s o x y c o r t i c o s t e r o n e a c e t a t e (DCA).  T e s t o s t e r o n e was  g i v e n e i t h e r as a s a l i n e  pension or d i s s o l v e d i n sesame o i l , 10 to 20 mgs. or three doses of 5 mgs.  sus-  per c c .  each were g i v e n subcutaneously,  Two the  i n i t i a l dose being g i v e n 48 hours before the i n i t i a l s t r e s s o f experiment to assure adequate blood l e v e l s . g i v e n as 0.4 (2 to 2.5  to 0.5  mgs.)  c c . of a 5 mg.  subcutaneously.  Cortisone  per c c . s a l i n e T h i s was  was  suspension  a d a i l y doee, begun  - 72 -  24 hours before l i g a t u r e 0.4  to 0 . 6  o f the l i m b .  c c . of a 5 mg.  per c c . suspension  cutaneously, a d a i l y dose s t a r t e d tion. (2.5  DCA mgs.)  to crush  doses were 0.1  g i v e n as  (2 to 3 mgs.)  sub-  48 to 72 hours p r i o r to l i g a -  c c . o f a 25 mg.  g i v e n subcutaneously  per c c .  suspension  each day beginning two  days p r i o r  injury. During  the 72 hours of o b s e r v a t i o n , the animals were  kept i n i n d i v i d u a l u r i n e and  Compound F was  metabolism cages designed  screening feces.  Urine was  for collecting  collected  f o r 24 hour  p e r i o d s i n s m a l l , open-mouthed j a r s placed c l o s e to the f u n n e l outlet.  Loss by e v a p o r a t i o n was  with deep food troughs standard  was  Cages were  equipped  i n which measured amounts of powdered  feed were p l a c e d .  e a s i l y without  minimal.  Animals could eat t h i s form o f food  i n t e r f e r i n g w i t h the u r i n e c o l l e c t i o n .  I f water  to be s u p p l i e d to the animals d u r i n g the t e s t , i t was  so w i t h the u s u a l b o t t l e t a n e o u s l y and  arranged  water i n t a k e and  avoided  Observations  i t was  a cheek o f  i n t e r f e r e n c e w i t h the u r i n e c o l l e c t i o n .  made were:  animal weight b e f o r e and  after  food consumed, u r i n e volume,  blood urea n i t r o g e n l e v e l s and  thought  caught i n a s m a l l pan  The method allowed  experiment, amounts of water and u r i n e pH,  so t h a t i t d i d not drop spon-  any water which d i d drop was  which could be e a s i l y emptied.  done  that a c o r r e l a t i o n  kidney h i s t o l o g y .  of kidney weight and  Later  body  s u r f a c e area would be of v a l u e , so t h a t i n l a t e r experiments kidney weights were r e c o r d e d .  U r i n e sediments were examined i n  - 73 -  early experiments but this procedure proved to be laborious and unrewarding and so was discarded. Urine volumes were recorded i n 24 hour periods, 9.00 a.m. to 9.00 a.m., and at the same time the gross appearance of the urine was noted.  Urine pH was determined with nitrazine  paper. Blood urea nitrogen levels (B.U.N.) were determined every 24 hours or immediately after death.  T a i l blood samples  were taken earlier, but this method was found to be impossible when dehydration was a factor.  Cardiac punctures were therefore  done every 24 hours for three days, the blood being drawn into a citrated syringe and measured i n a citrated pipette.  Occasional  d i f f i c u l t y was encountered i n the dehydrated animals and the occasional death resulted from hemopericardium and cardiac tamponade, but i n general the animals stood the procedure well when sharp one and one-half inch No.'25 needles were used and only 0.3 cc. of blood were withdrawn at a time.  For the urea deter-  mination, a modification (19) of Ormsby's diacetyl monoxime method (274) was used.  By this method, a Folin-Wu f i l t r a t e of  blood i s treated with diacetyl monoxime and concentrated sulphuric acid, heated i n a boiling water bath, the color emphasized by addition of potassium persulfate and the resulting yellow solution read i n a photoelectric colorimeter.  Urea nitrogen levels in  mg. per cc. can be easily calculated by constructing a standard curve and reading off the appropriate levels.  - 74  -  Three d i f f i c u l t i e s were encountered  w i t h t h i s method.  I t was found that s o l u t i o n s to be read were o c c a s i o n a l l y cloudy, so that readings were f a l s e l y high.  This f a u l t was found to  be incomplete p r e c i p i t a t i o n by s u l p h u r i c acid i n p r e p a r a t i o n of the Folin-Wu blood f i l t r a t e and when one drop of 10 per cent s u l p h u r i c acid was added to each blood sample, the f i n a l c l o u d i ness no longer occurred.  A second t r o u b l e arose when the f i n a l  s o l u t i o n s appeared red i n c o l o r , r a t h e r than yellow, t h e i r readings a l s o being f a l s e l y high.  I t was determined  that s a l i v a  blown i n t o s o l u t i o n s i n e x p e l l i n g the contents of p i p e t t e s , produced t h i s pink d i s c o l o r a t i o n . with absorbent cotton. of the method.  Henceforth, p i p e t t e s were plugged  The t h i r d problem was that of accuracy  As i n most procedures using small amounts of t e s t  substance, r e s u l t s allowed a wide range of e r r o r and normal f i g u r e s f o r r a t blood urea n i t r o g e n l e v e l s can only be stated as 50 to 100 mg. per cent.  I t follows that d i f f e r e n c e s of 10 to 20  mg. percent i n B.U.N, l e v e l s cannot be s i g n i f i c a n t , but t h i s f l e x i b i l i t y was nevertheless f e l t to be adequate f o r the purpose of these  experiments. H i s t o l o g i c a l sections of the l e f t kidneys were examined  a f t e r immediate post-mortem f i x a t i o n i n Zenker's or i n Herlant's f i x a t i v e , p a r a f f i n embedding and haemalum-phloxine s t a i n i n g . Kidneys of animals which died overnight were treated s i m i l a r l y but a kidney of any animal known to have died more than one hour p r i o r to f i x a t i o n was not considered v a l i d .  The hour of death  f o r the f i r s t s i x hours could be estimated roughly from the extent  - 75 -  of r i g o r mortis and other post-mortem f i n d i n g s .  I n a l l cases  ether was used f o r k i l l i n g animals remaining a l i v e at the t e r m i n a t i o n of an experiment. Kidney weights were taken i n the f o l l o w i n g way.  At  post-mortem, the e n t i r e decapsulated l e f t kidney was i n c i s e d and placed i n f i x a t i v e f o r 18 to 24 hours, at which time i t was b r i e f l y b l o t t e d dry and weighed i n m i l l i g r a m s .  Surface area  i n square centimeters was obtained from t a b l e s based on the animal weight i n grams at time of death.  The r e l a t i o n s h i p was  then calculated i n milligrams of kidney t i s s u e per square c e n t i meter of surface area.  REPORT OF EXPERIMENTS Experiment 1: Experiment IA considers the e f f e c t of 48 hours  dehydra-  t i o n , w i t h and without nembutal anesthesia, on the urea n i t r o g e n , urine output and kidney h i s t o l o g y o f the i n t a c t r a t .  Of twelve  r a t s f o u r were subjected t o dehydration alone, four to dehydration plus nembutal and four were normal c o n t r o l s .  Preliminary "test  runs" of the procedure were c a r r i e d out on these r a t s f o r 24 hour periods but they were allowed s u f f i c i e n t time f o r recovery before the 48 hours experiment.  Observations appear i n Table IA.  Figures f o r food and water are t o t a l s f o r 48 hours. I t i s evident t h a t , deprived of water, the r a t s ' intake of s o l i d  - 75 a.  food i s markedly diminished.  Weight change i n d i c a t e s that i n  48 hours of dehydration, animals l o s e about 10 per cent of t h e i r body weight.  Figures f o r urine volume show that the  dehydration i s not f e l t s i g n i f i c a n t l y u n t i l the second 24 hour period;  there appeared to be no trend i n pH values f o r u r i n e .  B.U.N, f i g u r e s are here low and show only a s l i g h t r i s e i n those animals dehydrated;  blood was taken by cardiac puncture  and no apparent errors were encountered during the analyses. H i s t o l o g i c a l examination revealed no s i g n i f i c a n t changes i n kidney s t r u c t u r e i n these animals. I t can be concluded that dehydration by removal o f water source i s accompanied by r e d u c t i o n i n s o l i d food intake and i s e f f e c t i v e i n reducing animal weight and urine output over a 48 hour period.  Urea n i t r o g e n l e v e l s r i s e s l i g h t l y ,  probably accountable f o r by hemoconcentration.  There was no  a l t e r a t i o n of kidney h i s t o l o g y . At a l a t e r date, the above experiment was repeated on twelve r a t s which had been r i g h t nephrectomied three days prev i o u s l y (Experiment I B ) ; four animals allowed free water were c o n t r o l s , while eight were dehydrated f o r 72 hours.  Food a v a i l -  able was 21 gms. each and kidney weights were recorded i n a d d i t i o n to the usual observations which appear i n Table IB. Results are e s s e n t i a l l y the same as those seen i n i n t a c t animals i n Experiment IA. s o l i d food w e l l ;  Animals allowed water ate  p r e v i o u s l y dehydrated animals had an increased  - 76 -  water intake when allowed water ( f o l l o w i n g the 72 hour readings) but handled t h i s water w e l l ;  a l l animals l o s t weight.  Urine  output f i g u r e s revealed normal f i g u r e s f o r c o n t r o l animals except at 96 hours, when f i g u r e s were unaccountably low. drated animals experienced a dehydration o l i g u r i a .  Dehy-  Blood urea  n i t r o g e n f i g u r e s a f t e r 24 hours dehydration are high (average 125 mg %, range 110 to 140, f o r c o n t r o l s ;  145 and 110 to 170  mg % f o r t e s t animals) and continue high at 48 hours (average 150 mg %, range 140 to 180, f o r c o n t r o l s ; mg % f o r t e s t animals).  155 and 140 to 170  At 72 hours, a marked drop i n B.U.N,  l e v e l s i s recorded i n s p i t e of the f a c t that dehydration c o n t i n ued i n t e s t animals (average 70 mg trols;  range 60 to 80, f o r con-  100 and 80 to 120 f o r dehydrated).  At 96 hours, l e v e l s  rose i n c o n t r o l animals but f e l l s l i g h t l y i n t e s t animals (average 115 mg  range 100 to 140, f o r c o n t r o l s ;  95 and 70 to  130 f o r dehydrated). I t i s apparent that these urea n i t r o g e n determinations may not be e n t i r e l y s a t i s f a c t o r y .  The high f i g u r e s i n c o n t r o l  animals at 24 hours may be accounted f o r by the f a c t that one kidney had been removed four days p r e v i o u s l y , w i t h those i n t e s t animals s l i g h t l y higher because of the 24 hours dehydration. At 48 hours, however, c o n t r o l l e v e l s continued to r i s e t o essent i a l l y the same l e v e l s as t e s t animals.  The sudden drop  observed at 72 hours can be explained only as an e r r o r i n absol u t e determination of urea n i t r o g e n f i g u r e s .  The r e l a t i o n  TABLE IA  TOTAL TOTAL FINAL WEIGHT FOOD (GMS) WATER ( c c )AND CHANGE (GMS)  URINE pH URINE VOLUME (cc) 24 hrs 48 hrs 24 h r s . ,48 h r s .  B.U.N. 48 hrs  42.3  51-7  318 (+ 14)  8.0  6.7  6.5  6.5  40  D.2  38.0  44.5  350 (+ 4)  9.0  5.5  6.5  6.5  40  D.3  35.3  56.0  330 (+ 12)  12.3  11.4  7-0  6.5  40  D.4  30.2  31.5  282 (0)  7.9  3.5  6.5  6.5  40  m  D.5  o  -  290 (-34)  8.4  3.4  6.5  6.0  50 mg %  «  D.6  0  —  290 (-32)  7.0  1.6  6.5  6.5  50  D.7  0  —  294 ( - 3 0 )  4.8  2.1  6.0  6.0  50  D.8  6.0  322 (-36)  13.2  3.2  6.5  6.5  50  D..9  16.5  270 (-28)  7.3  2.6  7.0  6.5  50  +  D.IO  15.0  310 (-22)  6.4  2.2  6.5  6.5  60  D.ll^  16.5  322 (-28)  6.4  2.5  7.0  6.5  50  D.12  16.3  295 (-25)  8.6  3.0  7.0  6.5  50  COM 'ROL  D.l  NEMB.  RAT  DEHYDRATION IN INTACT RATS  m a  —  —  •  —  —  TABLE IB  TOTAL TOTAI FINAL FOOD WATEf WEIGHT AND RAT CHANGE (GMS)  O ffi EH O O  Q W  W Q  DEHYDRATION IN RIGHT NEPHRECTQMIED RATS  URINE VOL. ( c c ) 24  48  72  96  URINE pH  B.U.N.  24  48  72  96  24  48  72  96  KIDNEY ' WEIGHT (GMS.)  265  21  97  l 9 G ( - 3 0 ) 10.2 6.0  10.2 3.6  6.5  6.5  7.0  7.5  150 180  80  110  214.6  266  21  89  208(-42) 10.8 14.2 12.2 2.2  7.0  6.5  7.0  7.0  110 140  60  110  208.1  26?  21  54  176(-30) 10.0  6.8 1.0  6.5  7.0  7.0  7.5  120 140  70  140  196.0  268  21  92  180(-30) 10.8 13.4- 3 0 . 4 1.0  7.0  7.0  6.0  140 150  70  100  201.7  269  21  41  214(-44)  4.8  1.2  0 . 6 12.8 6 . 5  6.5  6.0  6.5  140 160  80  110  213.9  270  21  47  234(-34)  6.4  1.6  1.0 10.8 7.0  6.0  6.0  6.5  110 150  80  80  223.0  271  21  46  230(-20)  6.2  1.2  1.2  3 . 2 7.0  6.0  6.0  6.0  150 140 120  90  220.9  272  21  42  226(-l6)  3.0  0.4  0.2  2 . 5 7.0  6.0  6.0  6.0  150 160 100  80  225.9  273  21  42  198(-22)  5.0  2.0  0.8  5.0 7.0  5.5  6.0  6.5  140 150  90  70  233.6  274  21  39  220(-28) • 5.4  1.8  7.0 .7.0  6.0  6.0  150 160 100  90  217.6  275  15  19  224(-30)  6.4  1.4  1.0  4.5 6.5  6.0  6.0_. 7.0  150 150 100  130  207.7  276  21  37  l84(-36)  4.0  0.8  0.8  5.2 7.0  6.0  6.0  170 170 110  120  209.9  7.2  7.5  -  79  -  between c o n t r o l s and dehydrated animals remains s a t i s f a c t o r y . The r i s e of B.U.N, i n c o n t r o l s at 96 hours, to exceed l e v e l s i n t e s t animals, c o r r e l a t e s w i t h the decrease i n urine output of controls at t h i s time and i t can be concluded that some degree of dehydration must have occurred a c c i d e n t a l l y .  96 hour  f i g u r e s f o r t e s t animals f e l l s a t i s f a c t o r i l y f o l l o w i n g the f u l l 2 4 hours of hydration. Examined m i c r o s c o p i c a l l y , kidney s t r u c t u r e remained unaltered a f t e r 72 hours dehydration; kidney weights showed no s i g n i f i c a n t a l t e r a t i o n a f t e r the s t r e s s . I t i s apparent that uninephrectomy r e s u l t s i n a tempora r i l y elevated B.U.N, l e v e l which i s accentuated moderately by a period of dehydration l a s t i n g 7 2 hours.  Experiment 2 ; Experiment 2 i s concerned w i t h the e f f e c t o f myoglobin i n j e c t e d i n t r a v e n o u s l y on B.U.N., urine output and kidney h i s t o l ogy i n the r a t .  Experiment 2 A consisted of four r a t s as normal  c o n t r o l s , four r e c e i v i n g intravenous i n j e c t i o n o f p h y s i o l o g i c s a l i n e and four Injected w i t h myoglobin d i s s o l v e d and suspended i n 1 cc. o f s a l i n e , 0 . 1 mg. per gram of body weight.  Experiment  2 B duplicated t h i s procedure, while Experiment 2 C used an increased dose of myoglobin i n s a l i n e , 0 . 1 5 mg. per gm. of body weight.  B.U.N's were determined on t a i l blood samples i n E x p e r i -  ment 2 A at 2 4 and 4 8 hours, but a l l other determinations were on  - 80 -  cardiac blood samples.  Observations appear i n Tables 2A, 2B  and 2C. D i f f i c u l t i e s were encountered w i t h the methods and procedures of t h i s experiment so that f i g u r e s i n red are considered not v a l i d .  These errors are considered i n the s e c t i o n on  "Discussion and Conclusions". In normal c o n t r o l s , food and water intake remained f a i r l y constant and a l l animals gained weight.  Urine output  i n the f i r s t measured 24 hour period averaged 7*3 cc. per r a t (range 3*2 to 10.5 cc.) and i n the second 24 hour period averaged 5*9 c c . (range 3*5 to 9.4 c c . ) . significant;  pH f i g u r e s were not  urea n i t r o g e n l e v e l s were w i t h i n normal l i m i t s .  S a l i n e controls i n a l l three experiments had l e s s constant food and water intakes but f i g u r e s are e s s e n t i a l l y the same as f o r normal c o n t r o l s ;  most animals gained weight.  Urine  volumes were comparable to the normal group, as were urine pH determinations.  Urea n i t r o g e n l e v e l s remained w i t h i n the normal  range. No s i g n i f i c a n t d i f f e r e n c e was detected i n animals given the  increased dose of myoglobin (Experiment 2C).  A l l test ani-  mals showed food and water intakes comparable to those of a l l saline controls;  f i v e animals l o s t weight and three gained  weight, of the eight animals weighed.  Urine volumes a l s o were  comparable, though i n two instances (P.22 and P.23) urine output  -  was increased.  81  -  This increase may represent an osmotic d i u r e s i s .  Urine pH f i g u r e s ranged from 6.5 to 7.5, but tended to the acid side at the time of myoglobin i n j e c t i o n .  B.U.N, f i g u r e s again  were w i t h i n normal l i m i t s . Renal h i s t o l o g y was not e s s e n t i a l l y a l t e r e d and though casts could be seen i n the c o r t i c a l tubules w i t h myoglobin i n j e c t i o n (Figure 6 ) , these were also present i n s a l i n e i n j e c t e d animals (Figure 7 ) .  Figure 6  Figure 7  Myoglobin i n j e c t e d intravenously appears to have no e f f e c t on the kidneys of i n t a c t r a t s as measured by urine output, blood urea n i t r o g e n and kidney h i s t o l o g y . may have been an "osmotic d i u r e s i s " .  I n two cases, there  The procedure of anesthe-  t i z i n g the animal and i n j e c t i n g i t w i t h s a l i n e or myoglobin may w e l l account f o r the greater v a r i a t i o n i n food and water intakes seen i n these animals.  TABLE 2A  TOTAL FOOD (GMS)  TOTAL WATER (cc.)  P.l  41.5  64.0  P.2  42.0  o P.3 is; P.4  « P.5 E-t  AX CONT:  RAT  O O  MYOGLOBIN IN INTACT RATS  FINAL WEIGHT AND CHANGE (GMS)  URINE VOLUME  URINE pH.  48 hrs.  72 hrs.  48 hrs  72 hrs  394(+12)  9.1  9.4  6.0  7.0  49.0  358(+12)  7.4  4.8  6.5  45.5  58.5  380(+l8)  10.5  8.9  44.5  51.0  304(+4)  8.1  0  1.5  342(-l6)  32.5 2.0  B.U.N. 24 hrs  hrs  120  90  70  7.0  60  80  40  6.5  6.5  80  70  60  8.3  6.5  7.0  100  90  50  11.6  2.8  7.5  7.0  70  80  60  424(-24)  12.6  10.3  7.0  6.5  110  90  70  324(-40)  6.4  2.4  6.0  6.0  180  70  50  90  60  19.0  P.7  0  P.8  23.0  26.5  370 (0)  2.2  7.2  6.5  6.0  70  P.9  24.0  36.0  360C-8)  4.2  2.8  7.0  6.5  100  P.10  24.5  24.5  340(-12)  6.0  4.4  7.0  6.5  P.11  28.0  29.0  352(-14)  6.2  5.0  7.0  7.0  9 P.12  20.0  18.5  330(-l6)  6.0  3.5  6.5  7.0  SALINE  p.6  •—^  0GL(  M  72  48 hrs  —  —  90 —  190  —  60 60 50 70  TOTAL FOOD (GMS)  P.13  42.0  57.0  P. 14  38.0  58.0  p.15  41.5  57.0  P.16  41.5  50.5  P.17  47.5  •94.5  o o P.18  45.0  69.5  P.19  39.0  60.5  P.20  45.5  57.0  P.21  43.5  59.5  P.22  26.5  64.0  o P.23 o P.24  44.5  87.O  43.5  54.0  3RMAL CONTROI JTROL. N(  RAT  SAL INE  TABLE 2B  M  ...  MYOGLOBIN IN INTACT RATS  TOTAL FINAL WEIGHT WATER AND CHANGE (ec) (GMS)  -  URINE! .VOLUME #8  URINE pH 48  B.U.N.  72  48  hrs  hrs  hrs.  hrs.  hrs  hrs  24 hrs  7-0  4.6  6.5  6.5  60  60  60  7.6  5.8  6.5  7.5  60  70  60  10.1  9.0  7.0  7.5  60  70  50  7.0  3.8  7.0  7.0  70  60  60  9.1  23.8  6.5  7.5  70 .  80  70  9.6  6.0  7.0  7.5  50  50  80  6.6  6.1  6.0  7-5  60  60  50  4.4  3.0  6.0  6.5  70  60  60  7-4  6.6  6.5  7.0  60  60  50  10.4  15.4  6.5  7.5  70  60  60  11.5  15.4  7.0  7.5  70  70  50  6.6  7-2  6.5  7.5  60  70  50  72  72  TABLE 2C  TOTAL WATER (cc)  MYOGLOBIN IN INTACT RATS  FINAL WEIGHT AND CHANGE (GMS)  URINE VOLUME 48 72 hrs hrs  URINE pH 48 72 hrs hrs  B.U.N. 48 72 hrs hrs  TOTAL FOOD (GMS)  P.25  41.0  59.0  248 (+8)  9.2  4.6  6.5  7.0  70  P.26  37.0  47.0  238 (+18)  4.4  3.8  6.5  7.5  60  70  50  P.2?  41.0  54.0  246 (+14)  3.2  ; 3.5  6.0  6.5  60  60  60  o P.28  41.0  58.5  252 (+14)  4.4  3.6  6.5  6.5  60  70  60  EH  P.29  35.0  53.0  252 (+8)  4.2  4.0  6.5  6.5  70  60  60  O O  P.30  40.0  66.0  248 (+10  10.8  8.6  7.0  7.5  60  70  50  P.31  40.0  63.5 "  256 (+10)  7.5  5.0  6.5  7.5  70  70  60  P.32  34.0  73.5  240 (+8)  14.2  9-0  6.5  7.5  70  80  70  P.33  40.0  58.0  254 (+14)  5.0  4.9  6.5  7.5  60  60  50  P.34  39.0  6o.o  240 (+8)  9.6  7.2  7.0  7.5  70  60  60  P.35  32.0  63.0  244 (-1)  6.6  8.6  6.5  6.5  70  70  50  P.36  25.0  40.0  232 (+4)  4.6  3.8  7.0  7.5  60  70  50  CONTROL  RAT  50  )GL0 BIN  !  SAL INE  *•  24 hrs  1  MYOGLOBIN AND DEHYDRATION IN INTACT RATS  URINE P H 48 72 hrs hrs  B.U.N. 48 72 hrs hrs  TOTAL WATER (cc)  24.0  54  276 (+6)  0.15  2.6  6.0  6.0  70  70  60  11.0  36  250 (-10)  0.2  2.2  6.0  6.0  70;  70  60  M.15  19.0  47  240 (+4)  0.1  2.0  6.0  6.0  90  70  70  M.16  23.0  49  286 (-14)  0.1  1.0  6.0  6.0  120  90  70  M.17  29.0  51  262 (+4)  0.1  1.4  6.0  6.5  80  90  70  •s M.18 o i—i  24.0  48  250 (-4)  0.2  1.6  6.5  6.5  90  60  70  M.19  17.0  46  238 (0)  1.0  3.1  6.0  6.0  70  90  70  M.20  21.0  49  290 (0)  0.6  2.1  6.0  6.5  50  90  70  M.21  22.0  54  264 (+6)  0.15  2.5  6.5  6.0  120  100  70  M.22  23.0  55  260 (+10)  0.5  3.8  6.0  6.0  90  70  70  M.23  16.0  43  250 (0)  0.1  2.4  6.0  140  80  70  M.24  24.0  54  284 (-2)  drop  0.8  -  6.0 5.5  80  80  60  RAT  FINAL WEIGHT AND CHANGE (GMS)  URINE VOLUME 48 72 hrs hrs  rOTAL POOD (GMS)  *• DEHYDRAT:  TABLE 3  24 hrs  —f-u  DEHYD RJ  o o If. 13' Q W M.14 EH  MYOGLOB  n  - 86 -  Experiment  3:  Experiment 3 considers the e f f e c t of 72 hours dehydrat i o n plus myoglobin i n j e c t i o n on the u r i n e output, B.U.N, and kidney h i s t o l o g y i n eight r a t s , w i t h an a d d i t i o n a l four animals, subjected to dehydration alone, as c o n t r o l s . dehydrated  Animals were  f o r 24 hours before the i n j e c t i o n , which was followed Dosage was again 0 . 1 5 mg  by a f u r t h e r 48 hours without water. of myoglobin  ( i n s a l i n e ) per gram of body weight and a l l blood  samples were by cardiac puncture.  Table 3 l i s t s the observa-  tions. Figures i n red are again not v a l i d .  Food and water  intajkes d i d not d i f f e r i n c o n t r o l and t e s t groups; change was a l s o e s s e n t i a l l y the same i n each group.  weight Figures  for u r i n e volume were recorded f o r the 24 t o 48 hour period following injection — —  i . e . , f o r the l a s t 24 hours of dehydration,  and f o r the subsequent 24 hours i n which f r e e water was  allowed.  Urine output during dehydration was markedly dimin-  ished i n a l l cases, as l i t t l e as 0 . 1 cc as recorded here. one case (M.19) an osmotic d i u r e s i s may have been seen.  In Urine  was n o t i c e a b l y more acid i n both groups here, being u s u a l l y pH 6 . 0 , and at no time was i t observed to be d i s c o l o r e d by the i n j e c t e d pigment.  Figures f o r urea n i t r o g e n show the e f f e c t of  dehydration (24 and 48 hour f i g u r e s ) , w i t h a f a l l a f t e r water intake was allowed (72 hour f i g u r e s ) .  These f i r s t two s e r i e s  of f i g u r e s a l s o are s l i g h t l y higher i n the t e s t animals  (average  - 87 -  80 mg  range 70 t o 120 mg % ) .  No e s s e n t i a l d i f f e r e n c e s were  seen i n the kidney h i s t o l o g y of the two groups.  There were no  casts. Intravenous myoglobin therefore would appear to have no s p e c i f i c e f f e c t on urine output, urea nitrogen l e v e l or k i d ney h i s t o l o g y even i n the presence of dehydration.  As i n  Experiment 1, there was a diminution of urine volume almost t o the point of a n u r i a , a s l i g h t e l e v a t i o n of B. U. N. and a s l i g h t l y more acid u r i n e .  These changes appear to be due to  dehydration alone and are not s i g n i f i c a n t l y changed by the addit i o n of myoglobin.  There was no pigmentation  of the urine  seen and no tubular casts even though urine was c o n s i s t e n t l y acid.  Experiment 4: The r o l e o f crush i n j u r y I n the genesis of "lower nephron nephrosis" was i n v e s t i g a t e d here by l i g a t i o n of the l e f t hind limb f o r f i v e hours.  Eight r a t s were so t e s t e d , w i t h  another four animals a c t i n g as normal c o n t r o l s .  Since i t was  assumed that the t e s t animals would not d r i n k f r e e l y a f t e r the crush i n j u r y , attempts were made t o match the water intake of the c o n t r o l animals to that o f the t e s t animals.  Observations  of water intake were therefore made at 24 hour I n t e r v a l s . order that the kidney h i s t o l o g y might be viewed  In  temporally,  animals M.32 and M.35 were s a c r i f i c e d 24 hours a f t e r l i g a t i o n , animals M.30 and M.34 a f t e r 48 hours and the remaining  four  - 88 -  72 hours a f t e r crush injury,, at which time c o n t r o l animals were also k i l l e d .  Observations are recorded i n Table 4.  I t can be seen from t h i s Table that animals s u f f e r i n g crush i n j u r y d i d not eat as much s o l i d food as d i d c o n t r o l a n i mals i n s p i t e of the f a c t that they drank more water.  It is  apparent that the matching of water intake was not accomplished. As a r e s u l t , the 'normal* controls were i n e f f e c t dehydrated to some e x t e n t , as evidenced by t h e i r consistent weight l o s s . Weight l o s s i n t e s t animals was nevertheless g r e a t e r .  The most  remarkable observations were, however, of the urine output. Test animals experienced a c o n s i s t e n t , immediate and marked d i u r e s i s , the average output f o r the f i r s t 24 hours being 2 3 . 1 cc.  compared to c o n t r o l average of 3.4 cc.  The p o l y u r i a was  evident during the f i v e hours of l i g a t i o n i t s e l f , the t e s t a n i mals p u t t i n g out an average of 10.3 cc of urine i n that time, the c o n t r o l animals averaging 0.6 cc.  This l a s t f i g u r e i s low  because volumes were recorded w i t h animals i n metabolism rather than i n funnels.  cages  The d i u r e s i s p e r s i s t e d to the 48 hour  observation and was even evident 72 hours a f t e r the i n i t i a l stress of crush. TJrine a c i d i t y again v a r i e d i r r e g u l a r l y between 6.0 and 7.5«  Urea n i t r o g e n f i g u r e s exhibited some e l e v a t i o n above  normal l i m i t s but cannot be said t o be s i g n i f i c a n t l y elevated as measured here i n t e s t animals.  The o c c a s i o n a l high value seen  i n the c o n t r o l group can be accounted f o r by p a r t i a l dehydration. A l s o , some of the lower f i g u r e s seen i n t e s t animals at 48 and  TABLE 4  LEFT HIND LIMB LIGATION IM INTACT RATS  WATER INTAKE (cc) FINAL WEIGHT URINE VOLUME AND CHANGE 24 72 24 48 48 Tot. (GMS) 72  M.25  37  9  24  3  36  298 (-30)  4.7  4.6  M.26  36  13  21  14  48  320 (-28)  2.1  o M.27 o  43  15  20  31  66  322 (-14)  M.28  35  7  12  24  43  M.29  29  39  18  42  M.30  3  20  11  M.31  7  33  o M.32  0  M..33  noN  [TRO  RAT  TOTAL FOOD (GMS)  B.U.N, (mg. %) 24 48  24  48  72  4.6  7.0  7.0  6.5  70  110 80  1.4  2.6  6.5  6.5  6.0  80  80 60  3.0  4.7  4.8  7.0  6.5  7.0  90  70 70  302 (-24)  3.8  2.8  2.4  7.6  6.5  7.0  90  100 60  292 (-22)  28.6  7.5  6.6  6.5  7.5  7.0  80  60 50  -  99 38  300 (-40)  24.4 1 3 . 2  -  6.5  7.5  15  20  68  270 (-34)  12.2 12.8  6.4  6.5  7.5  33  -  -  39  17  48  14  34  98  324 (-48)  M.34  0  36  32  68  314 (-22)  M.35  0  28  M.36:  13  22  tH  M Hi  URINE pH  -  -  31  6  37  65  -  -  296 (-48)  6.5  -  29.4 21.0 1 7 . 2  6.5  7.5  31.2 26.5  6.7  7.0  25.2  19.4  -  -  -  14.1 11.6  -  7.8  6.0  -  6.0  7.0  -  110  7.5 100  -  90  7.5  90  -  -  80 110  7.0 110  72  100 70 70  -  -  100 80 80  -  -  90 70  - 90 -  72 hours may he explained by the hemodilution f o l l o w i n g haemorrhage from self-amputated crushed limbs.  This habit of the  hydrated animals b i t i n g i t s i n j u r e d limb gave some d i f f i c u l t y i n o b t a i n i n g v a l i d observations. Post mortem f i n d i n g s were e s s e n t i a l l y negative - there was no gross evidence of kidney change - except f o r the injured limb, which was swollen, c o l d , pale to blue and edematous. H i s t o l o g i c a l changes were not remarkable  at any stage except  f o r s l i g h t granular changes i n the cytoplasm of tubules of Zone 3» w i t h s w e l l i n g and v a c u o l i z a t i o n of the n u c l e i (see F i g . 8 and compare w i t h F i g . 9 ) .  An o c c a s i o n a l cast was seen i n  Figure 8  Figure 9  medullary tubules (Figure 1 0 ) . L i g a t i o n of one hind limb f o r f i v e hours i n otherwise i n t a c t hydrated r a t s appears to have no permanent damaging e f f e c t on the kidneys.  There was no s i g n i f i c a n t e l e v a t i o n of  blood urea nitrogen and no remarkable  r e n a l tubular damage  - 91 -  Figure 10  histologically.  There was also no o l i g u r i a , and on the con-  t r a r y , a marked d i u r e t i c e f f e c t i n response to the i n j u r y was observed-, i n d i c a t i n g some a l t e r a t i o n i n "kidney f u n c t i o n .  Experiment 5: In Experiment 5A, eight male r a t s were subjected to the s t r e s s of l e f t hind limb l i g a t i o n f o r f i v e hours together w i t h 72 hours dehydration (24 hours p r e - l i g a t i o n , . 4 8 hours postligation).  Four a d d i t i o n a l animals acted as c o n t r o l s , being  only dehydrated.  Experiment 5B repeated t h i s procedure except  that the l i g a t i o n period was lengthened to f i v e and one h a l f hours.  Observations appear i n Tables 5A and 5B. I t i s apparent that there i s no e s s e n t i a l d i f f e r e n c e  between r e s u l t s of the f i v e and the f i v e and one-half hour l i g a t i o n periods 'so these e x p e r i m e n t s w i l l be considered together. /  TABLE 5A  FIVE HOURS LIGATION PLUS DEHYDRATION  TOTAL TOTAL FINAL WEIGHT WATER AND CHANGE FOOD (GMS) (GMS) (cc) 24 hrs  RAT  URINE VOLUME  URINE PH  B. U. N.  24  48  72  24  48  72  24  48  72  EH  iLIGiITED AND DEHYDRATED  DEHYD RA]  o M.37 o Q M.38  11  33  338 (+2)  1.2  1.0  5.6  6.0  6.0  6.0  80  50  50  12  33  346 (+2)  0.8  0.6  4.3  6.5  6.5  6.0  100  60  50  M.39  11  33  330 ( - 2 )  2.5  0.7  5.4  6.5  6.0  6.0  100  70.  60  M.40  7  22  342 ( - 2 )  0.4  1.2  1.6  6.0  6.0  6.0  80  90  60...  M.41  4  350  (-12)  2.8  7.3  6.9  6.0  6.5  .6.5  170  110  110  1.42  0  -  244 ( - 2 6 )  2.6  7.3  -  6.0  6.0  -  140  230  -  M.43  0  31  328 (-18)  1.5  8.7  15.4  6.0  6.0  7.0  190  180  160  M.44  0  31  (-15)  2.5  6.0  14.4  6.0  6.0  7.5  160  130  110  1.45  0  -  329  300 ( - 3 4 )  2.2  -  -  6.0  -  -  -  -  -  M.46  0  0  270 ( - 3 4 )  1.4  4.3  6.0  6-»0  -  220  230  -  M.47  0  -  298 ( - 3 0 )  1.2  . -  -  5.5  -  -  280  -  -  M.48 .  0  18  310 ( - 2 6 )  3.4  8.0  15.1  6.0  6.0  6.5  250  1  -  130  TABLE 5B  NTROLS  RAT  FIVE AND ONE-HALF HOURS LIGATION PLUS DEHYDRATION  FINAL WEIGHT TOTAL TOTAL FOOD WATER AND CHANGE (ec) (GMS) (GMS) 24 hrs  URINE VOLUME 24  48  72  0.2  URINE pH  B. U. N.  24  48  72  24  48  .72  9  29  254 (-30)  1.6  2.6  6.0  6.0  6.0  80  110  70  o o M.50  9  29  298 (-32)  1.8 d r i e d 1.8  6.0  -  5.5  100  100  70  M.51  0  20  304 (-50)  1.8  4.0  6.0  6.0  6.5  80  100  80  M.52  6  11  226 (-44)  0.6 dried 0.6  6.0  -  6.0  80  120  90  M.53  0  -  330 (-32)  0  M.54  0  35  318 (-62)  M.55  1  30.  Q M.56  1  Q M.57  DEHYDR:  M.49  0.8  -  -  -  -  -  -  2.6  4.4  24.5  6.0  6.0  7.5  170  220  180  292 (-52)  2.8  4.0  8.0  6.0  6.0  6.5  140  110  90  31  240 (-46)  2.0  4.7  11.0  6.0  6.0  7.0  130  140  90  0  28  274 (-48)  2.8  1.0  10.0  6.0  5.5  7.5  230  220  250  < M.58  0  0  272 (-50)  2.6  1.0  -  6.0  5.5  -  360  270  M.59  0  -  310 (-36)  2.5  -  -  6.0  -  440  -  -  M.60  0  0  270 (-52)  2.1  3.4  -  6.0  5-?  -  280  -  RATED  -  /—\  LIGA .TED  (—1  -  -  - 94 -  The d i f f i c u l t y i n matching food intakes and water intakes i s evident here.  Animals not allowed water w i l l eat  l i t t l e s o l i d food, and those also l i g a t e d w i l l eat v i r t u a l l y no s o l i d s at a l l .  For t h i s reason i t was thought wise to  l i m i t s o l i d food a v a i l a b l e to a l l animals i n f u r t h e r e x p e r i ments to 10 grams each. water i n t a k e s ;  S i m i l a r l y , i t was d i f f i c u l t  to c o n t r o l  some of the l i g a t e d animals - those obviously  i l l - would not d r i n k water when i t was made a v a i l a b l e , thereby hastening t h e i r deaths. In Experiment 5A the c o n t r o l group managed to r e t a i n i t s weight w e l l , while a l l other animals ( i n c l u d i n g c o n t r o l s of Experiment 5B) showed a s a t i s f a c t o r y weight l o s s (up to 16% of o r i g i n a l body weight). Urine volumes i n both experiments again showed the d i u r e t i c response, apparent p a r t i c u l a r l y at 48 hours and amplif i e d at 72 hours by a l l o w i n g water f o r the preceding 24 hours. Eight c o n t r o l animals at the 48 hour reading averaged 0 . 6 cc of u r i n e , while twelve t e s t animals averaged 5.0 cc f o r that 24 hour period - almost t e n times as much.  pH f i g u r e s showed  a s l i g h t tendency towards a c i d i t y a f t e r 72 hours but no d e f i n i t e trend can be s t a t e d . With regard to B. U. N. f i g u r e s , d e f i n i t e uremic l e v e l s were reached f o r the f i r s t time.  I n three instances cardiac  puncture was unsuccessful because the heart could not be located  - 95  -  by p a l p a t i o n or with the needle and i n a l l instances blood withdrawn was very t h i c k and dark.  In s p i t e of the f a c t that  the volume of urine excreted i n 24 hours by c o n t r o l animals was as l i t t l e as 0.2 cc, t h e i r B.U.N. l e v e l s did not r i s e above 120 mg %.  On the other hand, t e s t animals whose urine output  was always above 1.0  cc per 24 hours and rose as high as 8.7  cc.  had B. U. N. l e v e l s c o n s i s t e n t l y over 100 mg % and at times r i s i n g as high as 440 mg % during the dehydration  period  These f a c t s would presumably n e c e s s i t a t e a d i l u t e u r i n e i n t e s t animals, a c o n d i t i o n which was v e r i f i e d by the appearance of very p a l e , watery urine i n t e s t animals while that of c o n t r o l s was dark amber, almost syrupy. H i s t o l o g i c a l evidence of r e n a l t u b u l a r damage was present d e f i n i t e l y f o r the f i r s t time.  Sections were examined  under low and high powers, d i v i d i n g the r e n a l t i s s u e i n t o four zones f o r f a c i l i t y of examination.  These were :  Zone 1 - cortex proper, containing glomeruli and convoluted  t u b u l e s , proximal and d i s t a l , w i t h  t h e i r appropriate v a s c u l a t u r e .  This f i r s t zone  corresponds to Smith's d i v i s i o n "cortex". (See F i g . 1 1 ) . Zone 2 - corticomedullary r e g i o n , i n which there i s the t h i c k descending limb of Henle's loop. (Smith's "Medulla - outer band of outer zone.")  - 96  -  Figure 11. From Smith: "The Kidney" 1951 (333). Zone 3 - outer medullary p o r t i o n containing s e c t i o n s of both t h i c k and t h i n limbs of Henle's loop together w i t h bundles of venae r e c t a e . (Smith's "Medulla - inner band of outer zone."). Zone 4 - medulla proper, containing sections of Henle's t h i n loop, c o l l e c t i n g tubules and vasa r e c t a . (Smith's "Medulla - inner zone"). Zones 1, 3 and 4 are more e a s i l y defined i n the r a t kidney and received more a t t e n t i o n i n the examinations.  Zone  3, c o n s i s t i n g e x c l u s i v e l y of "lower nephron" received most attent i o n and was found on examination to e x h i b i t the c h a r a c t e r i s t i c pathologic damage seen throughout these experiments. C o n t r o l animals showed no h i s t o l o g i c a l a l t e r a t i o n i n kidney s t r u c t u r e , a f a c t which would be expected from observations recorded i n Experiment 1. (See Figure 12).  Figure 12  Of the 16 t e s t animals, eight showed minimal to moderate changes i n r e n a l tubules, to be described below; others had questionable changes.  two  Two animals died s e v e r a l  hours before t h e i r kidneys could be f i x e d , thereby e x h i b i t i n g post-mortem change; valid.  these sections could not be considered  The remaining four animals showed no h i s t o l o g i c a l  evidence of r e n a l t u b u l a r damage i n s p i t e of the f a c t that two had urea nitrogens of over 280 mg %, i n Figure 13,  T y p i c a l changes appear  most marked i n Zones 2 and 3 , but extending i n t o  Zone 1 as w e l l .  Proximal tubule c e l l s remain i n t a c t , whereas  d i s t a l tubules ( c h i e f l y the t h i c k p o r t i o n of Henle's loop) are i n an e a r l y stage of degeneration with granular, vacuolated cytoplasm, swollen, pale and vacuolated n u c l e i and the occasional pyknotic nucleus. one lumen.  A desquamated e p i t h e l i a l c e l l can be seen i n  These changes accurred i n an animal p u t t i n g out  3.4, 8.0 and 15.1  cc. of urine per 24 hours and whose B.U.N, rose  to at l e a s t 250 mg %.  S i m i l a r changes appear i n the d i s t a l  - 98 -  tubules of Zone 3 i n Figure 14.  Here the degenerating  tubules  are f u r t h e s t removed from the venae r e c t a e , which appear at the upper and lower margins of the f i g u r e .  This animal (M. 59)  died at 24 hours with a postmortem value f o r B. U. N. of 440 mg % and a urine volume f o r 24 hours of 2.5 c c .  Figure 13  Figure 14  From t h i s experiment i t appears that crush i n j u r y ( l e f t hind limb l i g a t i o n ) when coupled with severe dehydration can produce r e n a l tubular damage i n a f a i r proportion (50$ o f 16 r a t s ) o f otherwise i n t a c t male a l b i n o r a t s .  This damage i s  i n d i c a t e d by disordered f u n c t i o n of the kidney (elevated blood urea n i t r o g e n , increased volume of d i l u t e urine) and disordered s t r u c t u r e as w e l l (tubular degeneration).  The d i u r e t i c response  observed i s not one of the c r i t e r i a stated f o r r e n a l damage, being i n f a c t the opposite of o l i g u r i a or anuria, but i t nevertheless i n d i c a t e s a d i s o r d e r of r e n a l f u n c t i o n .  - 99  -  Experiment 6: The e f f e c t of crush i n j u r y i n the presence of i n t r a venously i n j e c t e d myoglobin i n normally hydrated animals was observed i n t h i s experiment.  Water intake of four normal  c o n t r o l animals was matched to that of a t e s t group of four animals and a c o n t r o l group of four animals i n which the b u f f e r s o l u t i o n alone, here used as a solvent f o r myoglobin, was injected.  Table 6 l i s t s the complete data on these animals.  Animal Mi.71 died at time of i n j e c t i o n and therefore i s not included i n the a n a l y s i s of observations. In s p i t e of the f a c t that animals were allowed free s o l i d s , the normal c o n t r o l group d i d not eat f r e e l y .  The  amounts consumed, however, compared favourably with the remaining animals.  Water intakes and weight changes a l s o compared  favorably i n the three groups.  The d i u r e t i c response to the  trauma of l i g a t i o n was again seen i n the seven l i g a t e d animals, no s i g n i f i c a n t d i f f e r e n c e between the myoglobin-injected and the b u f f e r - i n j e c t e d groups being evident.  Two c o n t r o l animals  d i d , however, show a pronounced d i u r e s i s at 72 hours which i s unexplained.  B. U. N. l e v e l s cannot be said to be elevated  s i g n i f i c a n t l y i n any of the groups;  a h i g h f i g u r e i n one con-  t r o l animal i s unexplained and must be presumed to be an e r r o r . Urea n i t r o g e n f i g u r e s may be low owing to hemodilution which was observed i n many animals during cardiac puncture at 24 hours. This was thought i n e a r l y experiments to be the r e s u l t of  TABLE 6  RAT  TOTAL FOOD (GMS)  TOTAL WATER (cc.)  LIGATION PLUS MYOGLOBIN INJECTION IN INTACT RATS  FINAL WEIGHT AND CHANGE (GMS)  URINE VOLUME . 24  B.U.N.  URINE pH  48  72  24  48  72  24  48  72  M.61  28  57  202 (-16)  3.8  5.0  22.2  6.5  6.0  6.0  90  70  90  M.62  28  51  220 (-6)  4.6  5.4  20.5  7.0  6.0  6.0  100  70  70  M.63  25  39  224 (-12)  4.4  6.8  8.6  6.5  6.0  6.0  110  70  60  M.64  26  40  206 (+6)  6.5  2.3  3.6  6.0  6.0  7.0  150  80  90  M.65  19  66  242 (-20)  16.4  16.5  12.4  6.5  7.0  6.0  100  80  80  O £  M.66  4  18  224 (-24)  6.0  8.0  5.4  6.0  7.0  7.0  120  80  80  O C  M.6?  13  75  238 (-2)  2 3 . 4 10.5  9.7  6.0  7.0  6.0  110  90  100 '  M.68  12  37  228 (-14)  11.2  8.8  4.2  6.5  7.5  6.0  120  90  80  M.69  23  62  215 (-5)  1 0 . 2 12.0  4.8  6.0  7.5  6.0  120  80  80  M.70  19  58  2-50 (-2)  13.6  6.4  4.2  6.0  6.5  7.0  120  80  80  M.71  -  -  -  -  -  -  -  -  -  -  21  55  8.5  8.7  6.0  7.0  7.5  120  90  80  «ajpc  oc  M |Z i-l <  2 & ^<  t-  O C  M l-  < c H  S M.72  -  -  236 (-14)  14.4  TABLE 7  RAT  Q  W EH HH  •=xi o Q  EH  fe W O Ex] O  tH  o  fe fe o o  TOTAL TOTAL WATER' FOOD (cc) (GMS)  DEHYDRATION, LIGATION AND MYOGLOBIN IN INTACT RATS  FINAL WEIGHT AND CHANGE (GMS)  URINE VOLUME  URINE pH  24  48  72  24  48  72  24  B. U. N. (mg. % ) 48 72  M.73  10  23  185  -29)  2.4  0.6  1.2  6.0  6.0  6.0  80  110  60  M.74  10  27  200  -30)  1.9  1-5  2.4  6.0  6.0  5.5  90  100  70  M.75  10  21  180  -16)  1.1  0.6  1.5  6.0  6.0  6.0  60  90  70  M.76  10  39  214  -26)  2.3  1.6  10.1  6.0  6.0  6.0  80  140  70  M.77  10  35  220  -30)  1.6  3.4  6.4  6.0  6.0  7.0  -  110  90  M.78  rf <t\ « o M.79  9  43  224  -20)  3.2  7.2 10.4  6.0  7.0  6.0  170  150  80  7.5  44  216  -42)  2.4  4.0  8.6  6.0  6.0  7.5  160  120  80  M.80  5.0  33  196  -40)  2.0  5.0  9.0  6.0  6.5  7-5  130  130  90  M.81  1.0  41  224  -44)  2.9  5.0 14.0  6.0  6.0  7.0  29O  150  100  M.82  0  34  208  -42)  2.3  7.4  15.8  6.0  7.0  7.5  160  220  160  M.83  7.0  59  224  -28)  4.9  9.8  15.1  6.0  6.0  7.0  -  200  110  M.84  2.0  25  218  -46)  3.0  7.8  9.2  6.0  6.0  7.0  220  170  120  M M EH EH  Q . M > H vA  W + Q  —g +  a o  HH  c5  o  a* CxJ  M  - 102 -  haemorrhage from chewed l i m b s , but these animals had not attacked t h e i r limbs.  The response appears to be one o f  hemodilution to the shock of limb l i g a t i o n , when h y d r a t i o n i s adequate.  Constantinides ( 8 l a ) states that i n the production  of severe shock i n r a t s by pinching exposed i n t e s t i n e s i n several p l a c e s , marked and r a p i d l y developing hemodilution rather than the expected hemoconcentration,  appeared.  Histo-  l o g i c a l l y , the kidneys showed no d e f i n i t e tubular damage when animals were k i l l e d at 72 hours. In Experiment 2 i t was shown that myoglobin I n j e c t i o n alone had no nephropathic e f f e c t ;  i n Experiment 4, i n which  l i g a t i o n of a limb was c a r r i e d out on normally hydrated  animals,  again no l e t h a l r e n a l damage r e s u l t e d , although a d i u r e t i c e f f e c t was n o t i c e d .  In the present experiment  these two f a c t o r s  together (myoglobin and crush i n j u r y ) a l s o produced no l e t h a l kidney damage but again r e s u l t e d i n a marked and d e f i n i t e diuresis.  I n a d d i t i o n , an apparent hemodilution  response,to  the trauma was observed.  Experiment 7: The three f a c t o r s , limb l i g a t i o n , dehydration and myoglobin i n j e c t i o n were combined i n t h i s experiment.  Four  animals were tested thus and compared w i t h four a d d i t i o n a l r a t s subjected to dehydration and l i g a t i o n alone. group of four animals was dehydrated  The c o n t r o l  s i m i l a r l y but was otherwise  - 103  untouched.  -  Dehydration and myoglobin i n j e c t i o n were accom-  p l i s h e d as i n Experiments? with a phosphate b u f f e r as the s o l vent. buffer.  The dehydrated-ligated group received no i n j e c t i o n of Observations are i n Table 7« Intakes of s o l i d s and water were f a i r l y w e l l matched  i n the three groups, as was the weight l o s s .  The l i g a t e d a n i -  mals, however, d i d lose more weight w i t h one animal l o s i n g of i t s o r i g i n a l body weight.  17$  Urine volume f i g u r e s again show  the d i u r e t i c response without any s i g n i f i c a n t d i f f e r e n c e between the two l i g a t e d groups.  Urea n i t r o g e n f i g u r e s i n l i g a t e d  animals were raised to uremic l e v e l s and although at f i r s t glance there seems to be a s i g n i f i c a n t e l e v a t i o n of the myoglobininjeeted group over the non-injected one, i t i s necessary because of the wide range of f i g u r e s and the s m a l l group of s t a t i s t i c s to apply s t a t i s t i c a l methods to these f i g u r e s i n order to reach accurate conclusions.  When t h i s i s done (see Table 7A) i t i s  found that,, at the 24 hour reading the d i f f e r e n c e between these two groups i s not s i g n i f i c a n t and could have occurred by. chance. On the other hand, 48 and 72 hour readings are found to d i f f e r s i g n i f i c a n t l y In the two groups at the 5% l e v e l - i . e . , i n of cases t h i s d i f f e r e n c e would not occur by  9%  chance.  The normal h i s t o l o g y of the kidney of animal M. 76 i s shown i n Figure 15, c o n t r a s t i n g w e l l w i t h a s i m i l a r area i n Zone 3 of animal M.80  (Figure 16) which shows various degrees  of degeneration i n the r e n a l tubules.  This animal was subjected  - 104 -  to l i g a t i o n and dehydration without pigment i n j e c t i o n .  A  s i m i l a r type of damage i s apparent i n animal M.82 ( F i g . 17)  Figure 15  Figure 16  which received myoglobin and here a rare cast i s shown.  In  a d d i t i o n , d i s t a l tubules i n the c o r t i c a l region of t h i s kidney also were degenerating (Figure 1 8 ) .  Figure 17  Results of t h i s experiment  Figure 18  ( l i g a t i o n + dehydration +  myoglobin) should be compared to those of Experiments  5 (ligation  + dehydration) and 6 ( l i g a t i o n + myglobin) as w e l l as w i t h the c o n t r o l animals.  There were no deaths and there was very l i t t l e  - 105 -  e l e v a t i o n of B.U.N, w i t h l i g a t i o n and myoglobin as the s t r e s s ; with l i g a t i o n and dehydration, uremic l e v e l s were reached i n twelve of s i x t e e n t e s t animals and there were no r e n a l deaths. I t would appear, then, that dehydration i s an e s s e n t i a l f a c t o r i n the development of acute t u b u l a r n e c r o s i s from crush i n j u r y and that i n j e c t i o n of myoglobin though not e s s e n t i a l f o r the development of the syndrome, adds s i g n i f i c a n t l y t o the t u b u l a r damage as i n d i c a t e d by urea n i t r o g e n l e v e l s and kidney h i s t o l o g y .  Experiment  8:  The e f f e c t of i n c r e a s i n g the area of crush was i n v e s t i gated i n four r a t s subjected t o b i l a t e r a l hind limb l i g a t i o n f o r four hours a f t e r being dehydrated  f o r 24 hours p r e v i o u s l y .  Dehydration was continued f o r the subsequent 48 hours.  Control  groups of four animals were run on dehydration alone as w e l l as dehydration plus l e f t hind limb l i g a t i o n f o r f i v e hours.  Table  8 c l a s s i f i e s the p e r t i n e n t data. The d i f f i c u l t y i n c o n t r o l l i n g food i n t a k e and to a l e s s e r extent water intake i n the l a s t 24 hours i s again evident, but a l l animals apparently suffered s i m i l a r l y as gauged by t h e i r losses of weight.  Figures f o r urine volumes, though incomplete  because of deaths, again show p o l y u r i a at 24 hours which i s a m p l i f i e d by the water intake at 72 hours.  I n a d d i t i o n i t was  noted that i n two dehydrated c o n t r o l animals wine-coloured urine was excreted though there was no evidence of e x t e r n a l b l e e d i n g .  - 106 -  This was apparently a true hematuria.  Urine pH f i g u r e s again  show the general trend towards a l k a l i n i t y w i t h d i u r e s i s . Urea n i t r o g e n f i g u r e s were among the highest yet recorded.  Dehydrated  c o n t r o l f i g u r e s were elevated at 24 hours  and maintained that l e v e l at 48 hours, though one animal rose to 180 mg %.  That t h i s e l e v a t i o n was due to dehydration alone  i s shown by the prompt r e t u r n to normal i n a l l cases at 72 hours, a f t e r the animals had been allowed water f o r 24 hours. In the u n i l a t e r a l l y l i g a t e d dehydrated c o n t r o l group, B. U. N. l e v e l s were g e n e r a l l y higher and p e r s i s t e d at an elevated l e v e l . f o r a longer time.  Only one animal reached an e x c e s s i v e l y high  l e v e l and that animal died apparently of r e n a l damage. In the b i l a t e r a l l y l i g a t e d dehydrated t e s t group, the r e s u l t s appeared to be not e s s e n t i a l l y d i f f e r e n t from the above group except that the damage appeared more f a t a l .  Three animals  died i n t h i s group, one (M.95) from the shock of haemorrhage from a lacerated foot and from excessive withdrawal of blood at cardiac puncture.  Two others (M.93 and M.96) died uremic deaths w i t h  elevated B.U.N's and periods of a n u r i a of from s i x to eight hours. The f o u r t h animal of the group survived but maintained an elevated B. U. N. l e v e l . H i s t o l o g i c a l sections showed e s s e n t i a l l y normal kidneys i n the dehydrated c o n t r o l s i n s p i t e o f the hematuria observed i n two of the four animals (Figure 1 9 ) .  T y p i c a l degenerative  changes were seen i n Zone 3 of animals M.89, M.90, M.92,  M.94,  TABLE 7 A  24  HOURS  DEHYDR. DEHYDR. + LIG'N, + LIG'N MYOGLOBIN  STATISTICAL ANALYSIS OF FIGURES IN TABLE 7  DIFF. FROM MEAN  "X"  II Y»  DIFF. FROM MEAN »x".  170  290  +17  +67  289  4489  160  160  +7  -63  49  3969  130  220  -23  -3  529  9 £^ = 8467  1=153  x  2  l.y.t  £x=867  M=223  110  150  -18  -35  324  1225  «  150  220  +.22  +35  484  1225  o  K  120  200  -8  +15  64  225  co  130  170  +2  -15  4  225  =R7P  2900  1=128  M=l85  90  100  +5  -22  25  484  CQ  80  160  -5  +38  25  1444  O  80  110  -5  -12  25  .144  CM  90  120  +5 ,  -2  25  4  «  1=85  v. I  1=122  ^  =100  2072  &x. = 17 if  = 53.12  <* * =12.06  /U  6My =37.60  ^  M  H  = 39.48 = 70  - 70/39.48 = 1.77  t  For s i g n i f i c a n c e a t 5% l e v e l , need 2.78 6  K  - 14.76 ^ =26.92  =8.53  <K =15.56  = 17.74 -PH = 57  = 3.21 For s i g n i f i c a n c e a t 5%, need 2 . 4 5 ; a t 3.71. cfx.  =5  *y  = 22.76  <^x = 2 . 8 9 <K= 13.16  ^  =13.47  i)M = 37  t = 2.75 For s i g n i f i c a n c e a t 5^» need 2 . 4 5 ; a t 1%, 3 . 7 1 .  TABLE 8  RAT J25  DEHYDRATION AND BILATERAL LIGATION IN INTACT RATS  TOTAL TOTAL FINAL WEIGHT WATER AND CHANGE FOOD (GMS) (cc) (GMS)  URINE VOL. ec. 24 48 72  URINE pH  B. U. N.  24  48  72  24  mg %  48  72  M.85  10  36  222 (-38)  2.2  1.8  3.3  6.0  6.0  6.0  110  110  70  M.86  10  34  244 (-28)  0.5  0.3  2.8  7-0  6.0  6.5  90  110  70  M.87  10  24  234 (-34)  1.6  1.0  3.4  6.0  6.0  7.0  100  100  90  M.88  10  28  216 (-28)  1.3  0.3  6.0  6.0  6.5  130  180  80  M.89  10  39  252 (-32)  4.1  5.4  8.2  6.0  7.0  7.0  140  120  110  M.90  6.5  36  230 (-30)  3.6  7.2  11.3  6.0  7.0  7.5  190  90  M.91  0  -  -  2.1  0  6.0  -  -  140  410  -  -  M.92  0  15  190 (-46)  1.5  8.1  6.0  6.5  7.0  210  150  160  + e M.93 H  0  232 (-28)  2.3  0  O  M.94-  0  224 (-32)  2.8  5.1  M.95  0  220 (-30)  0.7  M.96  0  222 (-28)  1-9  OCO  Mi_3 E-tO  ©a 0:  OM 1-W EH  DEHYDRA  EH  3.5  :  9.0  6.0 13-3  6.0 6.0  -  6.0  7.5  : :  190  220  220 20  290 260  36-48  36-48  210 6.5  DIED (HRS)  -  -  24-26  -  M.95 and M.96.  109  -  (Figures 20 and 2 1 ) .  Animals M.91 and  M.93  had been dead more than one hour so sections were not v a l i d . In a d d i t i o n , the medulla of animal M.95 casts i n Henle's loop;  Figure 21  showed numerous hyaline  no hematuria was noted i n t h i s animal  Figure 22  I t i s apparent that the more d r a s t i c trauma of b i l a t e r a l crush i n j u r y coupled with the e s s e n t i a l dehydration i s capable of producing a more f a t a l r e n a l damage than i s u n i l a t e r a l  - 110 -  l i g a t i o n and dehydration.  However, the improvement gained  by t h i s procedure, when balanced against the extra time involved and the impression that the procedure approached  the  area of shock deaths, was not f e l t to be s u f f i c i e n t to warrant i t s use.  . I t was instead f e l t advisable to approach the prob-  lem from the opposite p o i n t of view -- i . e . , reduce the r e n a l reserve of the animals.  Subsequent experiments accomplish  t h i s by p r i o r uninephrectomy on a l l animals. Also emphasized  i n t h i s experiment i s the f a c t that  dehydrated c o n t r o l animals r e t a i n water, when i t i s s u p p l i e d , much b e t t e r than do those animals whose limbs had been l i g a t e d .  Experiment  9t  The e f f e c t of reducing r e n a l reserve i s studied i n Experiments 9A and 9B, which deal w i t h male, r i g h t - n e p h r e c t omied r a t s subjected to 72 hours dehydration and f i v e hours ligation.  In 9A, twelve animals were nephrectomied one week  p r i o r to i n i t i a t i o n of dehydration;  four of these were simply  dehydrated, while the remaining eight were l i g a t e d as w e l l as dehydrated.  In 9B, nephrectomy was done three days p r i o r to  dehydration, but the c o n t r o l group of four and t e s t group of eight animals were treated as i n 9A.  Experiment 9B was  designed as a 36 hour experiment so that animals were k i l l e d at that time and correspondingly fewer observations are recorded (Tables 9A and 9B).  - Ill-  In Experiment 9A, a l l animals appeared f u l l y recovered f o l l o w i n g nephrectomy, having regained or exceeded t h e i r preoperative weights.  However, a l l were n o t i c e a b l y more s e n s i t i v e  to nembutal sedation. were not unusual.  Food, water and weight change f i g u r e s  Urine volumes again showed the d i u r e t i c  response p r i n c i p a l l y at 48 hours, and pH f i g u r e s the tendency towards a l k a l i n i t y w i t h d i u r e s i s .  A l l f i g u r e s were higher than  comparable f i g u r e s f o r dehydrated i n t a c t animals. Two dehydrated c o n t r o l animals showed e x c e s s i v e l y elevated B.U.N, l e v e l s and. at autopsy one of these had a granul a r kidney which showed a chronic i n t e r s t i t i a l inflammation histologically.  The kidney of the other animal was not examined.  Of the remaining eight t e s t animals, s i x reached and held high B.U.N, l e v e l s , two o f these as high as 470 mg % and 530 mg %. dehydrated  The remaining two behaved i n a way much l i k e the controls.  Animals M.105  and M.107  died a t 48 and 72 hours respec-  t i v e l y , as a r e s u l t of hemopericardium Animal 1.106  f o l l o w i n g heart puncture.  was k i l l e d at 53 hours p o s t - l i g a t i o n because i t  had been moribund f o r the e n t i r e day, was anuric and s u f f e r i n g severe r i g o r s .  Animal M.104 was c a r r i e d beyond the 72 hour  period but was k i l l e d at 100 hours because i t was moribund; i t s «  post-mortem B.U.N, was 280 mg % and i t was noticed that i t s bowel was f i l l e d w i t h t a r r y m a t e r i a l l i k e old blood.  This  TABLE 9A  RAT  HH  O  Q«  ,IGATION  DEH'  a  ITTO  o  cc; 1 DEH'  s  FOOD (GMS)  LIGATION AND DEHYDRATION IN RIGHT NEPHRECTOMIED RATS  WATER FINAL WEIGHT (cc) AND CHANGE (GMS)  URINE VOLUME cc. 48 24 72  B. U. N.  URINE pH 24  48  72  24  48  72 ,  M.97  5  30  184  (-26)  2.2  1.7  8.8  6.0  6.0  7.0  240  170 100  M.98  5  27  220  (-22)  1.5  1.2  6.0  6.0  6.0  7.0  110  100  M.99  5  30  226  (-14)  1.5  0.6  7.6  6.0  6.0  7.5  180  90 1 1 0  M.100  5  31  210  (-20)  1.2  0.8  9.8  6.0  5.5  7.0  120  90  M.101  5  31  226  (-14)  1.8  4.3  17.0  6.5  6.5  7.5  M.102  5  36  226  (-16)  2.0  1.9  9.1  6.5  7.0  M#103  0  46  196  (-28)  1.3  2.7 2 9 . 3  7.0  M.104  0  9  192  (-26)  1.4  1.2  M.105  0  -  180  (-44)  1.8  7.0  M.106  0  3  212  (-28)  1.0  0.6  -  M.107  0  41  222  (-34)  2.2  7.4  M.108  0  23  220  (-40)  1.2  0.8  DIED (HRS)  90  80  260  130 100  7.5  230  130  90  7.0  7.5  410  230  140  6.5  7.5  7.5  200  300  6.0  7.0  -  240  410  230  48  6.0  6.5  -  270  220 310  53  20.8  6.0  7.0  7.5  530  220  72  16.5  6.5  6.0  7.5  220  230 250  5.1  .  160  TABLE 9B  i  4  M.110  5  M.lll  4  M.112  4  M.113  0  M.114  0  GAT]  M.115  0  i—t  M.116  0  a  M.117  0  a  M.118  0  M.119  0  w  M.120  0  ) RATED JTROL  M.109  :DRA  RAT FOOD  H  W (xl Q  0  O  a  O 1  1  EH  LIGATION AND DEHYDRATION IN RIGHT NEPHRECTOMIED RATS  WATER FINAL WEIGHT (cc)  -  ATJn  HTT  A MCI r?.  URINE VOL. URINE pH B.U.N. 24  36  24  200 (-34)  1.1  dried  238  (-36)  1.9  254 (-40) 230 258  DIED (HR)  'KIDNEY WEIGHT  36  24  36  7.0  -  100  80  271.5 -  0.2  6.0  6.0  100  269.1  1.6  dried  6.0  -  80 140  280.0  (-42)  1.7  dried  7.5  110  90  258.9  (-24)  1.7  0  6.0  190  290  256 (-32)  2..2  0  248 (-24)  1.7  -  6.0  242 (-18)  2.9  248 (-40)  -  6.0  -  0.7  6.0  2.6  1.4  248 (-38)  2.2  264 (-38) 258  (-34)  mg per cm. 2  32-36  254.9  600  -  6.0  210  310  293.6  6.0  6.5  230  270  261.9  2.3  6.0  6.5  220  200  275.4  3.1  2.3  6.0  6.0  210  220  -  2.8  1.0  6.0  6.0  220  260  248.4  400  26  * 276.5 304.7  - 114 -  observation had been made before but not recorded and apparently i l l u s t r a t e s one of the three c l a s s i c a l r e a c t i o n s to s t r e s s , that of g a s t r o - i n t e s t i n a l haemorrhage. anemic at post-mortem.  This animal was g r o s s l y  I n a d d i t i o n , true hematuria was again  observed, t h i s time i n a t e s t animal (M.108). Of eight t e s t animals, then, s i x had elevated B.U.N's which were maintained.  S i x were a c t u a l l y p o l y u r i c while one  was o l i g u r i c and one anuric f o r the ten hours preceding death. H i s t o l o g i c a l l y , three showed d e f i n i t e Zone 3 lower nephron degeneration (Figure 23, compare- Figure 24); questionable changes;  one showed  the remaining four d i d not show h i s t o l o g -  i c a l evidence of r e n a l damage when k i l l e d at 72 t o 144 hours.  Figure 23  Figure 24  An i n t e r e s t i n g s e r i e s of observations was made on animal M. 104.  This dehydrated l i g a t e d animal e x h i b i t e d  o l i g u r i a , progressive uremia and g a s t r o - i n t e s t i n a l but not  - 115  bladder haemorrhage.  -  Figures 2 5 , 2 6 , 27, 28, and 29 show  d i l a t e d tubules and ischemic glomeruli of the cortex and Zones 3 and 4 (medulla) loaded with h y a l i n , granular and y e l l o w i s h (pigment?) casts.  This was the only observation, i n t h i s  series of experiments, i n which o l i g u r i a was so apparently associated w i t h frequent tubular casts and d i l a t a t i o n .  Figure 25  Figure 26  Figure 27  Figure 28  Figure 29  - 116 -  Results o f Experiment 9B were e s s e n t i a l l y the same as i n Experiment 9A.  One c o n t r o l animal reached a B.U.N, l e v e l  of 140 mg % while a l l t e s t animals were over 190 mg. %.  Two  of eight t e s t animals died 24 to 36 hours f o l l o w i n g l i g a t i o n , one being anuric f o r about 20 hours p r i o r t o death.  AH remain-  ing animals retained ( i n most cases at higher l e v e l s ) t h e i r elevated B.U.N's u n t i l k i l l e d at 36 hours.  H i s t o l o g i c a l examin-  a t i o n o f these kidneys revealed that f i v e of eight animals died with t y p i c a l degenerative changes i n Zone 3;  one kidney was  normal, one. showed questionable change and the e i g h t h i l l u s t r a t e d postmortem changes o n l y .  A l l these f i v e had elevated B.U.N.'s  and two died i n uremia.  S i x animals were p o l y u r i c and o n l y one  was anuric p r i o r to death. Kidney weights at death were recorded f o r the f i r s t time i n Experiment 9B and cannot be said to show any s i g n i f i c a n t Controls averaged  269.8 mg.  t e s t animals averaged  273.6 mg.  d i f f e r e n c e between the two groups.  2  per cm  (range 258.9 to  290.0);  254.9 to  304.7).  per cm (range 2  Comparing these r e s u l t s to those of Experiments 5A and 5B make i t apparent that r a t s w i t h reduced r e n a l reserve are more s u s c e p t i b l e to crush i n j u r y w i t h dehydration as i n d i c a t e d by urea nitrogen l e v e l s , tubular damage and m o r t a l i t y r a t e .  These  r e s u l t s compare favorably w i t h those of Experiment 8, i n which the area o f crush was increased i n i n t a c t animals.  - 117  -  10:  Experiment  In an attempt to reduce the number of t e s t s used and to observe the n a t u r a l course of the syndrome, t h i s experiment was concerned c h i e f l y w i t h the m o r t a l i t y i n r i g h t nephrectomized rats subjected to 72 hours dehydration and f i v e hours limb l i g a tion.  Because cardiac punctures would i n t e r f e r e w i t h spontan-  eous deaths, only postmortem B.U.N's were done i n s u r v i v i n g animals.  The weight of kidneys at death i n mg. per sq. cm. of  surface area was again recorded (Table 1 0 ) . Data i n the f i r s t f i v e columns are not s i g n i f i c a n t l y d i f f e r e n t from previous observations except f o r the remarkable weight l o s s seen, e s p e c i a l l y i n dehydrated c o n t r o l s , i n which i t i s commonly 30% of body weight. again. the  The d i u r e t i c response appears  B.U.N.'s were not obtained i n the f i r s t two animals nor  three t e s t animals dying spontaneously.  animals s u r v i v i n g were not elevated. 319.4  mg.  per cm.  mg. per cm.  2  2  Figures f o r t e s t  Kidney weights average  f o r c o n t r o l s (range 289-5 to 368.8) and 332.7  f o r t e s t animals (range 307.4 to 348.4) and though 2  t h i s i s a d i f f e r e n c e of some 13 mg. per cm. , i t cannot be s i g n i f i c a n t because of the wide range seen i n c o n t r o l animals. H i s t o l o g i c a l changes were not v a l i d i n the three t e s t animals which d i e d , and appeared absent i n the remaining animals, i n c l u d ing  controls. Three of eight animals died spontaneously.  M.139  was  found dead at 24 hours and appeared to have died about 11 to 15  - 118 -  hours a f t e r removal of l i g a t i o n ;  M.143 was a l s o found dead at  24 hours and judging by l a c k of r i g o r mortis had died 16 to 17 hours f o l l o w i n g l i g a t i o n removal; a f t e r the i n i t i a l s t r e s s .  M.144 died more than 48 hours  These three were f e l t to be " v a l i d "  deaths - i . e . from r e n a l f a i l u r e and not from shock - and t h i s m o r t a l i t y r a t e was taken as a standard f o r subsequent e x p e r i ments on treatment. I t can be concluded that 72 hours dehydration plus f i v e hours u n i l a t e r a l hind limb l i g a t i o n i n uhinephrectomied  male  a l b i n o r a t s of the Wistar s t r a i n produced death i n r e n a l f a i l u r e i n three of eight t e s t animals (37%) >  Disturbance of r e n a l  f u n c t i o n as indicated by p o l y u r i a and hyposthenuria was seen i n eight of eight (100$) t e s t animals.  Experiment 11: Female r a t s were used at t h i s point i n order to determine any sex d i f f e r e n c e i n the response t o the various therapeut i c agents.  In Experiment  11A, twelve uninephrectomied  animals  were used, s i x as c o n t r o l s and the remaining s i x ( a l t e r n a t e animals) were treated w i t h testosterone 5mg. i n o i l and 5 mg. i n s a l i n e subcutaneously at the time of l i g a t i o n .  A l l animals  were subjected t o 2 4 hours p r e - l i g a t i o n dehydration followed by 1  f i v e hours l e f t hind limb l i g a t i o n and 48 hours p o s t - l i g a t i o n dehydration.  Again, m o r t a l i t y was the main,factor observed i n  order to determine the e f f e c t o f testosterone i n p r o t e c t i n g  - 119  against acute r e n a l f a i l u r e .  -  Observations are recorded i n  Table 11A. ' Because i t was f e l t that the testosterone may not have had s u f f i c i e n t time i n which to a c t , Experiment 11A  was  repeated (Experiment 11B) g i v i n g 5 mg. of testosterone i n o i l 48 and 24 hours p r i o r to l i g a t i o n .  The procedure followed  was  otherwise the same and again m o r t a l i t y was the c h i e f f a c t o r observed (Table 11B). In Experiment 11A, f i g u r e s f o r u r i n e output and B.U.N, were comparable to previous r e s u l t s .  Kidneys of animals  r e c e i v i n g testosterone are not s i g n i f i c a n t l y heavier (at the 5$ l e v e l ) than those of c o n t r o l animals ( c o n t r o l s averaged 251.2 per cm.  2  w i t h a range of 2 2 2 . 8 to 259.95  266.9 rmg.% cm. , 2  range 2 3 8 . 8 to 3 0 2 . 5 ) .  mg.  t e s t animals averaged F i v e animals i n the  c o n t r o l group of s i x , and four i n the, t e s t group of f i v e , d i e d . This increased m o r t a l i t y plagued a l l subsequent therapy e x p e r i ments whether i n male or female animals and the problem w i l l be d e a l t w i t h i n the d i s c u s s i o n to f o l l o w .  I t i s , however, appar-  ent that testosterone proprionate given i n adequate dosage at the time of the i n i t i a l s t r e s s i s not e f f e c t i v e i n p r o t e c t i n g female r a t s against traumatic uremia and death i n acute r e n a l failure. H i s t o l o g i c a l examination of these kidneys r e v e a l e d , i n t e s t animals, three w i t h t y p i c a l lower nephron degeneration, one w i t h questionable changes, and the f i f t h animal i l l u s t r a t e d post  TABLE 10  •"OOD WATER WEIGHT AND (ce) CHANGE (GM) :GM)  URINE VOLUME (cc) 24 48 72 96  URINE pH 24  48  72  96  B.U.N. at 96 HRS  DIED  -  KIDNEY WEIGHTp. (mg/cm  M.133  5  16  128 (-66)  1.6 0 . 5  7.4 4.8  6.0 6.0 7.0 7.0  M.134  5  11  126 (-64)  0.8 0.5  5.3 4.3  6.5 6.0 6.0 6.5  M.135  5  14  114 (-50)  1.2 1.0  6 . 8 2.0  6.5 6.0 6 . 5 7.0  100  368.8  M.136  5  15  116 (-60)  1.4 0.4  5.0 3 . 8  6.0 5.5 7.5 7.5  80  289.5  M.137  5  66  128 (-46)  1.1 2.2 16.8 29.0 6.0 6.0 7.5 7.5  110  338.0  LIGJ1TI0  RAT  MORTALITY IN DEHYDRATED, LIGATED. UNINEPHRECTOMIED RATS  M.138  5  42  130 (-66)  2.0 8.8 18.7 7.2  90  348.4  M.139  0  —  138 (-26)  0.7  +  M.140  5  53  136  (-52)  1.9 6.6 13.9 7.6  6.0 6.0 7.0 7.5  80  307.4  M.141  5  41  132 (-50)  1.8 5.4 15.2 1D.6  6.0 6.5 7.0 7.5  110  310.3  M.142  5  37  122 (-50)  2.0 4 . 6 12.8 7-o!  6.0 6.5 7.5 7.5  130  341.8  M.143  0  -  160 (-32)  0.4  M.144  0  3  140 (-24)  1.1 1.0  P EH  K Q  S  C E-  is C  jij c  DEHYDR ATK  p  6.0 7.0 7.5 7.5  313.4 306.0  ; 12-24 337.8  -  -  - .; 7.0 - L  6.0 6.0  -  -  -  -  12-24  345.5'  -  ; 34-36  -  TABLE 11A  RAT  FOOD (GMS)  WATER (cc)  TESTOSTERONE IN CRUSH SYNDROME  WEIGHT AND CHANGE (GMS)  URINE VOLUME (cc) 24 48 72  URINE pH 24  48  72  B.U.N, KIDNEY at DIED WEIGHT DEATH (HRS) Mg/cnr  T  M.145  0  0  180 (-14)  0.7  -  6.0  -  -  300  21  302.5  C  M.146  0  0  188 (-18)  0.7  -  6.0  -  -  -  29  258.6  T  M.147  0  0  168 (-6)  0.8  -  6.0  •-  - -  370  24  272.7  C M.148  1  37  220 (-28)  1.3  0.3  24.5  6.0  6.0  7.0  170  T  M.149  0  0  200 (-24)  1.8  drop  -  5.5  7.5  -  -  56-72 263.7  e  M.150  0  0  178 (-20)  1.1  0  6.0  -  -  -  56-72 259.9  T  M.151  - .  -  -  -  -  -  c  M.152  0  0  200 (-22)  1.0  drop  6.0  -  -  -  T  M.153  3  50  198 (-24)  1.1  0.6 26.0  6.0  6.5  7.5  90  c  M.154 0  0  200 (-26)  0.7  -  6.0  -  -  T  M.155  0  0  152 (-21)  drop  -  -  -  C M.156  0  0  188 (-22)  1.0  -  5.5  -  -  -  -  .-  -  254.3  -  -  56-72 253.3  -  238.8  300  20  258.5  -  300  17  257.0  -  -  26  222.8  - 123 -  mortem changes.  A feature of the pathology p r e v i o u s l y men-  tioned i s here very evident.  The f a c t that the  degenerative  and pyknotic changes i n Zone 3 tubules are f a r t h e s t removed from congested venae rectae i s seen i n Figure 30 (low power) and Figure 31 (high power).  n»t  If V  Figure 30  ...  "  1  c*  '  •»«»•*  m' "  *  • *  • -  -* V  ' /  K>  •/  *•  Figure 31  In Experiment 11B, not one of the animals survived to be k i l l e d at 72 hours, so that once again the increased i t y rate i s i l l u s t r a t e d .  fatal-  Eight of the animals were "found  dead" 24 hours a f t e r l i g a t i o n was applied and so the problem of shock death rather than uremic death i s r a i s e d .  Four a n i -  mals (two t e s t and two c o n t r o l ) died a f t e r t h i s 24 hour period and are taken to be c e r t a i n uremic deaths;  two of these (the  t e s t animals) had r a i s e d urea nitrogen f i g u r e s . Three kidneys could be examined h i s t o l o g i c a l l y and a l l three showed changes t y p i c a l of acute tubular n e c r o s i s (see Figure 32) which were also apparent i n the d i s t a l tubules of the cortex.  These kidneys also e x h i b i t e d the f r e q u e n t l y  - 124 -  observed marked congestion of the medulla (Figure 3 3 ) . weights i n mg. per cm.  2  Figure 32  Kidney-  at death again show no p r e d i c t a b l e p l a n ,  Figure 33  average f i g u r e s being 271.6 mg. per cm.  2  f o r test animals  (range 260.5 to 280.0) and 277.2 mg. per cm.  f o r controls  2  (range 258.0 to 3 1 0 . 8 ) . In considering r e s u l t s of Experiments 11A and 11B i t must be concluded that testosterone proprionate given i n adequate doses to l i g a t e d , dehydrated, uninephrectomied female rats does not reduce t h e i r m o r t a l i t y r a t e .  Whether or not the  hormone has some p a l l i a t i v e e f f e c t as measured by decreased s t r u c t u r a l damage or lower urea n i t r o g e n l e v e l s i n t e s t animals cannot be stated.  Experiment 12 Experiment 11 was repeated here using twelve male animals and a dose of testosterone 5 mgs. i n o i l 72 and 24 hours p r i o r to l i g a t i o n , as w e l l as at time of l i g a t i o n , i n a l t e r n a t e animals.  Table 12 presents the observations.  TABLE 11B  RAT  FOOD (GMS)  TESTOSTERONE IN CRUSHED FEMALE RATS  WATER WEIGHT AND (cc) CHANGE (GMS)  URINE pH  URINE VOLUME (ce) 24 4b 72  24  48  72 -  B.U.N. at DEATH  DIED  KIDNEY WEIGHT (HRS) Mg/cm 2  -  24  269.8  24  310.8  -  24  269.4  T  M.157  0  0  196 (-18)  0.3  -  7.0  -  C  M.158  0  0  190 ( - 2 6 ) '  0.8  -  7.0  -  T  M.159  0  0  202 (-18)  0.2  -  C M.160  0  0  176 (-24)  0.4 0  -  -  T  M.l6l  0  0  194 (-26)  0.9  0  6.0  290  29  281.9  C  M.162  0  0  194 (-28)  1.4  0  6.0  26  258.0  T  M.163  0  0  180 (-18)  0.5  -  6.0  -  24  260.5  T M.164  0  0  170 ( - 2 0 J  0.7  -  -  -  24  282.0  T M.165  0  0  200 (-24)  0.8  0  6.0  300  28  282.0  C M.166  0  0  185 ( - 2 0 )  0.2  -  _•  24  258.2  T  M.167  0  0  190 ( - 2 0 )  0.4  -  6.0  24  264.9  C M.168  0  0  170 (-22)  1.2  -  6.0  24  267.4  -  -  -  -  -  32-48 2 8 6 . 9  TABLE 12  RAT  FOOD AND WATER  WEIGHT AND CHANGE  TESTOSTERONE IN CRUSHED MALE RATS  URINE VOLUME (cc) 24 48 72  URINE pH 24  48  72  B.U.N. at DEATH  DIED  KIDNEY WEIGHT mg/cm  24  266.4  24  214.8  2  M.253  0  208 (-14)  0  C M.254  0  206 (-20)  0.1 -  T M.255  0  192 (-16)  0  -  -  24  203.7  5E M.25.7  0  186 (-16)  0  -  -  24  230.1  C M.258  0  200 (-24)  0.5  -  24-  250.6  T M.259  0  194 (-16)  0.3 -  24  226.0  C M.260  0  202 (-20)  0.3 -  24  230.5  0 5 gm 60cc  210 (-18)  0.4 -  24  249.3  0  196 (-22)  T  -  -  -  -  C M.256  T  M.261  C M.262 T 0  M.264  5.0 6.0  188 (-24) 0.3  M.263  -  310  279.8  3.4 30.0 24  0.2 -  201.8  - 127 A l l animals appeared; markedly shocked when l i g a t u r e s were removed a f t e r f i v e hours and nine of t e n were dead when seen the f o l l o w i n g morning (24 hours a f t e r l i g a t u r e s were applied).  Two other animals died during the period of l i g a t i o n .  These r e s u l t s were t y p i c a l of those of l a t e r experiments i n which death occurred e a r l i e r and more f r e q u e n t l y . averaged and  235.1  235.5  mg. per cm.  mg. per cm.  2  2  Kidney weights  201.8 to 279.8) f o r c o n t r o l 203.7 to 266.4) f o r treated animals.  (range  (range  One untreated c o n t r o l animal r a n a 72 hour course showing a t y p i c a l c l i n i c a l p i c t u r e of acute r e n a l f a i l u r e i n the r a t , with an eventual d i u r e t i c response and a B.U.N., when k i l l e d , o f 310 mg. %.  H i s t o l o g i c a l l y , that kidney showed changes taken to  be regeneration f o l l o w i n g tubular damage:  areas of f l a t t e n e d ,  b a s o p h i l i c tubular c e l l s associated w i t h c e l l u l a r d e b r i s i n the lumens (see s e c t i o n on " D i s c u s s i o n " ) . Testosterone /propionate t h e r e f o r e would appear to be i n e f f e c t i v e i n maintaining or prolonging l i f e i n male r a t s experiencing shock and acute renal f a i l u r e .  Experiment 13t The e f f e c t of cortisone acetate on m o r t a l i t y i n uni-r nephrectomied, dehydrated and l i g a t e d female r a t s i s considered i n t h i s experiment.  Two mgs. of cortisone i n s a l i n e were  i n j e c t e d subcutaneously 24 hours p r i o r to l i g a t i o n and the dose repeated d a i l y u n t i l death.  Twelve animals were used, a l t e r n a t e  - 128  -  ones being treated w i t h the t e s t drug. Table  Observations are i n  13. M o r t a l i t y rate was again high, but most animals l i v e d  beyond the 24 hour p e r i o d .  A l l treated animals died i n the  24 to 28 hour p e r i o d , w h i l e two c o n t r o l animals died i n the 32 to 48 hour p e r i o d .  Of eight animals i n which B.U.N, l e v e l s  were determined at death, a l l were elevated to uremic l e v e l s and a l l showed h i s t o l o g i c a l changes of acute tubular n e c r o s i s . Representative areas of Zone 3 are i l l u s t r a t e d i n Figures 34 ( c o n t r o l ) and 35 ( t e s t ) .  Figure 34  F i g u r e 35  These animals were also a n u r i c f o r periods up to 24 hours p r i o r to death.  Kidney weights again revealed no s i g n i f i c a n t r e l a t i o n 2  ship of treated (average 250.7 mg. per cm. 272.2) to untreated (av. 246.4 mg. per cm.  - range 240.4 to 2  - range 235.5 to  260.5).  Cortisone acetate would appear to be i n e f f e c t i v e i n  -  -  129  reducing the m o r t a l i t y from or s e v e r i t y of acute tubular necrosis i n female r a t s subjected to uninephrectomy, dehydration and crush i n j u r y .  Experiment 14: The above experiment with c o r t i s o n e i s here repeated using, i n s t e a d , male animals.  Procedure and dose schedule  of cortisone acetate were e s s e n t i a l l y the same as i n that experiment.  Observations are i n Table 14. A l l twelve animals ( s i x t r e a t e d , s i x untreated) were  dead when observed 24 hours a f t e r l i g a t u r e s were a p p l i e d , so that no observations were made, other than weight l o s s and 24 hour urine volume.  No e x p l a n a t i o n f o r t h i s exaggerated increase  i n e a r l y m o r t a l i t y was apparent, but the problem i s considered i n the s e c t i o n on "Discussion .  Kidney weights show an i s o l a t e d  11  example of s i g n i f i c a n c e at the 1% l e v e l i n that treated animals averaged 243.6 mg. per cm.  (range 220.4 to 260.3)? while con-  t r o l s averaged 214.3 mg. per cm.  2  (range 191.8 to 236.3).  No  h i s t o l o g i c a l examinations were made because a l l kidneys had undergone postmortem change. No conclusions can be drawn from the experiment other than that cortisone appears to have no favorable e f f e c t i n prot e c t i n g male r a t s from death from shock and/or r e n a l f a i l u r e . I t becomes apparent that the standardized production of acute  TABLE 13  RAT  FOOD (GMS)  WATER (cc)  CORTISONE IN CRUSHED FEMALE RATS  WEIGHT AND CHANGE (GMS)  URINE VOLUME (cc) 24 -48 72  URINE pH 24  48  5.5  -  72  T  M.169  0  0  180 (-20)  1.4  C  M.170  0 '  0  180 (-20)  1.0  T  M.171  0  0  180 (-20)  0.5  - -  C  M.172  0  0  170 (-30)  1.2  0  T  M.173  0  0  178 (-18)  0.4  0  C M.174 0  0  184 (-24)  0.6  -  -  M.175  0  0  174 (-28)  1.3  0  6.0  C M.176  0  0  182 (-30)  0.7  0  6.0  M.177 0  0  184 ( -20 )  0.6  :0  5.5  -  -  5.5  -  -  -  -  T  T  -  -  5.5  -  6.0  -  C M.178  0  0  210 (-22)  0.6  0  T  M.179  0  0  194 (-34)  0.6  -  6.0  C M.I80  0  0  212 (-28)  1.1  0  6.0  -  -  -  -  B.U.N. at DEATH mg. %  DIED  190  25  243.7  400  26  260.5  230  27  240.9  270  48  235.5  370  251.4  -  27 12-24  248.6  290  27  272.2  -  32-48  500  28'  251.4  30  236.8  KIDNEY WEIGHT (HRS) Mg/cm 2  -  -  12-24  244.7  400  27  250.6  TABLE 14  FOOD (GMS)  WATER (cc)  T M.241  0  C M.242  CORTISONE IN CRUSHED MALE RATS  URINE pH  WEIGHT AND CHANGE (GMS)  URINE VOLUME (cc) 24 48 72  0  190 (-20)  0.3  -  -  0  0  192 (-22)  0.3  -  -  T M.243  0  0  198 (-16)  0.9  -  -  C M.244  0  0  232 (-16)  0.5  -  -  T M.245  0  0  180 (-16)  0.9  -  "  C M.246  0  0  190 (-16)  0.1  T M.247  0  0  204 (-22)  0.4  -  C M.248  0  0  184 (-14)  0.4  -  T M.249  0  0 •  228 (-22)  0.6  -  C M.250  0  0  190 (-18)  0.2  T M.25I  0  0  224 (-18) • • 0 . 5  -  -  C M.252  0  0  214 (-18)  -  -  RAT  0.3  24  48  _  _  72  _  _  DIED  KIDNEY WEIGHT (HRS) Mg/cm 2  12-24  248.6  12-24  209.1  12-24  220.4  12-24  231.1  12-24  238.0  12-24  191.8  12-24  260.3  12-24  196.1  12-24  252.9  -•  12-24  221.6  -  12-24  241.5  -  12-24  236.3  _  -  -  -  B.U.N. at DEATH  -  - 132 -  t u b u l a r n e c r o s i s and r e n a l f a i l u r e by these methods i s much l e s s p o s s i b l e than was i n d i c a t e d i n e a r l i e r experiments.  Experiment 15? A combination o f t e s t o s t e r o n e , w i t h i t s r e n o t r o p i c (333) and/or p r o t e i n - s p a r i n g a c t i o n s , and c o r t i s o n e , w i t h i t s c o - c a l l e d l i f e - m a i n t a i n i n g f a c t o r , might conceivably be e f f e c t i v e i n cases of acute t u b u l a r n e c r o s i s , where e i t h e r one o f these agents alone would f a i l .  These hormones were therefore  used together i n t h i s experiment, w i t h dosages arranged as i n Experiments 11B and 13.  Twelve female animals were again used  with a l t e r n a t e animals r e c e i v i n g the t e s t drugs.  Table 15  presents the observations. These animals were smaller than u s u a l , being 165 to 180 gm. i n weight, which f a c t may account i n part f o r the increased m o r t a l i t y at 24 hours. beyond the 24 hour p e r i o d ;  Only four animals l i v e d  three of these were untreated c o n t r o l  animals and one had been treated w i t h hormones;  a l l four had  elevated B.U.N.'-s; and a l l showed h i s t o l o g i c a l evidence of acute tubular n e c r o s i s . 2  cm.  Kidney weight f i g u r e s averaged 284.5 mg. per  (range 261.0 to 3 2 2 . 9 ) f o r t e s t animals, 277.4 mg. per cm  2  (range 251.7 to 287.5) f o r c o n t r o l s , hardly a s i g n i f i c a n t d i f f e r ence. Though these animals were perhaps too small f o r an  TABLE 15  RAT  FOOD WATER (GM) (cc)  TESTOSTERONE PLUS CORTISONE IN CRUSHED FEMALE RATS  WEIGHT AND CHANGE (GM)  URINE VOLUME 24 48 72  URINE pH 24 48 72  _  M.181  0  0  168 (-12)  0.5  -  -  C M.182  0  0  148 ( - 2 0 )  0.3  -  -  T  M.183  0  0  152 (-18)  0.6  C M.184  0  0  162 ( - 2 0 )  0.9  -  T  M.185  0  0  156 (-18)  1.1  -  C M.186  0  0  146 ( - 2 0 )  T  M.187  0  0  C  M.188  0  T  M.I89  T  2  -  12-24 261.0  -  12-24 2 8 7 . 5  -  12-24 282.1  -  -  -  6.0  -  410  -  6.0  -  -  12-24 265.4  0.6  6.5  -  -  12-24 283.9  152 (-16)  0.6  5.5  -  -  12-24 322.9  0  140 (-14)  0.7  -  5.5  "  230  25  251.7  0  0  146 ( - 2 0 )  0.8  -  6.0  -  440  26  296.8  C M.190  0  0  144 (-14)  1.6  -  6.0  -  320  27  285.7  T  M.191  0  0  160 (-14)  0.8  6.0  -  C M.192  0  0  154 (-18)  0.6  6.0  -  -  -  B.U.N. DIED KIDNEY at WEIGHT DEATH Mg/cm. Me. t  r  -  25  273.3  12-24 278.8 12-24 282.6  TABLE.16  RAT FOOD (GM)  TESTOSTERONE AND CORTISONE IN CRUSHED MALE RATS  URINE VOLUME WATER WEIGHT AND (cc.) (cc) CHANGE (GM) 24 48 72  0  200  (-28)  0.15  -  0  11  220  (-34)  0.95  -  M.196  0  0  212  (-40)  0.3  -  T  M.197  0  0  224  (-34)  0.4  C  M.198  0  0  210  (-36)  0.5  -  T  M.199  0  0  214 (-42)  0.5  -  C  M.200  5  55  228  (-38)  1.9  T  M.201  0  0  222  (-38)  0.1  C  M.202  0  7  194  (-36)  0.4  T HI. 2 0 3  0  0  208  (-32)  0.5  204  3  57  200  (-58)  0 . 5 5 14.7 2 0 . 8  T M.193  0  C M.194  '•-  T  M.195  C  C  pi.  URINE pH 24  48  —  —  ^ 5  -  -  -  72  —  -  B.U.N. at DEATH Mg.$ —  -  DIED (hrs)  KIDNEY WEIGHT^ Mg/cm.  12-24  281.9  36-48  261.6  12-24  -  12-24  285.0  12-24  269.5  12-24  318.4  -  295.9  12-24  286.4  5.5  6.5  7.0  180  -  -  -  0  5.5  -  -  -  -  -  -  12-24  296.9  5.5  6.5  7.0  200  -  276.7  1 7 . 2 9.2  30  281.9  - 135  -  adequate t e s t , i t would appear that testosterone and c o r t i s o n e together a f f o r d no p r o t e c t i o n against the lower nephron damage produced by uninephrectomy, dehydration and crush i n j u r y i n female r a t s .  Experiment  16;  Experiment 16 considers the e f f e c t of testosterone and c o r t i s o n e on the m o r t a l i t y i n uninephrectomied, and l i g a t e d male a l b i n o r a t s of the Wistar s t r a i n .  dehydrated Twelve  animals were used, but one c o n t r o l died during the l i g a t i o n period and was not replaced.  Dosage of testosterone was  increased to 5 mg. i n o i l 96, 48 and 24 hours before l i g a t i o n , as w e l l as 5 mg. at the time of l i g a t i o n ;  that of c o r t i s o n e  was, as before, 2 mg. d a i l y s t a r t i n g 24 hours before l i g a t i o n and continuing as a d a i l y dose.  The period of dehydration here,  however, was shortened to a t o t a l period of 48 hours - 24 before and 24 a f t e r l i g a t i o n - i n order to l e s s e n the s t r e s s and prolong l i f e to allow determination of whether or not the animals reached a s t a t e of acute r e n a l f a i l u r e . i n Table  Observations appear  16. Again, seven animals died w i t h i n the 24 hours f o l l o w i n g  l i g a t i o n i n s p i t e of being of adequate body weight;  one died  during the l i g a t i o n period of massive r e t r o p e r i t o n e a l haemorrhage f o l l o w i n g nembutal i n j e c t i o n ;  two animals (one t e s t and  c o n t r o l ) survived f o r 24 to 48 hours;  one  two remaining animals  - 136  -  (both c o n t r o l s ) survived f o r 72 hours and were k i l l e d .  These  had urea n i t r o g e n l e v e l s of 180 and 200 mg. % and though the f i r s t (M.200) h i s t o l o g i c a l l y appeared to show signs of healing lower nephron degeneration ( e p i t h e l i a l d e b r i s i n medullary tubules - see Experiments 12 and 17B), the second (M.204) had an e s s e n t i a l l y normal kidney.  Figures 36 and 37 show a t y p i -  c a l area o f Zone 3 degeneration and of Zone 4 (medulla) w i t h frequent c a s t s .  The two animals which recovered showed a  I  Figure 36  Figure 37  t y p i c a l d i u r e t i c response at 48 and 72 hours.  Kidney weights  averaged 288.4 mg. per cm^ f o r s i x treated animals (range to 3 1 8 . 4 ) , 281.0 mg. per cm  •  261.6  f o r four c o n t r o l s (range 269.5 to  295.9).  As i n Experiment 15, i t can only be concluded that testosterone and cortisone have no t h e r a p e u t i c value i n male r a t s w i t h acute tubular n e c r o s i s .  - 137 -  Experiment 17: The e f f i c a c y o f Compound F i n the treatment o f acute tubular n e c r o s i s i n uninephrectomied, dehydrated, l i g a t e d male r a t s i s tested i n Experiment 17A.  Observations are made on  urine output, blood urea n i t r o g e n , kidney h i s t o l o g y and mortali t y rate.  S i x animals were so t e s t e d , w i t h s i x untreated  controls.  Compound F was given as a d a i l y dose o f 2 mgs. i n  s a l i n e subcutaneously, s t a r t i n g 48 hours before l i g a t i o n .  Obser-  vations appear i n Table 17A. Because of the promise shown by Compound F i n t h i s experiment, i t was repeated i n Experiment 17B using a s l i g h t l y higher dosage o f t h i s substance, 3 mgs. d a i l y s t a r t i n g 72 hours before l i g a t u r e s were a p p l i e d . twelve males were so t r e a t e d .  A l t e r n a t e animals of a group o f Observations were made on urine  outputs and m o r t a l i t y r a t e but B.U.N's were determined only postmortem on animals f r e s h l y dead.  These appear i n Table 17B.  By 24 hours f o l l o w i n g l i g a t i o n , nine animals i n E x p e r i ment 17A had d i e d .  The three remaining animals survived f o r  72 hours t o be k i l l e d a t that time f o r kidney h i s t o l o g y . were treated animals.  These  Urine outputs were t y p i c a l o f the d i u r e t i c  response, and urea n i t r o g e n l e v e l s were elevated at 24 hours but at 72 hours were subsiding.  H i s t o l o g i c a l sections of these  three kidneys revealed what i s considered t o be h e a l i n g acute t u b u l a r . n e c r o s i s (see Figures 38-40 i n Experiment 17B).  Kidney  TABLE 17A  FOOD WATER (cc) RAT (MG)  COMPOUND F I N CRUSHED MALE RATS  URINE VOLUME WEIGHT AND cc. CHANGE (GM) 24 48 72  24  48  72  T  M.205 9  66  172 (-36)  1.5 5-9 2 6 . 4 6 . 0 6 . 0 6 . 5  C  M.206 0  0  224  (-24)  0.4  -  T  M.207 0  0  188 ( - 2 8 )  0.0  -  C  M.208 0  0  172 (-24)  0.5  -  T  M.209 0  0  174  C  M.210 0  0  180 (-28)  T  M.211 9  63  184  (-24)  (-44)  -  -  -  6.0  -  0.75 -  -  5.5  -  0.3  -  -  -  -  1.8 6 . 7 1 9 . 8  C  M.212  0  0  188 (-28)  0.4  -  T  M.213  0  0  186 (-30)  0.3  -  -  C M.214  0  0  170 (-28)  0.1  -  -  T  M.215  9  40  172 (-36*)  i . a 2 . 9 7.o  C  M.216  0  0  198 (-24)  0.6  B.U.N.  URINE PH  -  6 . 5 7 . 0 7.5  -  -  -  -  -  6.5 6 . 0 6 . 5  me.#. 2 4  240  48  -  72  -  -  190  -  2  231.2  12.24  251.2  12-24  201.0  12-24  202.3  12-24  197-7  130  -  260.5  -  12-24  262.4  12-24  247.2  12-24  210.9  -  12-24  203.4  150  -  225.4  12-24  212.5  - .  (hrs)  Mg/cm .  _  170  -  -  250  KIDNEY WEIGHT DIED  TABLE 17B  COMPOUND E IN CRUSHED MALE RATS  FOOD (GM)  WATER (cc)  0  99  218 ( -38)  2.6 16.6 47.4  5.5 6.0 7.5  200  C M.230 10  86  276 ( -40)  3.4 28.0 11.0  6.0 7.0 7.5  150  -  201.6  T M.231  9  77  174 ( -3D  1.4 21.3 18.6  170  -  229.2  C M.232  0  0  . 190 (-20)  0.2  -  -  6.0 6.5 7.0  -  12^24  208.1  T M.233  0  0  226 ( - 3 0 )  0.9  0  C M.234  0  3  230 ( -38)  1.2  0  T M.235  0  8  220 ( -32)  0.6  0.3  C M.236  0  0  196 ( -22)  0.7  -  •-  T M.237  0  0  190 ( -28)  0.4  -  -  C M.238  0  61  224 ( -52)  2.1  8.0 31.0  T M.239  0  0  190 ( -24)  0.7  0  C M.240  0  0  184 ( -24)  0.2  -.  RAT  T M.229  WEIGHT AND URINE VOLUME CHANGE (GM) (cc) (GM) 24 48 72  -  URINE pH , . B.U.N. 24 48  72  6.0  -  -•  6.0  -  -  6.0  -  6.0  -  -  6.0  -  -  mrt  •  DIED (hrs)  KIDNEY WEIGHT mg/cm.2  289.9  280  25  218.7  420  26  236.6  -  29-33  222.4  12-24  201.0  12-24  245.9  6.0 6.0 7.5  240  -  246.3  -  300  25  251.3  -  . 6.0  -  -  -  12-24  234.8  - 140 -  weights were again not s i g n i f i c a n t s 221.4  mg. per cm.  229.5 mg. per cm.  treated animals averaged  (range 197*7 to 262.4), untreated c o n t r o l s  2  (range 202.3 to 2 6 0 . 5 ) .  2  On the basis of Experiment 17A w i t h twelve male a n i mals, i t can be concluded that Compound F shows some promise i n the treatment of acute t u b u l a r n e c r o s i s induced by dehydration i n crushed animals.  Three of s i x treated animals survived  while none of s i x untreated animals s u r v i v e d . In experiment 17B, water was allowed animals a f t e r 48 hours dehydration, instead of the usual 72 hours, i n an attempt to prolong l i f e so that more s e r i a l observations could be made. I t was f e l t  that,  i f kidney damage was already present, free  water intake would not improve the u l t i m a t e outlook and so not i n t e r f e r e w i t h comparisons of m o r t a l i t y r a t e .  Kidneys were  here and subsequently f i x e d i n Herlant's s o l u t i o n because of t e c h n i c a l d i f f i c u l t i e s w i t h Zenker's f i x a t i v e and r e s u l t s amply j u s t i f i e d the switch. Urine outputs i n those animals s u r v i v i n g 48 hours or more showed a remarkable d i u r e t i c response and i n d i c a t e d that these animals w i t h damaged kidneys could not handle water intake satisfactorily.  I t appeared that the increased o r a l amount  was r a p i d l y flushed out through the kidney and l o s t , so that animals continued to lose weight. In the four animals s u r v i v i n g to 72 hours (two t r e a t e d ,  - 141 -  two untreated), B.U.N, l e v e l s remained elevated, i n d i c a t i n g continuing r e n a l damage, and r e n a l h i s t o l o g y showed a p i c t u r e of what has been described p r e v i o u s l y as healing acute tubular necrosis.  This healing p i c t u r e i s shown i n Figure 38 (Zone 2  of animal M. 229) are seen appearing  i n which new,  i n disorganized or degenerated areas; Figure  39 (Zone 3 of M. 229) tubules;  low b a s o p h i l i c cuboidal c e l l s  showing e p i t h e l i a l debris with n u c l e i i n  and i n Figure 40 (medulla of M. 229)  Figure 38  Figure 39  the e p i t h e l i a l casts.  Two  which again shows  Figure 40  treated and two untreated  animals  therefore survived to be k i l l e d at 72 hours with elevated urea nitrogen l e v e l s , evidence of healing tubular damage and  records  of d i u r e s i s again i n d i c a t i n g kidney d y s f u n c t i o n . Three other animals (two t r e a t e d , one untreated) died 25 to 36 hours a f t e r l i g a t u r e s were a p p l i e d , with B. U. N. l e v e l s elevated to 280 to 420 mg. %, 24 hour urine volumes at o l i g u r i c l e v e l s ( 0 . 7 to 1.2  cc) and h i s t o l o g i c a l acute tubular n e c r o s i s .  - 142 -  A t o t a l of eight animals died spontaneously, four having been treated w i t h Compound F and four untreated;  the remaining four  survived to 72 hours. Kidney weights averaged 242.9 mg. per cm. 218.7 to 289.9) f o r treated and 221.4 mg. per cm  2  (range (range 201.0  to 246.3) f o r untreated c o n t r o l animals. Conclusions to be drawn from t h i s experiment i n c l u d e the f o l l o w i n g :  1) Compound F does not appear to prolong the  l i f e of or reduce m o r t a l i t y i n male r a t s s u f f e r i n g from e x p e r i mental acute tubular n e c r o s i s .  Since t h i s agent d i d seem to  have some p a l l i a t i v e e f f e c t i n Experiment 17A, determination of i t s true value i n treatment of the syndrome must await f u r t h e r experimentation.  2) Animals w i t h evidence of r e n a l  damage are seen to handle o r a l intake of water i n an i n e f f i c i e n t and disadvantageous manner.  Experiment 18: A f o u r t h and f i n a l hormonal agent, desoxycoricosterone acetate, was tested i n male r a t s i n which dehydration and crush i n j u r y were used to produce acute tubular n e c r o s i s .  In particu-  l a r , m o r t a l i t y rate was noted, but observations on urine output, blood urea n i t r o g e n and kidney h i s t o l o g y were also made.  Again,  twelve animals were used, a l t e r n a t e ones being treated w i t h DCA 2.5 mgs. i n water subcutaneously each day beginning 48 hours before l i g a t i o n of the limb.  Table 18 l i s t s the observations.  TABLE 18:  RAT  FOOD (GM)  DESOXYCORTICOSTERONE IN CRUSHED MALE RATS  WATER WEIGHT AND URINE VOLUME (cc) CHANGE (GM) (cc) 24 48 72  URINE pH 24  48  72  5.5  DIED B.U.N. Mg. % 24 48 72  T M.217  0  0  236- (-24)  1.9  0  C M.218  0  0  250 (-12)  0.5  -  -  -  T M.219  0  0  228 (-36)  1  '°  2.0  -  6.0  6.0  C M.220  5  60  210 (-32)  2.9  2.3 25.9  6.0  T M.22I'  0  0  224 (-26)  2.3  0.7  -  5.5  5 . 5 7.5 5 . 5 -  420  C M.222  0  0  224 (-20)  2.1  0.1  -  5 . 5 -  240  T M.223  0  0  182 (-16)  1.0  0  -  5.5  C M.224  0  0  194 (-26)  1.2  0  T M.225  0  0  188 (-24)  0.7  0  C M.226  0  0  186 (-12)  0  -  T M.227  0  0  174 (-16)  0.9  0  C M.228  0  0  178 (-26)  1.5  0  -  300  -  -  -  5.5  • -  5-5  -  5.5 6.0  5-5  -  280  290  360  -  210  - 190  -  - -  QDNEY »ffiIGHT (Mg./cm~) 2  32-48  230.7  12-24  213.4  32-48  214.7  -  256.9  32-48  198.0  32-48  195.6  31  202.7  32-48  223.4  32-48  209.2  24  243.8  31-32  200.5  32-48  186.4  - 144 -  Only one animal, M . 220 to 72 hours;  (a c o n t r o l animal) survived  t h i s animal had an elevated  B.U.N,  and  kidney  h i s t o l o g y showed f o c i of regeneration i n Zones 2 and 3 as p r e v i o u s l y described.  I t has been pointed out before that  t h i s change was seen f r e q u e n t l y i n animals known to have suffered kidney damage but which e v e n t u a l l y recovered. c o n t r o l animal, M . 226,  A second  was observed to die i n convulsions  hours a f t e r the l i g a t u r e was applied and at that time the  24 B.U.N,  was 360 mg. % and kidney h i s t o l o g y was t y p i c a l of "lower nephron nephrosis" (Figure 41) together w i t h frequent proximal tubule vacuolization.  From t h i s observation i t becomes apparent that  acute tubular necrosis w i t h death i n uremia can indeed be produced w i t h i n 17 to 18 hours f o l l o w i n g removal of the crushing ligature.  Figure 41 F i v e other animals had  B.U.N's  elevated to from 210  to  420 mg. % and died at from 32 to 48 hours f o l l o w i n g l i g a t i o n . A l l apparently died i n acute r e n a l f a i l u r e w i t h a varying number of hours anuria preceding death.  Kidney h i s t o l o g y of the  two  - 145 -  t e s t animals was probably r e l i a b l e , though the animals could have been dead f o r one hour and t e n minutes when t h e i r kidneys were f i x e d ;  i t showed t y p i c a l acute t u b u l a r necrosis i n both  cases. Kidney weights f o l l o w i n g f i x a t i o n averaged 209.3 nig. per cm.  (range 198.0 to 2 3 0 . 7 ) f o r treated animals and 219.9  2  mg. per cm.  2  (range 186.4 to 256.9) f o r untreated.  I t can be concluded that DCA, given in^adequate dose, to male r a t s s u f f e r i n g from acute t u b u l a r n e c r o s i s , does not decrease t h e i r m o r t a l i t y rate nor prolong l i f e .  I t can also  be stated that death i n uremia from acute tubular n e c r o s i s r e s u l t i n g from dehydration and crush i n j u r y i n uninephrectomied animals can be produced 18 hours f o l l o w i n g release of l i g a t i o n .  DISCUSSION AND CONCLUSIONS  From these experiments s e v e r a l conclusions can be drawn which give r i s e t o some d i s c u s s i o n ;  but before present-  ing these points i t i s e s s e n t i a l t o r e c a l l the o r i g i n a l aim of the work.  I t was planned to produce a standardized "lower  nephron syndrome" i n r a t s by v a r y i n g three s t r e s s e s , dehydration, myoglobin i n j e c t i o n , and crush i n j u r y .  This  accomplishment  was to be followed by therapeutic use o f t e s t o s t e r o n e , c o r t i s o n e , desoxycorticosterane and Compound F i n a l l e v i a t i o n of the acute  - 146  renal f a i l u r e .  -  That t h i s i n i t i a l aim was accomplished i s  apparent i n Experiments 5» 7,  8, 9 and  10.  Several statements can be made about the f a c t o r s responsible f o r the production of acute tubular n e c r o s i s . Dehydration has been shown to be an e s s e n t i a l f a c t o r i n the production of traumatic uremia i n the r a t .  Even severe dehy-  d r a t i o n (Experiment I B ) , when alone, succeeded i n producing only s l i g h t uremia and o l i g u r i a w i t h almost immediate recovery on r e - h y d r a t i o n , without h i s t o l o g i c a l evidence of tubule damage. These r e s u l t s are probably adequately explained by simple hemoconcentration;  though prolonged dehydration could conceivably  produce shock, such a c o n d i t i o n was never observed i n animals (even uninephrectomied ones) dehydrated as long as 72 hours and therefore could p l a y no part i n the urea n i t r o g e n increase and oliguria.  The dehydration as employed here anteceded by 24  hours other stresses u t i l i z e d , and had s i m i l a r e f f e c t s i n i n t a c t animals and i n r i g h t nephrectomied animals.  This f i n d i n g that  the s t a t e o f . h y d r a t i o n i s an important f a c t o r i n the production of the syndrome i s i n agreement w i t h the works of L a l i c h 203)» Maluf (232)  (202,  and many o t h e r s .  I t i s a l s o obvious (Experiment 5) that there i s a second e s s e n t i a l f a c t o r which i n these experiments took the form of a crush i n j u r y .  Although release of a nephrotoxic agent  from the damaged t i s s u e (125,  162,  3 0 , 89) cannot be excluded as  the pathogenetic mechanism, shock w i t h r e n a l ischemia was  - 147 probably the chief cause of renal damage.  That shock actually  was present could only be assumed from the appearance of the animals immediately following removal of the crushing mechanism. These animals assumed a crouching position with eyes closed and fur ruffled, an attitude which was occasionally punctuated with attacks of rigors. anoxia — agent.  There was a second observation i n favour of  i . e . ischemia as a result of shock — as the damaging It was consistently noted that various degrees of post-  mortem change could i n no way be distinguished from the tubular damage seen i n kidneys of animals freshly dead as a result of ligation and dehydration.  Since postmortem autolysis must essen-  t i a l l y be primarily an anoxic change, then i t i s probable that the degenerative changes seen i n test animals i s also anoxic (see Figures 5 1 - 6 2 ) . The method of leg compression was used f i r s t by Bywaters and Popjak (74) i n order to simulate as closely as possible the c l i n i c a l crush injury.  They early noted that shock  occurred following the occlusive period, and used the method later in experiments with myoglobin (75).  Duncan and Blalock (116) also  used clamping of a limb to produce experimental shock i n dogs and noted the similarity to crush syndrome. Eggleton et a l (125,126), using eats and dogs, recorded low blood pressures following elastic rubber tube binding of limbs but was of the opinion that renal damage resulted from a released nephrotoxic agent.  Corcoran and Page  (85) also used a method of limb ligation i n their studies of the relationship of myoglobin to crush syndrome, and Keele and Slome (191) noted a marked reduction i n blood pressure following release of a  - 148 -  wrapped limb i n cats.  There would appear to be l i t t l e doubt,  therefore, that this method of limb compression can indeed produce shock i n experimental animals. It i s apparent therefore that the combination of severe dehydration and crush injury i s capable of producing renal tubular damage as evidenced by elevated blood urea nitrogen levels, altered urine output and histological changes.  In  experiments designed to test the mortality rate, this damage was sufficient to be fatal to 40 to 50% of test animals.  These  were the essential factors, such added refinements as prolongation of ligation, bilateral ligation, myoglobin injection and uninephrectomy being merely attempts to produce a more predictable and standardized result.  In the case of prolongation of  ligation and of bilateral ligation, these procedures either increased the early mortality so that animals died i n the shock phase or produced no more satisfactory tubule damage than did the simpler unilateral ligation.  In reducing the known high  renal reserve of the rat i n as physiological a way as possible by surgical removal of one kidney, i t was found that acute renal failure could be produced far more readily (Experiment 9). In examining the syndrome as produced experimentally in the rat and comparing i t to that i n the human (in which i t commonly runs a 7 to 14 day course) i t i s apparent that i t runs a fore-shortened course.  The corresponding events i n the rat  appeared to occur within one to three days following trauma,  - 149 -  those animals s u r v i v i n g f o r three days being c l i n i c a l l y f u l l y recovered.  This foreshortening gave r i s e to d i f f i c u l t i e s on  two accounts.  F i r s t , i t was o f t e n d i f f i c u l t to o b t a i n s e r i a l  observations since a f f e c t e d animals o f t e n died w i t h i n 24 hours; second, i t was o f t e n d i f f i c u l t to decide whether an animal died of shock i t s e l f or of r e n a l f a i l u r e , when i t succumbed w i t h i n 15 - 18 hours of the i n i t i a l trauma.  I t was f e l t , however, that  a f a i r l y d e f i n i t e sequence of events occurred, as observed c l i n i c a l l y i n the f i r s t 24 hours.  During the f i v e hour l i g a t i o n  p e r i o d , though animals were sedated they nevertheless behaved v i g o r o u s l y and i n a wide-awake f a s h i o n when that sedation  subsided.  But f o l l o w i n g removal of the l i g a t u r e they f e l l immediately i n t o a period which we c a l l e d "shock".  They crouched f a r back i n  t h e i r cages, eyes closed and f u r r u f f l e d , often developing marked tremor.  They remained i n t h i s state f o r from two to four hours  at which time t h e i r c o n d i t i o n could be described as "improved". That i s , there appeared to be a d e f i n i t e recovery from the i n i t i a l trauma which occurred at the time of l i g a t u r e removal.  One  encouraging and conclusive observation was made i n Experiment 18. This observation proved that i t was p o s s i b l e f o r a r a t (animal M.226)  to d i e i n acute r e n a l f a i l u r e w i t h acute t u b u l a r necrosis  17^ hours f o l l o w i n g removal of a f i v e hour u n i l a t e r a l l i g a t i o n . I t can be stated c a t e g o r i c a l l y that the pigment myog l o b i n i s not e s s e n t i a l to the production of acute tubular n e c r o s i s from crush I n j u r y i n the r a t (Experiments 5 , 6 and 7 ) .  This  -  150  -  statement i s i n contrast t o the o r i g i n a l work o f Bywaters and Stead ( 7 5 ) who, though unable to produce r e n a l f a i l u r e by i n j e c t i n g myoglobin alone o r by l e g compression alone, produced the syndrome by myoglobin i n j e c t i o n f o l l o w i n g l e g compression or f o l l o w i n g a c i d i f i c a t i o n o f the urine to pH 4 . 5 to 6 . 1 w i t h ammonium c h l o r i d e .  I n our experiments, i t can be noted that  though urine was c o n s i s t e n t l y o f pH 5 * 0 to 6 . 5 and animals were dehydrated, myoglobin i n j e c t i o n d i d not produce detectable r e n a l damage (Experiment 3 ) «  I t i s i n t e r e s t i n g to note that,,  although Bing (31j 3 2 ) found that 80 t o 1 2 0 gm. of ammonium c h l o r i d e given t o dogs to a c i d i f y urine d i d i t s e l f produce no r e n a l damage, Govan and Parkes ( 1 6 0 , l 6 l ) found that both ammonium and calcium c h l o r i d e produced r e n a l l e s i o n s and death i n rabbits.  I t would therefore appear that Bywaters' work ( 7 5 )  should be considered only w i t h r e s e r v a t i o n s .  Corcoran and Page  ( 8 5 , 8 6 ) have a l s o reported that crush syndrome i s reproducible by intravenous i n j e c t i o n of metamyoglobin a f t e r release o f compression from one crushed hind limb of r a t s .  They reported as  w e l l " p a r t i a l l y recoverable r e n a l i n j u r y " i n dogs subjected to myoglobin and metamyoglobin i n j e c t i o n i n a c i d u r i c dogs. ( 3 2 ) work, however, contrasts with these observations;  Bing's he f a i l e d  to produce any s i g n i f i c a n t impairment o f r e n a l f u n c t i o n by i n j e c t i o n of myohaemoglobin i n t o normal o r a e i d o t i c dogs. Whether or not myoglobin adds to the damage induced by crush and dehydration should be apparent i n Experiment 7»  -  151  -  In t h i s experiment a s t a t i s t i c a l l y s i g n i f i c a n t increased e l e v a t i o n of urea n i t r o g e n l e v e l s f o r myoglobin i n j e c t e d a n i mals over non-injected ones was found.  I t can be stated there-  fore that intravenously i n j e c t e d myoglobin adds to r e n a l damage induced by dehydration plus crush i n j u r y , although by i t s e l f or coupled with e i t h e r one of these f a c t o r s , the pigment i s nontoxic.  There was no h i s t o l o g i c a l evidence that the  aggravation  of r e n a l dysfunction was due to o b s t r u c t i v e c a s t s . The p o s s i b i l i t y that these observations of the e f f e c t of myoglobin are not v a l i d should be considered.  Corcoran and  Page ( 8 5 ) , i n i n j e c t i n g myoglobin remarked that the urine colored one to two hours a f t e r i n j e c t i o n . never seen i n our experiments.  was  This change was  A l s o , since the myoglobin was  i n part i n j e c t e d i n t r a v e n o u s l y as a suspension,  there remained  the p o s s i b i l i t y that t h i s p a r t i c u l a t e matter might have been f i l t e r e d by the lung c a p i l l a r i e s .  On the other hand, a good  p r o p o r t i o n ( 6 0 to 7 0 $ ) of the myoglobin was c e r t a i n l y d i s s o l v e d so that the e f f e c t i v e dose would at l e a s t be at the upper end of the range c a l c u l a t e d by Bywaters ( 7 5 ) and used a l s o by Corcoran and Page ( 8 5 ) .  And i n myoglobin-injected  p o s t - i n j e c t i o n p o l y u r i a was f r e q u e n t l y observed;  animals a since the  only v a r i a b l e was the presence of the hypertonic s o l u t i o n of myoglobin, t h i s f a c t i s best explained as an osmotic d i u r e s i s . That i s , the e a s i l y f i l t e r e d myoglobin molecules held water i n the t u b u l a r f l u i d to r e s u l t i n an increased urine flow. i s reasonable  It  to conclude therefore that myoglobin i n adequate  - 152  -  dosage passed through the kidneys. Because the c l i n i c a l picture of acute tubular necrosis includes a very apparent oliguria to anuria, this decreased urine output was thought to be a good standard of measurement i n the experimental production of the syndrome.  It soon became  obvious, however, that not only was i t d i f f i c u l t to measure urinary output accurately enough to distinguish dehydration oliguria from that of renal failure, but also the period of oliguria to anuria.in experimental acute renal failure was so abbreviated that i t s observation was barely significant.  For-  tunately, a new standard was available which was, strangely, exactly the opposite of oliguria.  Polyuria was observed to be  a striking, immediate and consistent response to the trauma of limb ligation.  This diuretic response was most marked i n  normally hydrated animals but was present also i n dehydrated; i t became apparent during the five hour ligation period and was continued for as long as 72 hours following ligation release. In dehydrated animals, though a comparative polyuria was present, the marked diuresis became very apparent when these animals were allowed free water;  they drank excessive quantities and excreted  similarly excessive quantities of urine.  Anuria was a feature  of only a few hours duration in those animals which died as a result of the trauma and kidney damage. This diuretic response to trauma has been mentioned seldom i n the literature.  Eggleton et a l (126) points out that  - 153 -  nephrotoxins inhibit water and chloride reabsorption to produce a polyuria at f i r s t , but adds that this phenomenon i s not observed in the crush syndrome in dogs.  Block et a l (41)  reported that polyuria was a striking feature i n dogs following a hypotensive period.  These workers suggest an explanation  which has long been used i n the polyuric phase of chronic glomerulo-nephritis —  decreased functioning renal tissue  requires that remaining nephrons eliminate the necessary nitrogenous wastes by increasing the volume of urine.  This explana-  tion may account for the late polyuria, but another mechanism must be responsible for the immediate diuresis observed.  It  seems plausible that only changes in renal hemodynamics and, thereby, changes in glomerular f i l t r a t i o n , can account for this immediate response to limb ligation.  Generalized renal  hyperemia or relative efferent arteriolar constriction can only be suggested, not proven, by this investigation. possibility may also be considered:  A third  hemodilution.  Hemodilu-  tion was observed frequently i n ligated animals but was usually associated with haemorrhage from bitten limbs.  It was, however,  also observed occasionally i n animals which showed no sign of external haemorrhage and i n those in which gastro-intestinal haemorrhage and hematuria were observed.  It has been observed  (81A) that i n producing severe shock i n rats by pinching the intestine in several places for short periods, marked and rapidly developing hemodilution appeared.  - 154- Perhaps the most probable explanation of the later polyuria i s one which accounts for the diuretic phase of human acute tubular necrosis, that of tubular damage to the extent that normal water reabsorption i s inhibited.  Surely anoxic  tubular damage can be such that the normal reabsorptive mechanism i s disrupted, just as mercury salts can be used either as diuretics or as poisons producing tubular necrosis and anuria. In any case, this diuretic response to trauma i n rats is a consistent observation and can be used as a definite indication of renal dysfunction which does not necessarily indicate recovery but i s only one stage i n the reaction of a damaged kidney. In those animals which showed eyidence of renal dysfunction either by polyuria, anuria or uremia, histological changes were exclusively i n the kidney tubules.  The intra-  capsular granular eosiniphilic granular debris and cubical metaplasia of capsular epithelium were not observed.  An occasional  glomerulus was observed, however, i n which the capsular space appeared to be dilated i n such a way as to incorporate the upper extremity of the proximal convoluted tubule, giving the appearance of cubical metaplasia of the capsular epithelium (Figure 42 and Figure 43, which shows a less obvious case of the same phenomenon).  This change occurred i n control and test animals  alike and i t is interesting to note that a similar though apparently true cubical metaplasia has been described as an action of DCA  (333).  V  Tubular changes could be recognized at two stages. In animals which died i n obvious acute r e n a l f a i l u r e , t u b u l a r c e l l s showed changes ranging from e a r l y degeneration to n e c r o s i s . Cytoplasm became granular and vacuolated, n u c l e i swollen, pale and vacuolated and i n severe cases, n u c l e i progressed to the s m a l l , dark pyknotic stage of degeneration.  In a l l cases the  basement membrane appeared to remain i n t a c t (See Figures 3 1 , 34,  32,  etc.). In those animals which showed signs of r e n a l dysfunc-  t i o n -- p o l y u r i a and uremia —  but went on to recovery, kidney  h i s t o l o g y was amazingly normal.  However, c o n s i s t e n t l y i n these  cases there were seen f o c a l areas of b l u i s h , granular degenerat i o n of tubules w i t h desquamation of these c e l l s to form c a s t s , and evidence of regenerating tubular e p i t h e l i u m .  In addition,  medullary tubules showed occasional casts of e p i t h e l i a l d e b r i s i n c l u d i n g pyknotic n u c l e i .  Such kidneys were taken to be  - 156  -  i l l u s t r a t i o n s of recovered, h e a l i n g and regenerating phases of the process. (See f i g u r e s 38 - 40). The l o c a l i z a t i o n of these t u b u l a r l e s i o n s w i t h i n the nephron i s i n t e r e s t i n g but not e s s e n t i a l .  I t was f i r s t empha-  sized i n the l i t e r a t u r e that the d i s t a l convolution was involved segment, hence Lucke's (213) rosis.  the  term Lower Nephron Neph-  Later reports (146) observed degenerative changes  o f t e n more advanced i n the proximal tubule and e v e n t u a l l y O l i v e r et a l (271) pointed out that the e s s e n t i a l l e s i o n of acute t u b u l a r n e c r o s i s ("tubulorhexis") could i n f a c t be located at any point i n the nephron.  Nevertheless, i t appears that  kidneys of animals subjected to haemorrhagic shock or crush i n j u r y more o f t e n develop l e s i o n s i n the lower nephron (59>  60,'  8 8 ) , while those subjected to r e n a l a r t e r y o c c l u s i o n show p r o x i mal tubule l e s i o n s (194, 195).  I n the experiments reported  h e r e i n , i n which r a t s were subjected to the s t r e s s of crush and dehydration, the s i t e of the l e s i o n was c o n s i s t e n t l y the d i s t a l tubule, c h i e f l y i n Zone 3 of the kidney but a l s o (though to a l e s s e r extent) i n the cortex.  The presence or absence of the  brush border i n proximal tubules can be used as a very f i n e index of damage to that u n i t and i n the kidneys examined t h i s s t r u c t u r e was c o n s i s t e n t l y present (Figure 44). There was one exception to t h i s statement i n an e x p e r i ment which was discarded because of a high incidence of chronic  - 157  -  Figure 44  kidney disease i n the rats used.  In three of four discarded  animals which had been subjected only to 72 hours dehydration, proximal tubules showed a high degree of so-called hydropic degeneration (Figures 45 and 46).  Figure  45  Figure 46  Chronic renal disease was encountered relatively frequently i n animals used. chronic inflammatory diseases seen.  Hydronephrosis (Figure 47) and  changes (Figures 48 and 49) were the chief  In one case, this last change appeared to  - 158  -  account f o r an e l e v a t i o n of the blood urea n i t r o g e n . c o n s i s t e n t l y the hydronephrotic  Almost  change occurred only i n the  r i g h t kidney, which was of course removed p r i o r to experimentat i o n so that i t was f e l t that t h i s d i d not i n t e r f e r e w i t h observations.  I n any case, i t i s u n l i k e l y that chronic disease  would i n t e r f e r e w i t h acute experiments such as were c a r r i e d out.  Figure 48  -  159  -  Figure 49  The s i m i l a r i t i e s between e a r l y (up to seven hours) postmortem change and acute tubular n e c r o s i s due to crush and dehydration have already been r e f e r r e d t o .  I t was noted that  the d i s t a l tubules q u i c k l y and s e l e c t i v e l y showed degenerative changes appearing very s l i g h t l y at two hours postmortem ( F i g ures 51> 52 and 5 3 ) and markedly by four hours (Figures 5 4 , 55 and 5 6 ) .  Medullary, glomerular and proximal tubule changes  occurred only at an advanced stage (Figures 57» 5 8 , 59? 6 0 , 6 1 and 6 2 ) .  The postmortem d i s t a l tubule degeneration was very  n o t i c e a b l e i n the c o r t i c a l r e g i o n as w e l l (Figure 5 0 ) . Two of the three c l a s s i c a l responses to s t r e s s were observed f r e q u e n t l y , that of g a s t r o - i n t e s t i n a l haemorrhage and enlarged, brown adrenal glands.  The g a s t r o - i n t e s t i n a l haemor-  rhage was o f t e n accompanied by hematuria and a secondary anemia  - 160 -  Figure 50  which may have contributed to renal ischemia. The problem of increased mortality to 72 hours of dehydration and five hours limb ligation encountered i n later experiments was a baffling one.  Referring to Experiment 1 0 ,  i t w i l l be seen that three of eight animals (37$) subjected to right nephrectomy, dehydration for 72 hours and ligation of left hind limb for five hours died spontaneously within a 72 hour period.  In subsequent treatment experiments with female  animals this mortality was increased to about 80$ and maintained at approximately this figure in male animals also used i n  Figure 51  Figure 52  Figure 53  - 161  Figure 57  Figure 60  -  Figure 58  Figure 59  Figure 61  Figure 62  r-  hormonal experiments.  162 -  I n Experiment 14, not only d i d 10G %  of c o n t r o l animals d i e , hut they d i d so w i t h i n 24 hours of the crush i n j u r y so that the problem of shock deaths i s amplified.  This problem has been discussed e a r l i e r . Two p o s s i b i l i t i e s can be considered as accounting  f o r t h i s phenomenon.  The t e n s i o n of the s t r i n g l i g a t u r e  could not be a b s o l u t e l y standardized and w i t h l a t e r experiment t h i s was undoubtedly t i g h t e r .  However, the v a r i a t i o n must  have indeed been s l i g h t and i n any case i t seems u n l i k e l y that simply tightness of o c c l u s i o n could a f f e c t the degree of syste i c shock since the mass of t i s s u e damaged and the d u r a t i o n of l i g a t i o n were the same i n a l l cases. t o r involved i s the time of year —  The second apparent fac  i . e . , during the course of  these experiments winter had become spring and summer and the environment was n o t i c e a b l y warmer and more humid.  This i s a  vague but not an unusual e f f e c t i v e f a c t o r i n a l t e r i n g e x p e r i mental observations and here may have reduced the r e s i s t a n c e of the animals by a l t e r i n g t h e i r water balance.  Interesting  i s the f a c t that the increase i n m o r t a l i t y was a gradual one. Hamilton, P h i l l i p s and H i l l e r (166) have r e f e r r e d to an increased m o r t a l i t y r a t e i n dogs subjected to r e n a l a r t e r y l i g a t i o n f o r varying periods when environmental temperature and humidity were increased. I t should be unnecessary to point out that as many  - 163  -  f a c t o r s as p o s s i b l e were kept constant throughout these e x p e r i ments.  Differences i n s t r a i n of r a t , body weight, sex,  d u r a t i o n of l i g a t i o n , mass of t i s s u e involved or sedatives used could not account f o r the change i n m o r t a l i t y r a t e . Results of treatment experiments were not encouraging. I t can only be concluded from the observations made that testosterone propionate, desoxyeorticosterone  acetate, cortisone  acetate and Compound F are of no value as therapeutic agents i n acute t u b u l a r n e c r o s i s .  I n the case of Compound F. some promise  was shown i n Experiment 17A so that some s l i g h t r e s e r v a t i o n s about t h i s agent are held and f u r t h e r experimentation Testosterone  is justified.  was used i n t h i s work i n the hope that i t s  " r e n o t r o p i c " a c t i o n might l e s s e n the damage induced i n the or hasten i t s recovery;  kidney  i t s e f f e c t on p r o t e i n metabolism ( a  s o r t of p r o t e i n - s p a r i n g a c t i o n i n the usual s t r e s s breakdown Of body p r o t e i n ) might a l s o reduce the i n t r i n s i c production of nitrogenous wastes.  Homer Smith (333)  states that testosterone  increases the hypertrophy of the remaining kidney a f t e r u n i l a t e r a l nephrectomy and t h i s growth i s l o c a l i z e d i n the t u b u l e s .  The  hormone a l s o appears to a f f o r d some p r o t e c t i o n against mercury b i c h l o r i d e poisoning (333). i n the dog that 1G0 mgs. r i s e i n Tm , D  Although i t has been shown  (333)  testosterone per day produces a r a p i d  corresponding  doses i n man  (90 to 300 mg. per  day)  produce no increase i n glomerular f i l t r a t i o n r a t e , r e n a l plasma  - 164  flow, T n i p ^ or Tm^  -  Smith also points out that the hormone  has been used i n the treatment of chronic n e p h r i t i s and i n cholera, i n which " b e t t e r s u r v i v a l " w i t h r e l i e f of o l i g u r i a and uremia and decrease i n albuminuria were reported.  In  our experiments, the r e n a l hypertrophy was apparent, e s p e c i a l l y i n Experiment 11A, but no reduction i n uremia or m o r t a l i t y rate was  observed. Disturbed  e l e c t r o l y t e balance, which accompanies  acute r e n a l f a i l u r e , has f r e q u e n t l y been named as the cause of death i n these cases.  In p a r t i c u l a r , an elevated blood  potassium l e v e l i s said to r e s u l t i n death by cardiac a r r e s t (181).  For t h i s reason, DCA  peutic agent.  would seem to be a u s e f u l thera-  As a m i n e r a l o c o r t i c o i d , i t i s known to promote  sodium and water r e t e n t i o n and potassium e x c r e t i o n so that plasma sodium increases while plasma potassium decreases ( 3 2 5 ) . This a c t i o n of DCA  i s thought to be a d i r e c t a c t i o n on the r e n a l  ( d i s t a l ? ) tubules to promote sodium reabsorption  (333)  but a l s o  on the c a p i l l a r y permeability and t i s s u e a f f i n i t y f o r water and electrolytes (325).  In dogs, DCA  has been shown (333)  to  expand the e x t r a c e l l u l a r f l u i d space at the expense of the i n t r a c e l l u l a r , to increase the glomerular f i l t r a t i o n rate and  renal  plasma flow and to increase T m  p A H i  Plasma potassium concentra-  t i o n i s i n i t i a l l y decreased.  However, whatever the mechanism  of death i n the r a t s i n our experiments, DCA d i d not prolong t h e i r l i v e s or lessen t h e i r uremia.  Hoff et a l (181) found  - 165  -  that although i n s u r g i c a l anuria ( b i l a t e r a l u r e t e r a l l i g a t i o n or b i l a t e r a l nephrectomy) the e l e v a t i o n of serum potassium i s such that cardiac damage i s the cause of death, i n mercuric c h l o r i d e anuria and chronic n e p h r i t i s , potassium l e v e l s do not r i s e to a f a t a l l e v e l and e l e c t r o c a r d i o g r a p h i c changes of potassium i n t o x i c a t i o n are not seen at death.  This work would  appear to minimize the r o l e of potassium r e t e n t i o n i n deaths i n acute r e n a l f a i l u r e and might a l s o e x p l a i n the f a i l u r e of  DCA  to prolong l i f e i n r a t s s u f f e r i n g from the syndrome. In using cortisone (17  hydroxy - 11 dehydrocortieo- »  sterone, Compound E) as a therapeutic agent i n the anuria syndrome (23)  traumatic  i t was hoped that the hormone would l e s s e n  the m o r t a l i t y by counteracting the shock of the e a r l y phase of the alarm r e a c t i o n (325, Selye (326)  326)  found that " c o r t i n " was h i g h l y e f f e c t i v e i n t h i s  regard, r e p o r t i n g that DCA a l (358)  seen i n response to limb l i g a t i o n .  alone had l i t t l e e f f e c t .  Ingle (185)  reported s i m i l a r f i n d i n g s i n r a b b i t s .  the other hand found that n e i t h e r DCA,  Weil et  cortisone nor  on  adrenal  c o r t i c a l e x t r a c t reduced the m o r t a l i t y rate of r a t s subjected to b i l a t e r a l hind limb l i g a t i o n .  Ingle (186)  reviews the  b i o l o g i c properties of c o r t i s o n e , p o i n t i n g out that i t can no longer be thought of as a simple g l u c o c o r t i c o i d .  Its effect  on e l e c t r o l y t e and water balance i s v a r i a b l e but i n acute e x p e r i ments i n r a t s an increased e x c r e t i o n of sodium, c h l o r i d e and potassium l a s t i n g one to three days has been reported.  Corti-  sone has been reported to maintain adequate c i r c u l a t i o n i n  - 166  -  adrenalectoraied'dogs subjected to trauma or hemorrhage; maintain renal function i n adrenalectomy;  to  and to increase  the PAH secretion i n normal males by up to 35% (186, 324). In high doses and prolonged administration i t produces hyalinization of glomerular capillaries, hypertension and elevation of plasma chloride and potassium (186, 324, 144, 2 3 ) . It can be seen from Experiments 13 and 14 that this hormone was of no value i n the alleviation of the renal effects of shock from limb ligation.  Life was not prolonged  and mortality rate and uremia were not diminished.  The com-  bination of testosterone and cortisone (Experiments 15 and 16) also showed no therapeutic effect. Compound F (17 hydroxyeorticosterene - 21 - acetate) was also used as a therapeutic agent, since i t has been shown, chiefly c l i n i c a l l y , to be of value where cortisone i s ineffective.  Compound F i s i n fact very similar to cortisone i n  chemical composition (325) and i n action (287).  It i s said to  be less active i n salt and water metabolism, having l i t t l e influence on these;  i t induces a slight negative nitrogen  balance and like cortisone i t also has a hypertensive effect i n rats and increases kidney mass (145)  Though i n Experiment 17A,  Compound F appeared to be of some considerable value, this phenomenon was not observed i n Experiment 17B.  Nevertheless  i t i s apparent that further experimentation with Compound F would be advisable.  -  167  -  In considering the failure of cortisone or Compound F to be of benefit to rats, i n acute renal failure i t i s perhaps worthy of note that Selye (326) points out that adrenal cortical hormones i n shock are more effective given i n divided doses, . and that pre-treatment i s useless and may be harmful.  Pre-  treatment may well depress the normal adrenal cortical activity. It i s very unlikely that the dosage used i n these experiments was sufficiently high or prolonged to produce any of the nephrotoxic actions referred to by Selye (324).  - 168 -  SUMMARY  A b r i e f review of the l i t e r a t u r e on traumatic  anuria  (acute tubular n e c r o s i s , lower nephron nephrosis) has been presented, i n c l u d i n g a complete b i b l i o g r a p h y .  Special attention  was paid to the pathology and pathogenesis of the syndrome, and i t was concluded that O l i v e r ' s recent work (271) probably comes c l o s e s t to presenting the true p i c t u r e .  He described  tubular  n e c r o t i c l e s i o n s f o r which the chemical toxins (mercuric  chlor-  i d e , carbon t e t r a c h l o r i d e ) were r e s p o n s i b l e , and t u b u l o r h e c t i c l e s i o n s which were c h a r a c t e r i s t i c of the shock kidney.  These  l e s i o n s could appear at any l e v e l i n the r e n a l tubule and were characterized by d e s t r u c t i o n of the basement membrane.  Pigment  casts were apparent i f i n t r a v a s c u l a r pigment release was associated w i t h the i l l n e s s . associates  The work of P h i l l i p s , Van Slyke and  (291, 292, 355, 356), of O l i v e r (271) and of Block et  a l (41) lead one to conclude that r e n a l ischemia i s the c h i e f pathogenetic mechanism, though i t i s obvious that s p e c i f i c ext r i n s i c r e n a l toxins play a major r o l e i n s p e c i f i c cases.  The  r o l e o f hemoglobin appears to be c h i e f l y i n the production of o b s t r u c t i v e casts l a t e r i n the course of the disease;  these  pigments are p r e c i p i t a t e d i n the lower nephron where urine i s concentrated and a c i d i f i e d , and dehydration and o l i g u r i a c o n t r i bute to t h e i r  formation.  - 169 -  Three hundred r a t s were studied i n eighteen e x p e r i ments concerning crush syndrome.  I t was concluded that the  most important s i n g l e f a c t o r tending to aggravate the r e n a l e f f e c t s of crushing i n j u r y i s the antecedent state of dehydration.  Myoglobin i s not an e s s e n t i a l f a c t o r i n the development  of r e n a l damage but tends to aggravate the e x i s t i n g uremia. Acute r e n a l f a i l u r e was seen to be a l a t e e f f e c t o f shock; animals developed acute tubular n e c r o s i s only i f i n i t i a l shock was severe, but not severe enough to produce death from c i r c u latory failure.  Development o f t h i s d e l i c a t e balanee o f  f a c t o r s was aided by reduction o f r e n a l reserve by u n i l a t e r a l nephrectomy.  A seldom described but d i s t i n c t and consistent  phenomenon was observed i n the development of marked, immediate and p e r s i s t e n t d i u r e s i s i n response to the trauma o f limb ligation.  This p o l y u r i a was of a d i l u t e urine and was taken as  an i n d i c a t i o n of i n i t i a l increased glomerular f i l t r a t i o n followed by decreased r e a b s o r p t i o n o f water because of tubular damage. I t was not an i n d i c a t i o n of a recovery phase as i s recorded i n the  c l i n i c a l syndrome. Testosterone propionate, desoxycorticosterone acetate,  cortisone acetate and Compound F d i d not appear to be promising as therapeutic agents, although i n one experiment Compound F showed some promise.  N e i t h e r d i d combined therapy w i t h t e s t o s -  terone and cortisone reduce the m o r t a l i t y rate or decrease uremia.  - 170 -  Although there was no doubt that the syndrome o f acute renal failure due to acute tubular necrosis could be produced i n large numbers of these relatively inexpensive laboratory animals by dehydration and limb ligation, production could not altogether be standardized and the syndrome ran such a short course that serial observations were d i f f i cult to obtain and separation of shock deaths was occasionally impossible.  It i s f e l t that future work might well make use  of some other laboratory animal, perhaps the dog or cat, and that an i n i t i a l stress of controlled hypotension or renal artery occlusion could be used.  It i s also our opinion that  further investigation into the value of Compound F as a therapeutic agent i n this syndrome i s j u s t i f i e d .  - 171  -  BIBLIOGRAPHY  : 27,  1  Abeshouse, B. S., J. 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