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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 in Partial Fulfilment of the Requirements for the Degree of MASTER OF SCIENCE in 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 pre sented, including a complete bibliography. Special attention was paid to the pathology and pathogenesis of the syndrome and it 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 in the renal tubule and were character ized 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, 356), of Oliver (271) and of Block et al (41); lead one to conclude that renal ischemia is the chief pathogen etic mechanism, though i t is obvious that specific extrinsic renal toxins play a major role in specific cases. The role of hemo globin appears to be chiefly in the production of obstructive casts later in the course of the disease; these pigments are precipitated in the lower nephron where urine is concentrated; and acidified, and dehydration and oliguria contribute to their formation. .. ' • ' Three hundred rats were studied in 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 is not an essential factor in the development of renal damage but tends to aggravate the existing uremia. Acute renal failure was seen to be a late effect of shock; animals developed acute tubular necrosis only If 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 in the devel opment of marked, immediate and persistent diuresis in 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 glomer ular filtration followed by decreased reabsorption of water because of tubular damage. It was not an indication of a recov ery phase as is recorded in the clinical syndrome. Testosterone propionate, desoxycorticosterone acetate, cortisone acetate and Compound F did not appear to be promising as therapeutic agents, although in 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 in 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 difficult to obtain and separation of shock deaths was occasionally impossible. It is felt 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 is also our opinion that further investigation into the value of Compound F as a therapeutic agent in this syndrome is 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 Page AIM i 65 METHODS AND MATERIAL 66 REPORT OF EXPERIMENTS 75 DISCUSSION AND CONCLUSION 145 SUMMARY 168 BIBLIOGRAPHY 171 TABLES AND EXPERIMENTS LIST OF TABLES * I l l REPORT OF EXPERIMENTS V -III- LIST OF TABLES TABLE Page IA Dehydration in Intact Rats 77 IB Dehydration in Right Nephrectomied Rats ....... 78 2A Myoglobin in Intact Rats 82 2B Myoglobin in Intact Rats 83 2C Myoglobin in Intact Rats 84 3 Myoglobin and Dehydration in Intact Rats 85 4 Left Hind Limb Ligation in 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 in Intact Rats 100 7 Dehydration, Ligation and Myoglobin in Intact Rats 101 7A Statistical Analysis of Figures in Table 7 . . . . 107 8 Dehydration and Bilateral Ligation in Intact Rats 108 9A Ligation and Dehydration in Right Nephrectomied Rats 112 9B Ligation and Dehydration in Right Nephrectomied Rats 113 10 Mortality in Dehydrated, Ligated Unlnephrec- tomied Rats 120 - IV - LIST OF TABLES continued TABLE Page 11A Testosterone in Crush Syndrome 121 11B Testosterone in Crushed Female Rats 125 12 Testosterone in Crushed Male Rats 126 13 Cortisone in Crushed Female Rats . 130 14 Cortisone in Crushed Male Rats 131 15 Testosterone Plus Cortisone in Crushed Female Rats 133 16 Testosterone and Cortisone in Crushed Male Rats .. 134 171 Compound F in Crushed Male Rats 138 17B Compound F in Crushed Male Rats 139 18 Desoxycorticosterone in Crushed Male Rats 143 - V - REPORT OF EXPERIMENTS OBSERVATIONS Page Experiment 1 75 Experiment 2 79 Experiment 3 86 Experiment 4 87 Experiment 5 • 91 Experiment 6 99 Experiment 7 • 102 Experiment 8 105 Experiment 9 • 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 in 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" clinical syndrome was described. Because the injuries sustained were con sistently the result of prolonged exposure to the pressure of destroyed brick and concrete structures, this syndrome was first named the Crush Syndrome (69) and was typified by apparent early recovery from the crushing injury followed by a state of progress ive, acute renal failure, with oliguria, anuria and uremia, frequently ending in death. In the ten years since that time much work has been carried on to investigate the possible patho geneses of the condition, with some progress being made. In this thesis, an attempt will be made to synthesize the great multitude of papers published on the subject, to present principally the experimental aspect of its pathogenesis and to formulate a workable pathogenetic basis for treatment of the syndrome in 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 in rats a syndrome resembling that seen in human crush injuries are reported, as well as the results of using cer tain agents to lessen the effect of the presumably temporary cessation of renal function. Since Bywaters (69) first described the crush syndrome, a similar clinical picture has been noted in a great many other conditions and has been described under various titles. "Traumatic Anuria" is perhaps a more general term, indicating that the anuria and its outcome is a result of various forms of trauma. The pathological picture has been taken into consideration together with a slightly different etiological agent in the description "Hemoglobinuria Nephrosis" (229) and in 1946, Lucke (213) summar ized the various conditions known to give rise to this syndrome and described the lesion in 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" is a better name, and more recently, other investigators (55) have tried to remain general in their description of the patho logical picture, at the same time avoiding the use of the undesir able term "nephrosis", by referring to i t as "Acute Tubular Necrosis". A l l these descriptions, varying in specificity and point of view, are descriptions of forms of acute renal failure which are closely allied and must be discussed in any consideration - 3 - of traumatic anuria itself. If one must be restricted by the narrowness of definition then one could describe the clinical syndrome and its 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 is obvious that this statement best defines "traumatic anuria", but to include a l l conditions likely to end in this picture one needs merely to add the various other etiologies such as intravascular hemolysis, extrinsic chemi cal toxins and so on. HISTORY As is the case with most "new" clinical entities, the syndrome of traumatic anuria and its 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 clinical t r i a l of blood transfusion continued through the subsequent years, notably in 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; in later editions, trauma and extensive surgery were added as causes of the subsequent renal damage. Adami (3) in 1909 added salicylic 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 litera ture, until its 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 first forty years of the century, for cases which we would now classify as lower nephron nephrosis or traumatic anuria were noted and carefully described. Bell (27) considered under the description of clinical 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 in the term "extra-renal (pre-renal) uremia" (133 )• This azotemia stands in contrast to that of primary renal disease in which morphological kidney damage is 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 listing prolonged vomiting, diarrhea (as in cholera), hepato-renal syndrome, diabetic acid osis, 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 in the kidney, Kimmelstiel (192) described cases of "acute hematogenous inter s t i t i a l nephritis" dying in 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 in infections and septicemia, conditions associated with hemolysis and the hepato-renal syndrome. In any case, Bywaters and Beall (69) in 194-1 noted that casualties brought in 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 first four such cases in the British Medical Journal of March 21, 1941. Bywaters soon discovered that the same entity had been described adequately in the German literature about the time of World War I, notably by Dr. Siego Minami (243) in 1923, though von Colmers (245) had also encountered i t with the German relief expedition to the Messina earthquake of 1909. Hackradt (245) in 1917 described a case of burial for nine hours with resultant renal damage in which he emphasized tubular damage but Minami's description of the tubule damage with pigment casts following "Verschuttung" (burial), resulting in 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 \* ./A d. w u « . sirs*. I Hartuna in r"»nw nutUrl 7>*Mr. K.rlmrw " » » H « m » k » •5.a ^Uu* ™ < « t * — ) •« ' " r " h r B <a*-4 O flit (!•*.'•. Kr-h.Wrrt. K - l »™l . -n wh F' V , iiMH t i l t ' XL* mm MdlMI «nH *• • k A f (,lrr rVttn-lk-n) • • • I i •U*krw 4 ™ /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 int t i i i* i « -h< i i Bi)<l Sitrm Htflk-n mwm ma Urn dar: - i jK | * *} * * " \VitodrniOB Hi. l iWi ? >•# KW% 1 mo * * V "Tt. * « « r r hlut^fullt *• l - r unH li.-bUhL NVI liaAV'O / 'VB „ S ^imnilimt:. Hi. U t M M 1 ( M M N • * .IMIIH mil »inH vi* 1 • i' ilk »!)•! rahal .hi - u h frinkm -hr ' • M ^ m / W ^ -ninmluni:. IH • i*«^™™t3r .-ni.-iii hm • • • / w l t i l i tnllw. uilM'liarf h> R I' I/I, 1 ' • ' " ' ^ - - « - " ' - ! ' " l - ' * l . T .Nl.ih Vj-nrhO.ll 1111* „ 1 ) l U , t , „ | i r „ , l J . . , v „ A W u - — • - -• T-r. - k**- komim- Maw i-rw , . K|4ih.u*t, .kn <;i--i>b rulu-*|Mlktr •wnil.ii ntiritn-n Ofa Figure 1 Bywaters and he compared them with paralytic myohemoglobinuria of horses. Although his description of pigment casts in the tubules of the renal pyramids and Bywaters1 rediscovery of the syndrome ani identification of the pigment as myoglobin (myohemo- globin, 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 this period, in the f i e l d of intravascular hemolysis. Blackwater fever and incompatible transfusions in particular were - 7 - involved and an end result of renal failure with uremia and pigment cast formation in 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 clinical entity. In 1941, then, investigation into the possibility of a common patho genesis 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) in 1925, Bywaters and Beall (69) at first carried on the idea of obstruction of renal tubules by pigment casts into their theory of the patho genesis, 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) in his work on black- water fever and he postulated some sort of short circuit of blood through the renal parenchyme (223). When Trueta and associates (353) in 1947 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 in 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 in 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 in trauma whieh causes a prolonged lowering of blood pressure, which in terms of Maegraith's concept (226) of renal anoxia, would damage the kidney in such a way as to produce the acute renal failure seen clinically. It can be seen, then, that the syndrome described by Bywaters (69) in 1941 was not new, but had been encountered in similar circumstances earlier in the same century and described adequately by Minami (243) in 1923• In addition, the same end result had been recognized and investigated in conditions of release of hemoglobin into the bloodstream, notably in incompati ble 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 years, s p e c i f i c descriptions such as crush kidney, hemoglobinuria nephrosis and lower nephron nephrosis were suggested. As might be expected, with the r e a l i z a t i o n that the kidney damage was the common end of multiple e t i o l o g i c a l factors, the pendulum has returned, so that at the present time the term suggested by B u l l , Joekes and Lowe (55)» acute tubular necrosis, seems more s a t i s f a c t o r y . Three main theories of patho genesis, to be discussed l a t e r , remain but these are perhaps being viewed i n t h e i r proper perspective as each contributing i n varying degrees to the end r e s u l t of acute renal f a i l u r e . AETIOLOGY In the years since the crush syndrome was rediscovered the concept has broadened to include many more et i o l o g i e s produc ing the same end r e s u l t . As mentioned previously, the s i m i l a r i t y between t h i s syndrome and the renal deaths encountered i n Black- water fever and incompatible transfusion was soon r e a l i z e d . These e t i o l o g i e s are so numerous and appear so diverse that i t seems advisable to name the syndrome on the basis of a common pathological picture. For t h i s reason, the term acute tubular necrosis seems s a t i s f a c t o r y . In Table A, an attempt has been made to group the causes of acute tubular necrosis under eight headings. Indicative of the confusion as to the pathogenesis o f the condition i s the rather large column under "Miscellaneous", and i t w i l l be noted that several e t i o l o g i e s appear under more than one heading, a fact which indicates that more - 10 - TABLE A INTRAVASCULAR HEMOLYSIS 1 Transurethral prostatectomy ... 2 1 9 , 2 0 7 , 9 4 , 3 6 7 . 2 Blackwater fever ... 3 7 1 , 1 3 7 , 2 2 3 . 3 Incompatible transfusion ... 1 2 , 2 2 9 , 9 6 , 4 5 , 3 6 5 , 1 6 , 1 0 6 , 1 0 9 , 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 ... 307 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 ... 333* 10 Paralytic myohemoglobinuria ... 7 2 , 1 9 9 . 11 Toxins: Favism ... 3 3 3 , 3 1 0 , 137- 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 ... 87,88,175,229,100,355,280,249,250,251, 2 5 2 ; 1 0 2 2 2 8 ' 3 , 3 1 7 , l 6 4 : 2 6 , 5 9 , 6 0 , 6 1 , 9 3 , 2 0 8 , 3 0 5 ; 1 1 6 , 1 6 2 , 1 6 3 , 3 4 7 , 7 8 , 3 2 9 , 2 9 7 , 3 4 8 , 2 9 1 , 1 1 1 , 3 1 9 . 3 Burn shock ... See "Burns". 4 Crush injury ... 63,64,65,66,67,69,70,71,72,74,75,25,173, 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 . 5 Welch infection 6 Septic abortion ELECTROLYTE IMBALANCE 1 Pyloric obstruction ... 54, 82, 133,187,28,333,214,221. 2 Alkalosis ... 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. .. 362 ... 178 ...51 - 12. - TABLE A continued 4- Potassium chlorate ... 2 7 1 5 Sodium tetrathionate ... 332 7 Potassium dichromate ... 174 MASSIVE DESTRUCTION OF TISSUE 1 Burns ... See "Intravascular hemolysis" and "Shock". 2 Prolonged labor ... 3 7 3 , 372 3 Toxemia of pregnancy ... 8 3 , 360 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 ... 198 2 Electroshock ... 152 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 ... 2, 104, 2 9 0 , 142, 1 3 2 . 9 Allergy ... 2 1 1 , 142, 1 3 2 . 10 Myelomatosis ... 254 11 Lymphosarcoma ... 289 12 Volkmann's ischemic contracture ... 1 6 5 . - 13 - than one pathogenetic factor is involved. Also, in attempting to find a common factor in these many causes, i t is 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 is impossible to review in detail the many interesting intricacies of individual entities leading to the end picture of acute tubular necroses. However, i t was felt that the many references to these etiologies encountered in the literature might well be included in the Table for future reference. INCIDENCE A brief review of the incidence of the syndrome as present ed in the literature is advisable in order to place i t in its true perspective as a clinical entity. Bywaters (64) stated the incidence of ischemic muscle necrosis (crush syndrome) as one to five per cent in air-raid casualties. Presumably these were cases requiring hospital care. Douglas (114), in a very complete work, considered a random group of casualties, 764 in 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$) developed crush syndrome. That i s , six (0.79%) of the total of 764 casualties were cases of crush syndrome and one of the six (16.6%) died. The works of Lauson et al (208), of - 14 - Cournand et al (93) and of Burnett et al (59, 60) present very- complete renal function studies in cases of trauma with or without shock, but these records are mainly in the acute phase of trauma. Darmady (99) also found that blood urea nitrogens done in 79 battle casualties were elevated in 35$ 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. Snyder et al (335) considered 1411 battle casualty deaths and found 68 deaths from lower nephron nephrosis, with 31 other deaths in 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 in random cases in which impair ment of renal function was not clinically apparent. Gaberman et al (146) searched widely for cases of the "renal anoxia syndrome" and found few 22 cases in two years of admissions to two large Chicago hospitals. Although these incidence figures are not high and the frequency of the syndrome in hospital practice will not be great, i t is apparent that in times of violence cases of traumatic shock will increase this incidence to perhaps 1% of cases treated. An understanding of its pathogenesis and an adequate regime of treat ment therefore become of some importance. - 15 - PATHOLOGY With the great number of etiologies recorded, i t is apparent that many minor variations of essential kidney pathology would be expected. There must be, however, a basic common factor in the pathologies in order that the syndrome be described as an entity in itself. 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" in 190? cannot essentially be improved upon today, this pathologic entity received less and less emphasis in 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 its tubular obstruction by blood and hemoglobin casts, and also as "acute interstitial nephritis" and "pure tubular degeneration" as in mercury poisoning. Again, Kimmelstiel (192) described a number of cases in which he empha sized the focal interstitial edema and infiltration by naming the renal pathology "acute hematogenous interstitial nephritis". In 1942, Bywaters (71) followed his report of the new crush syndrome with a f u l l description of its pathology. The most outstanding gross feature of the kidneys was the almost con stant 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 appear ance of intracapsular granular eosinophilic debris and occasion ally cubical metaplasia of the capsular epithelium. Again, the essential lesion was in the renal tubules; Bywaters describes a catarrh of the proximal tubule and descending loop of Henle, while in 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, in the distal convolution and collect ing tubule. Bywaters' first report (69) emphasized the severe degeneration of proximal tubules, but in his more extensive consideration (71) he localized the severe changes to the distal convolution. It will be seen later that, in Oliver's (271) opinion, the renal lesions in crush syndrome were perhaps most accurately noted by Dunn, Gillespie and Niven (117)' Bywaters, of-course, noted the similarity of this patholo gy to that described in transfusion reactions and Blackwater fever as hemoglobinuric nephrosis, as did Dunn et al (117) to the lesions described by Dunn and Poison (120) with uric acid neph r i t i s , and McFarlane (218) with phosphate nephritis. Mallory (229) carefully listed the characteristics of the hemoglobinuric kidney. Grossly, the kidney is enlarged with a pale cortex and purplish pyramids, but may be normal. Microscopically, the glomeruli are again largely normal. The first change noted is - 17 - fatty vacuolization of the ascending loop of Henle, becoming severe degeneration by three days, with herniation and rupture. Little change is seen in the proximal segments. Prominent are the casts and these are of two types: pigment casts, staining red-orange in Hematoxylin-Eosin sections, are seen in the lower nephron, while hyalin casts are encountered higher up. A controversial feature described is dilatation of the proximal tubules, which appears to be more prominent in formalin fixed material. It will be seen later in the discussion of patho genesis that the problems of frequency of casts and presence or absence of tubule dilatation are key points in the argument. In addition, a focal and diffuse inflammatory infiltration of the interstitial tissue is seen with a granulomatous reaction around herniated casts. A report which has dominated the literature on this syndrome since its publication in 194-6 is that of Lucke (213), who recognized the similarity in clinical picture and renal pathology in these various clinical entities. He described the common pathological picture and because he believed i t was essentially a lesion of the distal convoluted tubule, he named it 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 in continuity. By his microdissection technique, complete individual nephrons are dissected out and stained. Ten years of careful investiga tion (271) of human material — 54 kidneys of crush injuries, burns, transfusion reactions, Blackwater fever, obstetrical deaths, surgical shock, paroxysmal cold hemoglobinuria, sulfona mides, mercuric chloride, diethylene glycol, carbon tetrachloride, potassium chlorate and mushroom poisoning — and of animals subjected to induced shock or toxins, led Oliver and his co workers (271) to conclude that there are two essential tubular lesions in 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 in the proximal convolution and are evenly distributed in a l l nephrons of a damaged kidney. Such agents as mercuric chloride, potassium chlorate, diethylene glycol and carbon tetra chloride produce the typical nephrotoxic lesion, but i t must be remembered that the second essential tubular lesion may also be observed in these cases, presumably because the toxins also induce shock. (2) Tubulorhexis, in which there is a localized destruc tion of the entire tubular wall. The basement membrane disinte grates and there may be intralumenal material such as pigment casts in a fortuitous distribution. There may be an interstitial granulation tissue reaction associated, and regeneration, though - 19 - i t may begin, is 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 its junction with the collecting tubule. Maximum development is usually in the terminal portion of the proximal tubule which is in the outer stripe of the outer zone of the medulla. The distribution of the lesion in any one kidney is 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 is as well based on a pathogenetic concept which is 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 is true that some observers have searched for more significant changes in the renal corpuscles of these kidneys. Goormaghtigh (155» 157, 158), 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 precip itate in 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 in 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 in classifications of renal diseases. PATHOGENESIS That an understanding of the pathogenesis is the key to successful treatment of a disease entity is an obvious truth which is no less a fact in 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 in the most acceptable theories of today. In doing this i t is advantageous to group ideas into three categories: (1) simple mechanical obstruction of tubules; - 21 - (2) toxic action of agents on renal tubules; and (3) renal ischemia. It will be seen that these three theories of patho- genesis overlap a good deal and are in no way mutually exclusive. Obstruction: The earliest theory put forth, chiefly because first 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 al (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 in the thin loop of Henle. They observed dilatation of tubules, presumably as a result of the plugging, and believed that this in turn impinged on adjacent patent tubules thereby increasing the obstruction. They recognized the fact that a lowered blood volume (shock) in Blackwater fever aids production of anuria and precipitation of hemoglobin casts. A paper which appears to be the basis of many present- day opinions was published in 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 in one. Experimentally, they concluded - 22 - that the hemoglobin released intravascularly was excreted by the kidney as oxyhemoglobin, but in 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 tubu lar 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 first supported by De Gowin et al (108) in 1937* but in the following year, De Gowin (106) observed that in 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 degeneration and dilatation of tubules. De Navasquez (109) 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. He believed that hemoglobinuria did no harm with pH at 5.5 to 6.3, and that i f the glomerular flow was sufficient, urine flow would wash out any casts formed. This opinion, i t will 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 in the precipitation of hemo globin 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 is 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, is not the case. Ayer and Gould (10) concluded that necrosis of the dis tal convolution was the only progressive change seen in the renal pathology and that casts do not produce the structural and func tional 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 is a normal variation. A number of investigators (137»32, 377, 202-206) record their belief that precipitation of pigment is 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 in the kidney is lipid vacuolization in the ascending loop of Henle (229). - 24 - Maegraith and Findley (223) in a preamble to their conclusion that a redistribution of renal blood flow is responsible for anuria, l i s t four objections to the simple pigment cast obstruction theory: (1) Casts are not extensive enough in distri 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 is reversible. In contrast to these opinions, there are several argu ments for the obstruction theory. Corcoran and Page (85) conclude that renal damage in pigment nephropathy is due to three factors — obstruction by casts, ingestion of pigment by cells and a cytotoxic action of hematin on distal tubules. They refer to Oliver's work (270) of the same year, in which he points out that a renal lobule which in histological section appears only partially obstructed by pigment casts may be in fact completely occluded since the casts form at different levels, as seen by microdissection. Experimen tally, Flink (135) on the basis of studies in 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) comes out strongly in favor of the obstruction theory: "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 filtration." The answer to these strongly held opinions is probably the compromise stated so convincingly by Oliver in 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 con tributing to the anuria by obstructing the tubules which normally conduct fluid. 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 is 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 experi mental injection of hemoglobin solutions, i t is obvious that the - 26 - theory applies equally well to those conditions i n which myoglo bin i s the released pigment. Muscle pigment entered the discussion with the reporting of the crush syndrome by Bywaters and B e a l l (69), who noted the loss of t h i s pigment from pale, edematous, crushed s k e l e t a l muscle and were able (70) to i d e n t i f y i t i n the urine of these injured patients. M i l l i k a n (242) reviewed the properties of muscle hemo globin thoroughly and reported that i t was f i r s t i s o l a t e d and c r y s t a l l i z e d by Theorell i n 1932. Myoglobin, with a molecular weight of 17,500 (as compared to hemoglobin's 68,000) has a renal threshold o n e - f i f t h that of hemoglobin, i s very soluble and i s ea s i l y oxidized to the 'met' form. According to Morgan (253) i t i s extremely soluble i n phosphate buffers at pH 6.6. It occurs i n red muscle and has a c h a r a c t e r i s t i c spectrum. It has the t y p i c a l hemoglobin oxygen-carrying capacity but probably acts c h i e f l y i n storing oxygen rather than transporting i t . Its i s o - e l e c t r i c point has been reported as 6.78 (7). Newman and Whipple (265) stated that t h i s pigment was not taken up by renal tubule c e l l s , a fact with which Yuile and Clarke (375) agree. These workers found that the pigment was rapidl y cleared from the blood — 25 times more ra p i d l y than hemoglobin — and i t s thresh old value was 20 mg. per cent. When Bywaters and Beal l (69) f i r s t reported the crush syndrome they suspected that the c i r c u l a t i n g muscle pigment might be responsible for the kidney damage. They soon i d e n t i f i e d the - 27 - myoglobin spectrographically i n the urine of a i r - r a i d casualties (70), though they could not i d e n t i f y i t i n the plasma because of i t s rapid clearance. Bywaters and Dible (72) reviewed seven reported cases of acute p a r a l y t i c myohemoglobinuria i n man, and added an eighth case i n which the kidney pathology was the same as that seen i n the crush syndrome. Kreutzer, S t r a i t and Kerr (199) reported a ninth case i n which the pigment was again i d e n t i  f i e d spectrographically i n the urine. It was natural that experimental work involving the i n j e c t i o n of myoglobin solutions would follow these observations. Bywaters and Stead (75) prepared such solutions of human myo globin by Theorell's method (242) and injected amounts calculated to approximate that released i n a t y p i c a l crush i n j u r y (150-200 mg. per Kg.). Using rabbits, they found that myoglobin alone produced no kidney damage i n eight animals; myoglobin injections following leg compression produced o l i g u r i a , uremia and renal dys function with casts i n six of s i x animals; and the pigment injected into.animals with ammonium chloride a c i d i f i e d urine resulted i n four deaths i n 27 animals, with a r i s e i n urea nitrogen of 100 to 860 mg. % i n 15 rabbits. Corcoran and Page (87» 88) were also able to produce kidney damage i n rats, comparable to that seen i n human crush syn drome, by i n j e c t i n g myoglobin following limb l i g a t i o n f o r f i v e hours. Their dose was 75 to 180 mg. per Kg. and i n experiments on dogs (85) with urine a c i d i f i e d by die t and sodium acid phosphate they produced p a r t i a l l y recoverable renal 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 itself 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 al (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 conclu sions can be drawn as to the role of myoglobin in the production of renal damage. It seems probable, however, that the muscle pigment will contribute in much the same way as hemoglobin Itself does, be i t obstructive or toxic. As Oliver has stated (271), i t is difficult to say that pigment cast obstruction does not contribute to renal dysfunction when microdissected speci mens show the tubes to be plugged. This effect may well be a later phenomenon, but appears to be a definite one. Whether or not the pigments have a cytotoxic effect is best considered under the discussion of the nephrotoxic theory of renal dysfunc tion. 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 - First, 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 will not flow. So i t is 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 hemo lysis has been considered by Foy et al (137) to be a possible cause of oliguria in Blackwater fever. With 50% of the blood hemolyzed, plasma protein would be increased by 8%, thus increasing the plasma osmotic pressure to counteract the hydro static pressure, reduce filtration and result in oliguria. They concluded, however, that this change made no contribution to oliguria because both albumin and globulin levels in plasma dropped in proportion to the hemoglobin rise so that the total osmotic pressure remained unchanged. Third, a decreased hydrostatic pressure could also con ceivably result in reduced urine output. This is an obvious cause of anuria in 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 con tinues to be n i l , so that obviously no urine can form. This mechanism, then, involves the renal ischemia theory of pathogene sis and will be discussed under that heading. - 30 - A fourth mechanism has been discussed as early as 1925 by Dunn and Jones (119) in their experiments with oxalate neph r i t i s . This is the "back diffusion theory". They believed that the urea retention and oliguria seen in "experimental tubular nephritis" could be explained on the theory that the damaged tubule cells are unable to prevent indiscriminate reabsorption of glomerular filtrate with urea from the tubules into the connect ive tissue and vessels of the kidney. They carried this idea into their work on uric acid nephritis (120) and recalled i t in a consideration of two cases of crush syndrome in 1941 (117). In 1929» A. N. Richards (303) had reported that in frogs made anuric with mercuric chloride, glomeruli were more active and remarked, "The only explanation which I can reach is that under these abnormal conditions the osmotic pressure of the blood pro teins is unobstructed by the normal qualities of the tubular epithelium and is able to draw a l l or nearly a l l of the glomeru lar filtrate back into the blood stream." The idea appealed also to Nicholson et al (266) in their work with sodium tartrate nephrosis, as i t did as well to Hayman et al (17D in uranium nephrosis and to Bywaters and Dible (71). In more recent functional studies, Redish et al (299) determined clearances and tubular maximums in a case of sulphonamide anuria and found, in the third week, that these values were negative. They became positive at six weeks. They concluded that the i n i t i a l decreased clearances (mannitol) were not indicative of the true glomerular - 31 - filtration rate and that some back diffusion had occurred. Similarly, a negative Tm p A H indicated back diffusion of PAH (para-amino hippuric acid) through the tubules. This same conclusion was reached by Govaerts (159) in comparing urea and creatinine clearances in mercury and bismuth poisoning cases and uranium and oxalate poisoning in dogs. It would seem reasonable to conclude that anuria is first the result of reduced or absent renal blood flow in the in i t i a l stages of shock. If glomerular flow is 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 is involved, tubular obstruction is undoubtedly a factor. Nephrotoxin: The nephrotoxic theory, of renal damage in the syndrome has, like the obstruction theory, been held for many years. Implied in this theory is that a substance toxic to the kidney is released into the circulation in trauma, shock, crush, intravascular hemolysis or destruction of tissue, resulting in the clinical picture of acute tubular necrosis. Two possible modes of action of the toxin are considered: f i r s t , a direct cytotoxic action whereby tubular cells are poisoned and put out of action; and second, a vasospastic action in which case the renal ischemia theory of pathogenesis is 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 in the belief that various intracellular compon ents are released from damaged or shocked tissues, which, in 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) in 1935 that rats fed an excess of inorganic phosphate developed lesions in the terminal portions of the proximal convolution were confirmed by McFarlane (218) in 1941, though the affected portion according to this observer was lower in 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 factor from striated muscle. 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 in 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 phos phate 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 adenosine- like action, and believed that adenosine triphosphate may play a role in the renal failure death in rabbits. Bollman and Flock (44), however, in 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 con cluded that adenosine triphosphate was not released from ischemic muscle; the adenosine triphosphate was hydrolyzed and only non toxic products were liberated. Green continued in his opinion, however, reiterating in 1945 (163) that adenosine triphosphate is toxic to the kidney and in crush syndrome its effect is magni fied by that of myoglobin. But later (164), in discussing a case of traumatic uremia, he concludes that renal anoxia (vaso spasm) and toxic metabolites (myoglobin) are responsible for the renal damage. A second approach considered the toxicity of thoracic due.t lymph in crush injury......trauma and tourniquet shock. Blalock (38) concluded that lymph from dogs suffering five hours - 34 - of crush to one limb contained some factor lowering blood pres sure and producing casts i n urine. Katzenstein et a l (190) also found that blood pressure was lowered following i n j e c t i o n of thoracic duct lymph taken from animals subjected to tourni quet shock. The eff e c t was, however, variable. The work of Eggleton (124, 125? 126) on crush i n j u r y i n the cat i s i n t r i g u i n g and has not been altogether discounted. She f i r s t reported i n 1942 that crush syndrome could be repro duced by a sudden release of compression whereas gradual release prevented development of impaired renal function. An int a c t l i v e r appeared to be esse n t i a l for the d e t o x i f i c a t i o n of an agent gradually released from the ischemic muscle. She was able to show (125) that an extract of ischemic muscle depressed the creatinine clearance while an extract of fresh muscle did not; that extracts of muscle dead for from four to ten hours proved to be toxic; and that the extract appeared to be a break down product of a large protein molecule formed anaerobically. Also i n 1944, Mlrsky and F r e i s (244) injected t r y p s i n i n t r a p e r i t o n e a l l y into rats and rabbits and produced renal damage. They concluded that t h e i r findings supported the theory that extensive tissue damage released p r o t e o l y t i c enzymes which i n turn released "catabolic factors" responsible for renal and hep a t i c i n j u r y . Most searches f o r these biochemical factors have been - 35 - concerned with the etiology of shock and many agents have been described since Chambers, Zweifach et al (78) demonstrated a vaso-excitor material (VEM) in early shock and a vasodilator material (VDM) in later shock. This work has been carried on to present times (329)? but both Reinhard et al (302) and Frank and his co-workers (139) have discounted the importance of this VEM-VDM mechanism in shock. They do, however, substantiate the importance of an intact, functioning liver in the prevention of irreversible shock. Page (297> 302, 34-8) has also been active in this field, having described a serum vasoconstrictor, sero tonin, which he believed was the ultimate effector mechanism in renal ischemia. He believed the primary stimulus to renal vaso constriction was neurogenic. Frank et al (138, 139) found also that in dogs suffering haemorrhagic shock and treated by peritoneal irrigation or arti 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 irreversibility of shock, and that there is no circulating depressor substance in irreversible shock. To attempt to untangle the voluminous literature on the subject of humoral agents in shock is beyond the scope of this thesis, but one other lead is 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 is evident that - 36 - the newer investigations in the field of nephrotoxins have been directed to the discovery of an agent which is responsible first for the abnormal vascular responses of the body in 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 in what will 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 cell stroma was responsible for the symptoms observed in 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 in 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 analo gous to that responsible for anaphylactic shock". Reid (301) was the first to suggest a vasospastic action of hemoglobin when he noted a marked but transient decrease in kidney volume - 37 - following injection of distilled water or laked red cells. Mason and Mann (237) continued this work and found that associa ted with decreased kidney volume there were fewer blood filled 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 in the work of 0'Shaughnessy et al (276) who found that a 5% solution of hemoglobin in Ringer's was toler ated well as a blood substitute in doses up to 50 gm. of hemo globin. 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 circu lating 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 is 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 is not toxic, but with renal ischemia produced by renal artery constriction, added hemoglobin plays a significant part in the severity and mortality of the illness. Yuile et al (377) believed that a specific renal vasoconstrictive action of hemoglobin was not an - 38 - important factor in 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 clear ances postulated again a vasoconstrictor effect of hemoglobin. The cytotoxic action of hemoglobin has also received recent support in the work of Rosoff and Walter (309) who suggest that "heme" competes with cytochromes in the oxidative processes of the tubular cells, resulting in damage, degeneration and necrosis of these cells. In summarizing present opinion on the nephrotoxic theory, i t is 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 in that these agents have been reported to be both cytotoxic and vaso spastic. 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 is 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 is disrupted and the clinical picture can progress from one of i n i t i a l shock to acute renal failure with its olguria, uremia and death. Proponents of this theory believe that shock is the i n i t i a l event in a l l cases, whether i t be fromutrauma, crush, haemorrhage, dehydration, burns or 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 filtration 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 metabo lism, suffer varying degrees of anoxic damage. When the hypo tensive or anoxic period is prolonged, this damage is severe and even though renal blood flow may be restored, degeneration contin ues to necrosis and the kidney recovers neither structurally nor functionally. Death in acute renal failure ensues. The disput able point in this theory i s , what is responsible for the prolonga tion of renal ischemia beyond the period of i n i t i a l low blood pressure? There appear in the literature cases of acute tubular necrosis, especially in the field of gall bladder surgery (hepato renal syndrome), in which either no or only a very short period of hypotension was recorded and yet renal tubular degeneration occurred. It will be seen that nervous, humoral and hormonal agents have been described as producing renal arteriolar con striction to account for the prolonged ischemia, together with 4G - the complicated Arterio-venous shunt suggested by Trueta (353). Fishberg (133) in 1937 believed that a decreased renal blood flow, the result of decreased blood volume and cardiac output was the primary pathogenetic factor in 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 in 194-5, Maegraith et al (226) came out strongly for the renal anoxia theory. These workers point out that pigments are not always present and therefore are not essen t i a l ; that the nephrotoxic theory demands a wide variety of toxins; that circulatory collapse alone is common to a l l cases. They leave open the question of what causes the circulatory col lapse, but foresee the work of Trueta by suggesting the possibil ity of a redistribution of renal blood flow or glomerular bypass (225). In his classical review of lower nephron nephrosis in 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 first 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 is 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 in the distal tubule. In spite of these objections, much of the recent work on acute tubular necrosis has been concerned with its shock aspect. Page (280) believed that the entire cardiovascular musculature was altered in shock and pointed out that experimentally i t is always necessary to produce shock in rats before injecting myo globin solutions, i f lower nephron nephrosis is to develop. Marshall and Hoffman (233) in 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 in many cases associated with the deposition in the renal tubules of various derivatives of hemoglobin and, myoglobin." This defini tion would.appear to be a most satisfactory one in 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 al (146) who review the problem, report 22 cases and suggest an etiological classification. 42 Clinical 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 in 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 in alimentary haemorrhage (31) and particularly in traumatic shock (102, 99, 93, 208, 303, 322, 355, 356) and Herbut (175) lists 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), in which anuria and uremia developed but recovery occurred. It is to be expected that, with the idea that a period of hypotension was the prime factor in initiating the renal dys function, much experimental work with circulatory shock in animals and functional studies in patients suffering shock were reported. Corcoran and Page (84), working with haemorrhagic hypotension in dogs, showed that the renal blood flow f e l l and filtration decreased; that renal blood flow was distributed unequally; 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 al (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, hemoglobin- emia and an unknown substance, myoglobin or a toxin from ischemic muscle. Keele and Slome ( 1 9 D j using cats, found that the renal blood flow reduction was greater, in proportion, than the lower ing of blood pressure by crushing the limb. They therefore believed that reduction of renal blood flow was not due only to reduction in blood pressure. Selkurt (322) confirmed this finding in dogs in 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 likely that a true? acute tubular necrosis was obtained, in view of Oliver's recent work (271). Selkurt concluded that in shock the kidney received proportionally less of the cardiac output (5% instead of the usual 20%) due to intra- renal 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 in 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 in blood pressure with renal arteriolar con striction. If blood pressure dropped below 60 to 100 mm., renal blood flow and function ceased. If the i n i t i a l loss of - 44 - blood was not too great, the blood pressure was restored by- peripheral vasoconstriction, which is slower than the renal response and kidney function was restored to less than the pre- haemorrhagic 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 glomeru lar filtration, 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 in 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 in uremia, in dogs deaths appeared to be almost consistently from shock. Though they believed i t almost impossible to get uremia in these animals, i t will be seen that, in Oliver's examination of kidneys from these dogs (27D characteristic tubular lesions were indeed seen. Functional studies in clinical cases tend to show the same reduction of renal blood flow in shock. The work of Cournand and Lauson (93, 208) is again classical in the clinical field. Determinations of glomerular filtration rates and renal plasma flows in cases of trauma, haemorrhage, peritonitis, burns and head injuries with and without shock showed that "the rate - 45 - of glomerular filtration and effective renal plasma flow are significantly reduced in 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 filtration 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 in which there is renal vasoconstriction as compensation for the low blood pressure, and a renal damage phase of shock kidney in which reversible to irreversible renal failure is seen. That i s , there must be a degree of shock sufficient to result in 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 in shock is much less than the increased extraction in 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 - renal artery resulted i n anuria for a period longer than the closure, presumably because the tubules were more sensitive to anoxia and function was interfered with. Starr (337) produced albuminuria i n animals and man by renal artery c o n s t r i c t i o n and by adrenaline and ephedrine i n j e c t i o n and emotional upset. He was unable to recognize renal s t r u c t u r a l damage i n the animal experiments. McEnery et a l (217) reported elevated blood urea l e v e l s , o l i g u r i a and anuria and c o r t i c a l p a l l o r with medullary congestion i n kidneys following temporary clamping of the renal blood supply. The more recent era of renal artery occlusion began soon after the crush syndrome received so much attention. S c a r f f and Keele (312) describe uremia and renal pathology s i m i l a r to the crush kidney (but with proximal tubule degeneration) a f t e r clamp ing the renal pedicle for up to two hours. They conclude, "Thus there i s a p o s s i b i l i t y that the kidney l e s i o n i n cases of crush injury might be due to renal ischemia ... ". Selkurt (320), with shorter periods of ischemia, records reduced i n u l i n , diodrast and PAH clearances and tubule damage " s i m i l a r to mild uranium poisoning"; he l a t e r confirmed these findings by d i r e c t determina tions of renal blood flow and believed that afferent a r t e r i o l a r c o n s t r i c t i o n decreased the glomerular f i l t r a t i o n rate (321). Badenoch and Darmady (11), i n occluding the renal artery of rabbits temporarily, obtained elevated blood urea l e v e l s and renal tubular damage which appeared s i m i l a r to that seen i n human traumatic uremia. Koletsky and Gustafson (195) pointed out that the renal - 47 - lesion obtained by clamping the renal pedicle l£ to 2 hours in rats was not the same as seen in human or experimental crush syndrome or in rats with tourniquet shock; here the lesion was proximal tubule degeneration whereas in crush i t was lower nephron nephrosis. Later work (194) demonstrated that healing of the necrotic epithelium was possible. Scheibe et al (313) , 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 al (291, 292) is out standing. In noting that renal artery compression depressed urea clearance, they suggested that three possibilities were appar ent: decreased renal blood flow, decreased plasma filtration or increased reabsorption of urea by devitalized tubules. They clamped the left 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 pro gressively, indicating progressive tubular damage, and concluded that resultant urea retention was probably due to back diffusion. They emphasized in 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 (308, 41) has con firmed and elaborated on these conclusions based on renal artery occlusion in dogs. These investigations leave l i t t l e doubt that renal - 48 - anoxia plays a prominent role i n the production of renal tubular damage i n many of the c l i n i c a l e n t i t i e s presenting a picture of acute renal f a i l u r e . They also indicate that active renal 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 factor i n the development of the anoxia, despite the statement of Schroeder and Steele (316) that there i s l i t t l e renal vasoconstriction, i n shock. The agent causing that vasoconstriction has not been i d e n t i f i e d . Two mechanisms (146, 50) have been suggested, the nervous and the humoral (including endocrines and pigments). Darmady (99) re ported that as long ago as 1859» Bernard observed that stimulation of peripheral and splanchnic nerves produced renal vasoconstric t i o n . Results of controlled experiments i n dogs i n which the nerve t r a c t s were interrupted at various l e v e l s and i n various ways, p r i o r to muscle trauma shock, led Swingle, Kleinberg et a l (346) to conclude that pain impulses t r a v e l l i n g i n the dorsal spinothalamic tracts may be responsible for the various phenomena of shock. Wolff (366) i n discussing the mechanism of r e f l e x anuria, believed that the pain or discomfort of cystoscopy pro duced s t i m u l i which reduced renal blood flow and resulted i n anuria. Donnelly (113) also was led to conclude that there was some factor which allowed rapid cessation and resumption of kidney function, and Brod and S i r o t a (52) remarked on a r e f l e x renal ischemia from manipulating excitable rabbits. It was Trueta's work, begun i n 1942 (20) and published i n f u l l i n 1946 (353)» that emphasized the importance of neurogenic renal vasoconstric t i o n o r i g i n a t i n g i n damaged limbs. They believed that "noxious agents" stimulated nerves peripherally 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 toxin. 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. This concept w i l l be discussed f u l l y below. Several other reports (200,210, 361) appeared to support the idea that chronic stimulation of the renal nerves was responsible for renal vasoconstriction, and Cort ( 9 2 , 91) stated that trauma produced a sciatic-splanch nic reflex which could be interrupted by sympathetic block to allow diuresis. Smith (333)? however, summarized opinion on nervous control of renal hemodynamics by stating 'categorically, "the physiological control of the renal circulation remains almost a complete mystery," and points out that, in animals under local anesthesia, spinal anesthesia or denervation of the kidney does not produce renal hyperemia and that renal blood flow is normally determined by "autonomous intrinsic activity of the renal arterioles and is not dependent upon tonic activity in sympathetic pathways." Nevertheless i t would appear probable that the immediate response of the kidneys to drastic blood loss is neurogenic in nature ( 3 2 9 ) . Several humoral agents are known to constrict renal arterioles but whether they are responsible for this phenomenon in the traumatic anuria syndrome i s not known. Smith (333) considers a number of these (adrenalin (316, 2 6 0 ) , renin or angiotonin (321, 42) and histamine) and notes that fright, exer cise and pain a l l decrease the renal plasma flow. Corcoran and Page (84) consider that humoral vasoconstriction is 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. In later work, Taylor and Page (34-8) 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 in the discussion of the role of pigments in the production of anuria, i t has been suggested that hematin ( 8 5 ) and hemoglobin itself ( 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 vaso constriction 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 vasocon- striction may be due to a humoral agent. Investigations into the pathogenesis of shock itself, such as carried out by Shorr et al ( 3 2 9 ) and Frank et al (140, 1 3 9 ) may produce progress in - 5 1 - this direction. Trueta's corticomedullary shunt of renal blood flow: It was i n 1947 that Trueta et a l (353) published in f u l l results of experiments begun in 1942 (20, 141) to study the problem of arterial spasm in response to trauma. A com plete review of this very thorough work is impossible at this point, but in essence the theory is based on morphological differences between cortical 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 lary glomeruli, bypassing the cortical ones and the main mass of tubules. Trueta et al believe that a reflex neurovascular - 52 - mechanism is responsible for these profound alterations of intra- renal 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) in response to tourniquets, sciatic nerve stimulation, haemorrhage and various drugs (adrenalin, pituitrin, pitressin, ephedrine and staphylo coccus toxin); they made observations on dogs, cats, rats, guinea pigs but chiefly in rabbits. Because of this shunt, cortical glomeruli (which constitute 85% of renal glomeruli) are rendered ischemic which in turn renders at least an equal percent age of renal tubules ischemic (see Figure 2 ) , accounting for the renal dysfunction seen in traumatic uremia, crush syndrome, bi lateral cortical necrosis and other renal diseases. Trueta's shunt theory implies the following: (1) that so called juxta-medullary glomerular differences are present in spec ies other than rabbits. Trueta states that these differences are seen most often in rabbits, but less so in the dog, cat, guinea pig and rat, and that the differences are less evident in 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 in operation, allows blood to bypass tubules whose function is essen t 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 in the renal vein when the shunt was said to be in operation and therefore implied that the arterio-venous oxygen difference was decreased in 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 ob servation that the renal blood supply was reduced by neurogenic renal vasoconstriction. It is on these four points that Trueta's shunt theory has been most severely criticized. It is interesting that, many years before Trueta suggest ed his corticomedullary shunt, observations were made on kidney pathologies which support his ideas. Renal pathology in fatal blood transfusions was frequently described as an enlarged kid ney 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) in 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 is interest ing, too, that in 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 in 1946 (225) they pointed out that the renal blood flow must be redistributed in the kidney and from the histological appearance, suggested a corticomedullary diversion. The shunt theory received some early support from the work of Simken et al (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 by passes existed in the kidney or its capsule. Experiments were carried out in rabbits, dogs and human kidneys. The controversy of arterio-venous anastomoses has been apparent for many years and is reviewed adequately in Smith's book (333)• Arcadi and Farman (8 ) were able to duplicate Trueta's work by India ink injections in rabbits, as ware Goodwin et al (154), using tourni quets or sciatic nerve stimulation in 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 con trol of the renal circulation in which renal ischemia started in 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 vaso constriction. Black and Saunders (35) also supported Trueta's observations with the reservation that, before the shunt is accepted, three criteria must be satisfied: (1) low inulin and PAH clearances with increased C i n to Cp^g ratio, (2) PAH - 55 - extraction less than 80% and (3) absence of gross changes in general circulation, since efferent arteriolar constriction, rise in renal vein pressure or f a l l in systemic pressure could produce a picture simulating"the shunt. Evidence against the shunt also appeared early and has continued to accumulate since Barclay et al (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 is not reduced in the syndrome, the change being merely a short-circuiting of blood rather than a reduction in flow; he did not measure the renal blood flow, however. Based on many reports of functional studies carried out in animals (257» 302a, 258, 183» 259, 260) and in man (347, 302a, 331> 18) i t appears conclusive that in 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. in fact a true reduc tion of renal blood flow. Moyer et al (258) substantiated this claim by direct measurement of renal blood flow. Further inroads into Trueta's theory have been made in 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 in 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 al (40), using neoprene, state that "the only consistently exist ing vessels which directly communicate between the renal arter ies 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 substantia ted 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 is diffuse, not localized as Trueta suggests. He notes that the proximal nephron is affected as well as distal. No criticism of the work of Trueta et al can be complete without a consideration of the conclusions reached by Maxwell, Breed and Smith (238) on.the significance of the renal juxta- medullary circulation in man. These men point out (1) that, since the proximal segment is responsible for excretion, when blood is perfusing the vasa recta which are in contact only with - 57 - distal and thin segments PAH clearance should be low; (2) that with the bypass, filtering surface is 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 in 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 is against diversion of blood through uncleared channels; (4) juxtamedul- lary circulation in man has no unique functional significance. It is seen in the rabbit only as a species difference. a • On the basis of evidence cited above, the conclusions of Maxwell et al are justified. Observations made on renal blood flow and arterio-venous oxygen differences are not compati ble with Trueta's concept of a corticomedullary shunt of renal blood in the traumatic anuria syndrome. The thoroughness of Trueta's experiments, however, convince one that the phenomenon is of frequent occurrence at least in rabbits, and the occurrence - 58 - of clinical cases of bilateral cortical necrosis adds to one's conviction that the Trueta shunt may indeed have a place in the scheme of things as an extreme in a series which includes any thing 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 is of prime pathogenetic importance in the development of acute renal failur in shock, burns and crush. The work of Cournand's group (93» 208) showed conclusively that in shock from trauma with or without haemorrhage, peritonitis, abdominal injury and burns, "the rates of glomerular filtration 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 in 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 in formation when, by clamping the renal artery to produce ischemia they produced renal dysfunction as measured by clearance tech niques and tubular lesions described by Oliver (271) as being identical to those seen in shock or crush kidneys. Both the functional and structural changes seen in the kidney in human cases of shock from any cause, therefore, have been observed in animals subjected to experimental shock and in animal kidneys with obvious ischemia induced by clamping of the renal artery. - 59 - The pathogenetic si g n i f i c a n c e was apparent to Block et a l (41), when they undertook to carry out t h i s complete cycle of experi ments i n dogs. They subjected 28 dogs to periods of hypotension of 70 mm. of mercury for periods of from s i x to 26 hours and studied kidney function 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 early histology i n t h i r t e e n . They also studied the e f f e c t of renal artery occlusion from three to s i x hours on 24 dogs and 23 rats (one to three hours occlusion), as well as the e f f e c t of epinephrine on eight dogs. In a l l cases, they observed changes i n renal tubules from degeneration to complete c o r t i c a l necrosis, but found that death i n renal f a i l u r e was rare; i t required almost complete ischemic destruction of the kidney. They state that these results were independent of the nerve supply and that there was no evidence for Trueta's shunt. It i s also of importance to consider the means by which renal ischemia i s produced. In cases of shock i t i s obvious that an i n i t i a l period of hypotension i s responsible f o r the cessation of renal blood flow (291) . Renal vasoconstriction follows and appears to be primarily neurogenic, secondarily humoral ( 3 4 8 ) . A t h i r d possible source of anoxia i s a decreased oxygen extraction by the kidney; most investigators point out that, on the contrary, arterio-venous oxygen differences are increased — that i s , that oxygen extraction by the kidney i n shock i s increased rather than decreased (258, 259> 302a, 183, 111) . - 60 - Normal renal oxygen extraction, however, i s s i n g u l a r l y i n e f f i c  ient and arterio-venous oxygen differences are so s l i g h t that they may be within the error of methods of determination ( 3 9 , 2 3 8 ) . Summary of Pathogenesis In c r y s t a l l i z i n g an opinion on the pathogenesis of acute renal f a i l u r e due to acute tubular necrosis, one i s impressed by the conclusions reached by O l i v e r et a l (271) i n 1951 and by the work of P h i l l i p s , Van Slyke, et a l , on which to a large extent, Oliver's opinions were based. Observations made c l i n i c a l l y by Lauson, Cournand et a l (208, 93) are also convincing and the confirmation of experimental conclusions afforded by the work of Block, et a l , i n September 1952 (41/) appears to complete the picture of pathogenesis. It i s at f i r s t e s s e n t i a l to emphasize the existence of two pathogenetic mechanisms of prime importance. F i r s t l y , 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 1 e n t i t i e s , s p e c i f i c e x t r i n s i c toxic agents are responsible f o r d i s r u p t i o n of tubular c e l l metabolism, producing the acute tubular necrosis. Common among these agents are bichloride of mercury, carbon t e t r a  chloride and uranium s a l t s . O l i v e r (271) has named the lesions they produce "nephrotoxic tubular necrosis" and described con vin c i n g l y the c h a r a c t e r i s t i c pathological l e s i o n seen i n these poisonings and the differences from the second type of tubule damage. - 61 - Secondly, there i s a large group of c l i n i c a l conditions i n which shock i s a common factor and which frequently (146, 114, 335) give r i s e to the syndrome which we prefer to c a l l "acute renal f a i l u r e due to acute tubular necrosis". Such conditions have been described e a r l i e r i n this thesis but can be l i s t e d b r i e f l y ( 2 5 2 ) : trauma, haemorrhage, crush, burns, p e r i t o n i t i s , hepatorenal syndrome, retroplacental haemorrhage and others. There i s no doubt that t h i s second pathogenetic mechanism, renal ischemia, i s of prime importance here. A resume of the probable pathophysiological phenomena may well be as follows: i n shock induced from any cause the i n s u l t may be so severe or the resistance to i t so low that the i n d i v i d u a l progresses to the so- called i r r e v e r s i b l e stage and dies i n peripheral c i r c u l a t o r y f a i l u r e ; or the i n d i v i d u a l may respond well to the stress and recover uneventfully from the hypotensive shock period; or he may make an apparent recovery from the acute shock period only to pass into what might be called a l a t e e f f e c t of shock, a state of acute renal f a i l u r e with o l i g u r i a , anuria and, once again, either recovery or death i n uremia. This t h i r d p o s s i b i l i t y , acute renal f a i l u r e , i s t y p i f i e d by cases i n which massive haem orrhage i n i t i a t e s the renal f a i l u r e . With the haemorrhage there i s an immediate f a l l i n systemic blood pressure which, i f s l i g h t , may s t i l l allow glomerular f i l t r a t i o n due to the compensatory renal efferent a r t e r i o l a r c o n s t r i c t i o n . But i f the systemic blood pressure f a l l s below 60 to 1G0 mm. of mercury (291) renal blood flow and function cease. ' Accompanying th i s early hypo-- 62 - tensive period there i s renal vasoconstriction which i s part of a generalized compensatory vasoconstriction which may r e s u l t i n the i n d i v i d u a l compensating enough to survive the acute shock period following the haemorrhage. Blood pressure can be restored either by t h i s compensation or by transfusion and the kidney c i r c u l a t i o n returned to something less than pre-haemorrhagic l e v e l s . In spite of t h i s apparent return to normal, some cases go on to tubular degeneration and necrosis with c l i n i c a l acute renal f a i l u r e . Because i n both c l i n i c a l and experimental cases clearance techniques and d i r e c t measurements show renal blood flow to be diminished, and because experimental occlusion of the renal artery 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 tubular necrosis, the damage has been blamed primarily on ischemia. Whether the i n i t i a l period 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 pro duce the damage, or whether the early (probably neurogenic) renal vasoconstriction i s prolonged either by nerve impulses or by humoral agents to prolong the anoxemia cannot be stated d e f i n i t e l y . The role of i n t r i n s i c nephrotoxic agents, presumably released from damaged or ischemic tiss u e s , i n the pathogenesis of this syndrome can be less convincingly stated. The status of the various e x t r i n s i c chemical agents has been mentioned previously. However, the presence of a protein breakdown product of ischemic muscle (125) or a toxin from massively des troyed t i s s u e s , to disrupt the metabolism of tubular c e l l s would - 63 - appear to be unnecessary to explain the renal damage since in 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 vaso constrictive 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 is therefore implied that cellular anoxia is res ponsible for the tubular damage, which in turn accounts for the renal dysfunction. The mechanism of this dysfunction is prob ably "back diffusion" (303, 117), in which the dead tubule cells act as a membrane through which tubular fluid, urea and other wastes diffuse back to the interstitial fluid and thence into the circulation. The place of pigment cast obstruction in the development of renal dysfunction, so evident in the intravascular hemolyses, has been given its proper place by Oliver et al ( 2 7 D and Block et al (41). Pigment cast obstruction is at least unnecessary. Clinical lower nephron nephrosis is seen in cases in which no pigment release is involved and acute tubular necrosis can be produced experimentally without appearance of hemoglobin or related substances. Again, clinically, intravascular release of pigments usually occurs in cases in which there is associated shock — transfusions, Blackwater fever, post-operative - 64 - 1 transurethral prostatectomy, crush, burns — so that ischemia is probably contributing more to the renal failure than is tubule obstruction. But, as Oliver has pointed out, in cases in which pigment casts are prominent, one cannot ignore the fact that the involved tubules are plugged and will not carry urine. If there is back pressure in 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 (289, 364). Although i t appears possible to produce renal shutdown from induced hemo- globinemia (232, 168, 241), i t is 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 in shock or severe dehydration. Associated with pigment cast obstruction is the prob lem of precipitation of hemoglobin, myoglobin or related pigments. It is probable that a variety of factors (15, 376, 240), urine pH, glomerular filtration, urine salt content, tubular reabsorp- tion, combine in the lower nephron and collecting tubules to produce conditions favorable for heme pigment precipitation. EXPERIMENTAL AIM As was stated e a r l i e r , i t was thought advisable to repeat i n a systematic way some of the work done by other investigators i n order to determine the role of c e r t a i n factors i n the production of traumatic anuria. Preliminary experiments,-, then, were carried out with the aim of producing "lower nephron nephrosis" i n a standard way i n a substantial proportion of test r a t s . Once th i s standardization was accomplished, further experiments were designed to test the e f f i c a c y of c e r t a i n hor monal agents i n the a l l e v i a t i o n of the kidney damage. A sampling of the experimental l i t e r a t u r e on t h i s sub ject has shown that three factors play a prominent role i n the pathogenesis of acute tubular damage. The pigment cast obstruc t i o n theory can be associated cl o s e l y with the nephrotoxic theory, of pathogenesis and so the f i r s t variable factor chosen was the role of myoglobin (muscle hemoglobin) i n the production of kidney damage. It was not considered c r u c i a l to th i s work whether the pigment produced i t s e f f e c t by a toxic action or by obstruction, though observations on th i s problem w i l l be made. The second factor considered was what may be called c l i n i c a l shock, and here - 66 - a crush i n j u r y to the limb of the test animal was the means of production. Again, whether the pathogenetic mechanism was one of release of nephrotoxic materials from damaged tissues or simply one of production of prolonged hypotension was not consid ered, though some conclusions w i l l be drawn. The t h i r d factor controlled in.the following experiments was the hydration of the animals, since dehydration (202, 203, 232) has been shoira to emphasize, i n some way, the tubular damage to the kidney. It can be seen that the aim of the experiments reported below has not been primarily to investigate the pathogenesis of acute tubular necrosis, but rather to standardize the production of the syndrome i n the white rat, and to investigate the thera peutic p o s s i b i l i t i e s of various hormones. Statements as to the mechanism involved, based on these experiments, w i l l therefore be impressions rather than conclusions drawn from controlled ex periments. METHODS AND MATERIALS Bothemale and female white rats of the Sprague-Dawley and Wistar s t r a i n s , weighing from 200 to 350 grams, were used i n order to bring out any sex dif f e r e n c e . These animals are convenient generally 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 hardiness; s p e c i f i c a l l y , they are of use because the ske l e t a l muscle contains l i t t l e , i f any, myoglobin so that i n experimental work designed to test the role of - 67 - myoglobin release and of crush i n j u r y , these two factors can be conveniently separated. Dehydration was accomplished merely by withdrawing water f o r periods ranging from 24 to 72 hours. The e f f e c t i v e  ness of t h i s method was e a s i l y ascertained by the massive weight loss recorded (up to 20% of body weight i n 48 to 72 hours) and by the production of urine which was low i n volume and high i n concentration, of feces which were small, hard and dry as well as reduced i n amount. Urine s p e c i f i c g r a v i t i e s were not deter mined, but gross observation of color, and i n some cases v i s c o s i t y , gave a rough index of that f a c t o r . Myoglobin was obtained commercially i n a p u r i f i e d c r y s t a l l i n e form and was administered intravenously into the femoral vein a f t e r d i s s o l v i n g i t i n a phosphate buffer of pH = 7*35 ( 1 7 0 ) . D i f f i c u l t y was encountered i n dissol v i n g the protein which had been dehydrated so completely to t h i s c r y s t a l  l i z e d form. It was not thought advisable to use solutions of pH too f a r removed from i t s i s o e l e c t r i c point of 6.78 (7)> since i n j e c t i n g a solution of very acid or al k a l i n e pH would introduce a complicating factor. The p o s s i b i l i t y of di s s o l v i n g the pigment i n rat serum was considered but again i t was thought advisable to avoid the added question of s e n s i t i v i t y reactions to necessarily heterologous serum as well as the d i f f i c u l t y i n keeping such a solution s t e r i l e . I t was found f a i r l y s a t i s f a c  tory to make up a solution of 25 mg. per cc. of buffer which was - 68 - kept 24 to 48 hours i n an oven at 65% C, with frequent shaking. Complete d i s s o l u t i o n was not obtained, but i t was estimated that 60 to 70 per cent of the protein dissolved and the remainder could be suspended by vigorous shaking p r i o r to i n j e c t i o n . Dosage administered ranged from 0 . 1 to 0.15 mg. per gram of body weight, a figure based on that calculated by Bywaters ( 7 5 ) , and used also by Corcoran and Page ( 8 5 ) . This amount necessitated the i n j e c t i o n of a volume up to 1.6 cc. i n a 250 gm. r a t , but i f given slowly, no i l l effects were observed. Injections were made with a #25 hypodermic needle with the rat under ether anesthesia, at times considered to simulate as closely as possible the time relationships encountered i n human "crush syndrome" — i . e . , immediately on release of the crushing l i g a t u r e . Ligation was carried out on the hind limb of the animal, either l e f t or both, i n the manner i l l u s t r a t e d i n Figure 3* Figure 3 - 69 - • i Under ether anesthesia, the limb was clipped of hair from ankle to groin i n order that the l i g a t u r e would not s l i p . Using heavy twine, the limb was then wrapped t i g h t l y from ankle to as high on the limb as possible without i n t e r f e r i n g with the urethral outlet or incurring the r i s k of loosening. This meant that the ligature was usually t i e d at what was e s s e n t i a l l y the "mid-thigh" l e v e l . The ligature was l e f t i n place for i n t e r v a l s of four to fi v e and one half hours, being protected by adhesive tape wrapping i n case the animal attempted to b.ite loose the s t r i n g . During th i s time the rat was sedated with pentobarbital 2.5 mg. per 100 gm. of body weight (5 mg. per cc.) given i n t r a p e r i t o n e a l l y as needed. The f i v e hour period was spent i n large glass funnels which allowed for more easy observation and handling as well as for convenient c o l l e c t i n g of urine. With the period of crush completed, animals were removed from funnels, the l i g a t u r e was removed under the remaining nembutal sedation or ether anesthesia, the injured limb was massaged u n t i l i t l o s t i t s "doughy" consis tency and the foot appeared bright red, and the animal was then placed i n a metabolism cage for observation. The bladder was not emptied by compression at the completion of the f i v e hours crush because only the t o t a l 24 hour urine volume was to be recorded. In animals which were normally hydrated, d i f f i c u l t y was encountered i n that they would bite the injured limb, r e s u l t  ing i n haemorrhage and an at times severe anemia. Attempts were made to protect the limb i n some way to prevent the b i t i n g but not r e s t r i c t the swelling. Loosely applied 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. Dehydration animals were observed to not bite the crushed limb and frequently these were l e f t unwrapped. The effectiveness of the li g a t u r e i n producing a t y p i c a l crush injury was observed i n the almost immediate swelling of the limb (Figure 4) and at autopsy by the appearance of subcutaneous and muscle edema as well as di s c o l o r a t i o n of the muscle (Figure 5 ) . Figure 5 Because production of the syndrome was not completely s a t i s f a c t o r y i n the inta c t animal, and because the rat i s known - 71 - to have a formidable renal reserve, i t was decided to reduce this reserve i n as ph y s i o l o g i c a l a way as possible. Right nephrectomy was therefore carried out i n l a t e r experiments. Under ether anesthesia, the right kidney was approached poster i o r l y and decapsulated i n order to assure that the associated suprarenal gland remained i n s i t u ; the pedicle was t i e d , the kidney removed and the wound closed with a single black s i l k suture and skin c l i p s . A single subcutaneous i n j e c t i o n of 9,000 units of p e n i c i l l i n i n o i l was given post-operatively. Animals under 200 gms. weight were given four days i n which to recover before the stress of experiment began; those over 200 gms. were allowed only three days. Too long a period of recovery allowed for compensatory hypertrophy of the remaining kidney, while the period a l l o t t e d them allowed for complete recovery as s i g n i f i e d by gain i n weight. Test hormones used were a c r y s t a l l i n e preparation of testosterone propionate, a. saline suspension of cortisone acetate a saline suspension of compound F (17 hydroxycorticosterone — 21 - acetate) and a watery suspension of desoxycorticosterone acetate (DCA). Testosterone was given either as a saline sus pension or dissolved i n sesame o i l , 10 to 20 mgs. per cc. Two or three doses of 5 mgs. each were given subcutaneously, the i n i t i a l dose being given 48 hours before the i n i t i a l stress of experiment to assure adequate blood l e v e l s . Cortisone was given as 0.4 to 0.5 cc. of a 5 mg. per cc. saline suspension (2 to 2.5 mgs.) subcutaneously. This was a d a i l y doee, begun - 72 - 24 hours before ligature of the limb. Compound F was given as 0.4 to 0 .6 cc. of a 5 mg. per cc. suspension (2 to 3 mgs.) sub- cutaneously, a d a i l y dose started 48 to 72 hours p r i o r to l i g a  t i o n . DCA doses were 0.1 cc. of a 25 mg. per cc. suspension (2.5 mgs.) given subcutaneously each day beginning two days pr i o r to crush i n j u r y . During the 72 hours of observation, the animals were kept i n i n d i v i d u a l metabolism cages designed for c o l l e c t i n g urine and screening feces. Urine was collected for 24 hour periods i n small, open-mouthed jars placed close to the funnel ou t l e t . Loss by evaporation was minimal. Cages were equipped with deep food troughs i n which measured amounts of powdered standard feed were placed. Animals could eat this form of food e a s i l y without i n t e r f e r i n g with the urine c o l l e c t i o n . I f water was to be supplied to the animals during the t e s t , i t was done so with the usual bottle arranged so that i t did not drop spon taneously and any water which did drop was caught i n a small pan which could be e a s i l y emptied. The method allowed a cheek of water intake and avoided interference with the urine c o l l e c t i o n . Observations made were: animal weight before and a f t e r experiment, amounts of water and food consumed, urine volume, urine pH, blood urea nitrogen lev e l s and kidney histology. Later i t was thought that a c o r r e l a t i o n of kidney weight and body surface area would be of value, so that i n l a t e r experiments kidney weights were recorded. Urine 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 in 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. Tail 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 in a citrated pipette. Occasional difficulty was encountered in the dehydrated animals and the occasional death resulted from hemopericardium and cardiac tampon ade, but in 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 filtrate of blood is treated with diacetyl monoxime and concentrated sulphuric acid, heated in a boiling water bath, the color emphasized by addition of potassium persulfate and the resulting yellow solution read in 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 with t h i s method. It was found that solutions to be read were occasionally 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 sulphuric acid i n preparation of the Folin-Wu blood f i l t r a t e and when one drop of 10 per cent sulphuric acid was added to each blood sample, the f i n a l cloudi ness no longer occurred. A second trouble arose when the f i n a l solutions appeared red i n color, rather than yellow, t h e i r readings also being f a l s e l y high. I t was determined that s a l i v a blown into solutions i n expelling the contents of pipettes, pro duced t h i s pink d i s c o l o r a t i o n . Henceforth, pipettes were plugged with absorbent cotton. The t h i r d problem was that of accuracy of the method. As i n most procedures using small amounts of test substance, results allowed a wide range of error and normal figures for rat blood urea nitrogen le v e l s can only be stated as 50 to 100 mg. per cent. I t follows that differences of 10 to 20 mg. percent i n B.U.N, leve 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 for 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 afte 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 for 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 findings. In a l l cases ether was used for k i l l i n g animals remaining a l i v e at the termi nation of an experiment. Kidney weights were taken i n the following way. At post-mortem, the entire decapsulated l e f t kidney was incised and placed i n f i x a t i v e for 18 to 24 hours, at which time i t was b r i e f l y blotted dry and weighed i n milligrams. Surface area i n square centimeters was obtained from tables based on the animal weight i n grams at time of death. The relationship was then calculated i n milligrams of kidney tissue per square c e n t i  meter of surface area. REPORT OF EXPERIMENTS Experiment 1: Experiment IA considers the effect of 48 hours dehydra t i o n , with and without nembutal anesthesia, on the urea nitrogen, urine output and kidney histology of the in t a c t r a t . Of twelve rats four were subjected to dehydration alone, four to dehydration plus nembutal and four were normal controls. Preliminary "test runs" of the procedure were carried out on these rats 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 for food and water are t o t a l s for 48 hours. I t i s evident that, deprived of water, the r a t s ' intake of s o l i d - 75 - a. food i s markedly diminished. Weight change indicates that i n 48 hours of dehydration, animals lose about 10 per cent of t h e i r body weight. Figures for 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 for urine. B.U.N, figures 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 structure i n these animals. I t can be concluded that dehydration by removal of water source i s accompanied by reduction i n s o l i d food intake and i s eff e c t i v e i n reducing animal weight and urine output over a 48 hour period. Urea nitrogen l e v e l s r i s e s l i g h t l y , probably accountable for by hemoconcentration. There was no a l t e r a t i o n of kidney histology. At a l a t e r date, the above experiment was repeated on twelve rats which had been right nephrectomied three days pre viously (Experiment IB); four animals allowed free water were controls, while eight were dehydrated for 72 hours. Food a v a i l  able was 21 gms. each and kidney weights were recorded i n addition 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 intact animals i n Experiment IA. Animals allowed water ate so l i d food w e l l ; previously dehydrated animals had an increased - 76 - water intake when allowed water (following 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 figures revealed normal figures f o r control animals except at 96 hours, when figures were unaccountably low. Dehy drated animals experienced a dehydration o l i g u r i a . Blood urea nitrogen figures a f t e r 24 hours dehydration are high (average 125 mg %, range 110 to 140, for controls; 145 and 110 to 170 mg % for test animals) and continue high at 48 hours (average 150 mg %, range 140 to 180, for controls; 155 and 140 to 170 mg % for test animals). At 72 hours, a marked drop i n B.U.N, levels i s recorded i n spite of the fact that dehydration contin ued i n test animals (average 70 mg range 60 to 80, for con t r o l s ; 100 and 80 to 120 for dehydrated). At 96 hours, levels rose i n control animals but f e l l s l i g h t l y i n test animals (average 115 mg range 100 to 140, for controls; 95 and 70 to 130 for dehydrated). It i s apparent that these urea nitrogen 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 figures i n control animals at 24 hours may be accounted for by the fact that one kidney had been removed four days previously, with those i n test animals s l i g h t l y higher because of the 24 hours dehydration. At 48 hours, however, control levels continued to r i s e to essen t i a l l y the same levels as test animals. The sudden drop observed at 72 hours can be explained only as an error i n abso lute determination of urea nitrogen figures. The r e l a t i o n TABLE IA DEHYDRATION IN INTACT RATS RAT TOTAL FOOD (GMS) TOTAL WATER (cc) FINAL WEIGHT AND CHANGE URINE VOLUME (cc) URINE pH B.U.N. (GMS) 24 hrs 48 hrs 24 hrs. ,48 hrs. 48 hrs D.l 42 .3 51-7 318 (+ 14) 8.0 6.7 6.5 6 .5 40 'R OL  D.2 38.0 44 .5 350 (+ 4) 9.0 5.5 6.5 6 .5 40 CO M D.3 35.3 56.0 330 (+ 12) 12.3 11.4 7-0 6.5 40 CO M D.4 3 0 . 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 (-30) 4.8 2.1 6.0 6 .0 50 D.8 6.0 322 (-36) 13 .2 3 .2 6.5 6.5 50 NE MB . 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 m a D.12 16.3 — 295 (-25) 8.6 3 .0 7.0 6.5 50 TABLE IB DEHYDRATION IN RIGHT NEPHRECTQMIED RATS TOTAL FOOD TOTAI WATEf FINAL WEIGHT AND CHANGE (GMS) URINE VOL. (cc) URINE pH B.U.N. KIDNEY ' WEIGHT (GMS.) RAT 24 48 72 96 24 48 72 96 24 48 72 96 265 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 O ffi 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 EH O O 26? 21 54 176(-30) 10.0 7.2 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 Q W 272 21 42 2 2 6 ( - l 6 ) 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 W Q 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 l 8 4 ( - 3 6 ) 4 . 0 0.8 0 . 8 5 .2 7.0 6.0 6.0 7 .5 170 170 110 120 209.9 - 7 9 - between controls 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 controls at 96 hours, to exceed lev e l s i n test animals, correlates with 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 accidentally. 96 hour figures f or test animals f e l l s a t i s f a c t o r i l y following the f u l l 2 4 hours of hydration. Examined microscopically, kidney structure remained unaltered a f t e r 72 hours dehydration; kidney weights showed no si 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 stress. I t i s apparent that uninephrectomy results i n a tempor a 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 with the effect of myoglobin injected intravenously 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 rats as normal controls, four receiving intravenous i n j e c t i o n of physiologic saline and four Injected with myoglobin dissolved and suspended i n 1 cc. of sa 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 , 0.15 mg. per gm. of body weight. B.U.N's were determined on t a i l blood samples i n Experi 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 with the methods and pro cedures of t h i s experiment so that figures i n red are considered not v a l i d . These errors are considered i n the section on "Discussion and Conclusions". In normal controls, 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 rat (range 3*2 to 10.5 cc.) and i n the second 24 hour period averaged 5*9 cc. (range 3*5 to 9.4 cc. ) . pH figures were not s i g n i f i c a n t ; urea nitrogen le v e l s were within normal l i m i t s . Saline controls i n a l l three experiments had less con stant food and water intakes but figures are e s s e n t i a l l y the same as for normal controls; most animals gained weight. Urine volumes were comparable to the normal group, as were urine pH determinations. Urea nitrogen le v e l s remained within the normal range. No s i g n i f i c a n t difference was detected i n animals given the increased dose of myoglobin (Experiment 2C). A l l test a n i  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 also were comparable, though i n two instances (P.22 and P.23) urine output - 81 - was increased. This increase may represent an osmotic d i u r e s i s . Urine pH figures 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, figures again were within normal l i m i t s . casts could be seen i n the c o r t i c a l tubules with myoglobin i n j e c t i o n (Figure 6 ) , these were also present i n saline injected animals (Figure 7) . effect on the kidneys of in t a c t rats as measured by urine output, blood urea nitrogen and kidney histology. In two cases, there may have been an "osmotic d i u r e s i s " . The procedure of anesthe t i z i n g the animal and i n j e c t i n g i t with saline or myoglobin may well account for the greater v a r i a t i o n i n food and water intakes seen i n these animals. Renal histology was not e s s e n t i a l l y altered and though Figure 6 Figure 7 Myoglobin injected intravenously appears to have no TABLE 2A MYOGLOBIN IN INTACT RATS RAT TOTAL FOOD (GMS) TOTAL WATER (cc.) FINAL WEIGHT AND CHANGE (GMS) URINE VOLUME URINE pH. B.U.N. 48 hrs. 72 hrs. 48 hrs 72 hrs 24 hrs 48 hrs 72 hrs AX  C ON T:  P . l 41 .5 64.0 394(+12) 9.1 9.4 6.0 7.0 120 90 70 AX  C ON T:  P.2 42.0 49.0 358(+12) 7.4 4 .8 6.5 7.0 60 80 40 o P.3 45 .5 58.5 380(+l8) 10.5 8.9 6.5 6.5 80 70 60 is; P.4 44 .5 51.0 304(+4) 8.1 8.3 6.5 7.0 100 90 50 « E-t P.5 0 1 .5 342 ( - l6) 11 .6 2.8 7.5 7.0 70 80 60 O O p.6 19.0 32.5 424(-24) 12.6 10.3 7.0 6.5 110 90 70 SA LI NE  P.7 0 2.0 324(-40) 6.4 2.4 6.0 6.0 180 70 50 SA LI NE  P.8 23.0 26.5 370 (0) 2.2 7.2 6.5 6.0 70 90 60 •—^ P.9 24.0 36.0 360C-8) 4.2 2 .8 7.0 6.5 100 — 60 M P.10 24 .5 24 .5 340(-12) 6.0 4.4 7.0 6.5 — 90 60 0G L(  P.11 28.0 29.0 352(-14) 6.2 5.0 7.0 7.0 — 50 9 P.12 20.0 18.5 330(- l6) 6.0 3.5 6.5 7.0 190 — 70 TABLE 2B MYOGLOBIN IN INTACT RATS TOTAL TOTAL FINAL WEIGHT URINE ! .VOLUME URINE pH B.U.N. RAT FOOD (GMS) WATER (ec) AND CHANGE (GMS) #8 hrs. 72 hrs. 48 hrs 72 hrs 24 hrs 48 hrs 72 hrs ON TR OI  P.13 42 .0 57.0 7-0 4 . 6 6.5 6.5 60 60 60 3R MA L C P. 14 38.0 58.0 - 7.6 5.8 6.5 7.5 60 70 60 3R MA L C p.15 41 .5 57.0 - 10.1 9.0 7.0 7.5 60 70 50 JT RO L.  N(  P.16 41 .5 50.5 - 7 .0 3.8 7.0 7 .0 70 60 60 JT RO L.  N(  P.17 47 .5 •94.5 - 9.1 23.8 6.5 7.5 70 . 80 70 o o P.18 45 .0 69.5 - 9.6 6.0 7.0 7 .5 50 50 80 IN E P.19 39.0 6 0 . 5 - 6.6 6.1 6.0 7-5 60 60 50 SA L P.20 45.5 57.0 4 . 4 3 .0 6.0 6.5 70 60 60 P.21 43.5 59.5 - 7-4 6.6 6.5 7.0 60 60 50 M P.22 26.5 64.0 - 10 .4 15.4 6.5 7.5 70 60 60 o o P.23 44.5 87.O 11.5 15.4 7.0 7 .5 70 70 50 P.24 43.5 ... 54.0 - 6.6 7-2 6.5 7.5 60 70 50 TABLE 2C MYOGLOBIN IN INTACT RATS TOTAL TOTAL FINAL WEIGHT URINE VOLUME URINE pH B.U.N. RAT FOOD (GMS) WATER (cc) AND CHANGE (GMS) 48 hrs 72 hrs 48 hrs 72 hrs 24 hrs 48 hrs 72 hrs CO NT RO L P.25 41.0 59.0 248 (+8) 9.2 4 .6 6.5 7.0 70 50 CO NT RO L 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 IN E P.31 40.0 63.5 " 256 (+10) 7.5 5.0 6.5 7.5 70 70 60 !SA L 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 BI N P.34 39.0 6o.o 240 (+8) 9.6 7.2 7.0 7.5 70 60 60 )G L0  P.35 32.0 63.0 244 (-1) 6.6 8.6 6.5 6.5 70 70 50 P.36 1 25.0 40.0 232 (+4) 4 .6 3 . 8 7.0 7.5 60 70 50 TABLE 3 MYOGLOBIN AND DEHYDRATION IN INTACT RATS RAT rOTAL TOTAL FINAL WEIGHT URINE VOLUME URINE P H B.U.N. —f-u POOD (GMS) WATER (cc) AND CHANGE (GMS) 48 hrs 72 hrs 48 hrs 72 hrs 24 hrs 48 hrs 72 hrs o o If. 13' 24 .0 54 276 (+6) 0.15 2.6 6.0 6.0 70 70 60 Q W EH M.14 11.0 36 250 (-10) 0 . 2 2.2 6.0 6.0 70; 70 60 DE HY D R J M.15 19 .0 47 240 (+4) 0 . 1 2.0 6.0 6.0 90 70 70 DE HY D R J M.16 2 3 . 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 o i — i M.18 24 .0 48 250 (-4) 0.2 1.6 6 .5 6.5 90 60 70 RA T:  M.19 17 .0 46 238 (0) 1.0 3 .1 6.0 6.0 70 90 70 *• DE HY D M.20 21 .0 49 290 (0) 0 . 6 2.1 6 .0 6.5 50 90 70 *• DE HY D M.21 22.0 54 264 (+6) 0.15 2.5 6.5 6.0 120 100 70 n M .22 23 .0 55 260 (+10) 0 . 5 3 . 8 6 .0 6.0 90 70 70 MY OG LO B M.23 16.0 43 250 (0) 0 . 1 2.4 6.0 6.0 140 80 70 MY OG LO B M.24 24 .0 54 284 (-2) drop 0 . 8 - 5.5 80 80 60 - 86 - Experiment 3: Experiment 3 considers the effect of 72 hours dehydra t i o n plus myoglobin i n j e c t i o n on the urine output, B.U.N, and kidney histology i n eight r a t s , with an additional four animals, subjected to dehydration alone, as controls. Animals were dehydrated for 24 hours before the i n j e c t i o n , which was followed by a further 48 hours without water. Dosage was again 0 .15 mg of myoglobin ( i n saline) 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 did not d i f f e r i n control and test groups; weight change was also e s s e n t i a l l y the same i n each group. Figures for urine volume were recorded for the 24 to 48 hour period following i n j e c t i o n — i . e . , for the l a s t 24 hours of dehydration, — and for the subsequent 24 hours i n which free 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. In one case (M.19) an osmotic diuresis may have been seen. Urine was noticeably more acid i n both groups here, being usually pH 6 . 0 , and at no time was i t observed to be discolored by the injected pigment. Figures for urea nitrogen show the effect of dehydration (24 and 48 hour f i g u r e s ) , with 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 series of figures also are s l i g h t l y higher i n the test animals (average - 87 - 80 mg range 70 to 120 mg % ) . No ess e n t i a l differences were seen i n the kidney histology of the two groups. There were no casts. Intravenous myoglobin therefore would appear to have no s p e c i f i c effect on urine output, urea nitrogen l e v e l or k i d  ney histology even i n the presence of dehydration. As i n Experiment 1, there was a diminution of urine volume almost to the point of anuria, a s l i g h t elevation of B. U. N. and a s l i g h t l y more acid urine. 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 addi t i o n of myoglobin. There was no pigmentation of the urine seen and no tubular casts even though urine was consistently acid. Experiment 4: The role of crush i n j u r y In the genesis of "lower nephron nephrosis" was investigated here by l i g a t i o n of the l e f t hind limb for f i v e hours. Eight rats were so tested, with another four animals acting as normal controls. Since i t was assumed that the test animals would not drink f r e e l y a f t e r the crush i n j u r y , attempts were made to match the water intake of the control animals to that of the test animals. Observations of water intake were therefore made at 24 hour Intervals. In order that the kidney histology might be viewed temporally, animals M.32 and M.35 were s a c r i f i c e d 24 hours after 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 af t e r crush injury,, at which time control animals were also k i l l e d . Observations are recorded i n Table 4. It can be seen from t h i s Table that animals suffering crush i n j u r y did not eat as much s o l i d food as did control ani mals i n spite of the fact that they drank more water. I t i s apparent that the matching of water intake was not accomplished. As a r e s u l t , the 'normal* controls were i n effect dehydrated to some extent, as evidenced by t h e i r consistent weight l o s s . Weight loss i n test animals was nevertheless greater. The most remarkable observations were, however, of the urine output. Test animals experienced a consistent, immediate and marked d i u r e s i s , the average output for the f i r s t 24 hours being 23 .1 cc. compared to control average of 3.4 cc. The polyuria 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 test ani mals putting out an average of 10.3 cc of urine i n that time, the control animals averaging 0.6 cc. This l a s t figure i s low because volumes were recorded with animals i n metabolism cages rather than i n funnels. The diuresis persisted 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 varied i r r e g u l a r l y between 6.0 and 7.5« Urea nitrogen figures exhibited some elevation above normal l i m i t s but cannot be said to be s i g n i f i c a n t l y elevated as measured here i n test animals. The occasional high value seen i n the control group can be accounted for by p a r t i a l dehydration. Also, some of the lower figures seen i n test animals at 48 and TABLE 4 LEFT HIND LIMB LIGATION IM INTACT RATS RAT TOTAL FOOD WATER INTAKE (cc) FINAL WEIGHT AND CHANGE URINE VOLUME URINE pH B.U.N, (mg. %) (GMS) 24 48 72 Tot. (GMS) 24 48 72 24 48 72 24 48 72 M.25 37 9 24 3 36 298 (-30) 4.7 4.6 4.6 7.0 7.0 6.5 70 110 80 [TR O M.26 36 13 21 14 48 320 (-28) 2.1 1.4 2.6 6.5 6.5 6.0 80 80 60 o o M.27 43 15 20 31 66 322 (-14) 3.0 4.7 4.8 7.0 6.5 7.0 90 70 70 M.28 35 7 12 24 43 302 (-24) 3.8 2.8 2.4 7.6 6.5 7.0 90 100 60 M.29 29 39 18 42 99 292 (-22) 28.6 7.5 6.6 6.5 7.5 7.0 80 60 50 no N M.30 3 20 11 - 38 300 (-40) 24.4 13.2 - 6.5 7.5 - 110 100 no N M.31 7 33 15 20 68 270 (-34) 12.2 12.8 6.4 6.5 7.5 7.5 100 70 70 o tH M.32 0 33 - - 39 - 25.2 - - 6.5 - - 90 - - M..33 17 48 14 34 98 324 (-48) 29.4 21.0 17.2 6.5 7.5 7.5 90 100 80 M H i M.34 0 36 32 - 68 314 (-22) 31.2 26.5 - 6.7 7.0 - 80 80 - M.35 0 28 - - 31 - 19.4 - - 6.0 - - 110 - - M.36: 13 22 6 37 65 296 (-48) 14.1 11.6 7.8 6.0 7.0 7.0 110 90 70 - 90 - 72 hours may he explained by the hemodilution following haemor rhage from self-amputated crushed limbs. This habit of the hydrated animals b i t i n g i t s injured limb gave some d i f f i c u l t y i n obtaining v a l i d observations. Post mortem findings were e s s e n t i a l l y negative - there was no gross evidence of kidney change - except for the injured limb, which was swollen, cold, 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 for s l i g h t granular changes i n the cytoplasm of tubules of Zone 3» with swelling and vacuolization of the nuclei (see F i g . 8 and compare with F i g . 9 ) . An occasional cast was seen i n Figure 8 Figure 9 medullary tubules (Figure 1 0 ) . Ligation of one hind limb for f i v e hours i n otherwise intact hydrated rats appears to have no permanent damaging effect on the kidneys. There was no s i g n i f i c a n t elevation of blood urea nitrogen and no remarkable renal tubular damage - 91 - Figure 10 h i s t o l o g i c a l l y . 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 effect i n response to the i n j u r y was observed-, indicating some a l t e r a t i o n i n "kidney function. Experiment 5: In Experiment 5A, eight male rats were subjected to the stress of l e f t hind limb l i g a t i o n f or f i v e hours together with 72 hours dehydration (24 hours pre-ligation,.48 hours post- l i g a t i o n ) . Four additional animals acted as controls, 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 half hours. Observations appear i n Tables 5A and 5B. I t i s apparent that there i s no essential difference between results 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 experiments /will be considered together. TABLE 5A FIVE HOURS LIGATION PLUS DEHYDRATION RAT TOTAL FOOD (GMS) TOTAL WATER (cc) 24 hrs FINAL WEIGHT AND CHANGE (GMS) URINE VOLUME URINE PH B. U. N. 24 48 72 24 48 72 24 48 72 E H o o M.37 11 33 338 (+2) 1.2 1.0 5.6 6.0 6.0 6.0 80 50 50 Q M.38 12 33 346 (+2) 0 . 8 0 . 6 4 .3 6.5 6 .5 6.0 100 60 50 DE HY D R A]  M.39 11 33 330 (-2) 2.5 0 . 7 5.4 6.5 6.0 6.0 100 70. 60 DE HY D R A]  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 RA TE D 1.42 0 - 244 (-26) 2.6 7.3 - 6.0 6.0 - 140 230 - RA TE D M.43 0 31 328 (-18) 1.5 8.7 15.4 6.0 6.0 7.0 190 180 160 AN D DE HY D M.44 0 31 329 (-15) 2.5 6.0 14 .4 6.0 6.0 7.5 160 130 110 AN D DE HY D 1.45 0 - 300 (-34) 2.2 - - 6.0 - - - - - AN D DE HY D M.46 0 0 270 (-34) 1.4 4 .3 6.0 6-»0 - 220 230 - IT ED  M.47 0 - 298 (-30) 1.2 . - - 5.5 - - 280 - - iL IG i M.481. 0 18 310 (-26) 3 . 4 8.0 15.1 6.0 6.0 6.5 250 - 130 TABLE 5B FIVE AND ONE-HALF HOURS LIGATION PLUS DEHYDRATION RAT TOTAL FOOD TOTAL WATER FINAL WEIGHT AND CHANGE URINE VOLUME URINE pH B. U. N. (GMS) (ec) 24 hrs (GMS) 24 48 72 24 48 72 24 48 .72 NT RO LS  M.49 9 29 254 (-30) 1 .6 0 . 2 2.6 6.0 6.0 6.0 80 110 70 o o M.50 9 29 298 (-32) 1.8 dried 1.8 6.0 - 5.5 100 100 70 DE HY DR : M.51 0 20 304 (-50) 1.8 0 . 8 4 . 0 6.0 6.0 6.5 80 100 80 DE HY DR : M.52 6 11 226 (-44) 0 . 6 dried 0 . 6 6.0 - 6.0 80 120 90 M.53 0 - 330 (-32) 0 - - - - - - - - RA TE D M.54 0 35 318 (-62) 2.6 4 .4 24 .5 6.0 6.0 7.5 170 220 180 RA TE D M.55 1 3 0 . 292 (-52) 2.8 4 . 0 8.0 6.0 6.0 6.5 140 110 90 Q M.56 1 31 240 (-46) 2.0 4 . 7 11.0 6.0 6.0 7.0 130 140 90 Q /—\ M.57 0 28 274 (-48) 2.8 1.0 10.0 6.0 5.5 7.5 230 220 250 (—1 < M.58 0 0 272 (-50) 2 .6 1.0 - 6.0 5.5 - 360 270 - .TE D M.59 0 - 310 (-36) 2.5 - - 6.0 - - 440 - - LIGA  M.60 0 0 270 (-52) 2.1 3 . 4 - 6.0 5-? - - 280 -- 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 ligated w i l l eat v i r t u a l l y no solids 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 available to a l l animals i n further experi ments to 10 grams each. S i m i l a r l y , i t was d i f f i c u l t to control water intakes; some of the ligated animals - those obviously i l l - would not drink water when i t was made ava i l a b l e , thereby hastening t h e i r deaths. In Experiment 5A the control group managed to r e t a i n i t s weight w e l l , while a l l other animals (including controls of Experiment 5B) showed a sat i s f a c t o r y weight loss (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 ampli fi e d at 72 hours by allowing water for the preceding 24 hours. Eight control animals at the 48 hour reading averaged 0 .6 cc of urine, while twelve test animals averaged 5.0 cc for that 24 hour period - almost ten times as much. pH figures showed a s l i g h t tendency towards a c i d i t y after 72 hours but no d e f i n i t e trend can be stated. With regard to B. U. N. figures, d e f i n i t e uremic lev e l s were reached for the f i r s t time. In three instances cardiac puncture was unsuccessful because the heart could not be located - 95 - by palpation or with the needle and i n a l l instances blood withdrawn was very thick and dark. In spite of the fact that the volume of urine excreted i n 24 hours by control animals was as l i t t l e as 0.2 cc, t h e i r B.U.N. levels did not r i s e above 120 mg %. On the other hand, test 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. levels consistently over 100 mg % and at times r i s i n g as high as 440 mg % during the dehydration period These facts would presumably necessitate a d i l u t e urine i n test animals, a condition which was v e r i f i e d by the appearance of very pale, watery urine i n test animals while that of controls was dark amber, almost syrupy. H i s t o l o g i c a l evidence of renal tubular damage was present d e f i n i t e l y for the f i r s t time. Sections were examined under low and high powers, d i v i d i n g the renal tissue into four zones for f a c i l i t y of examination. These were : Zone 1 - cortex proper, containing glomeruli and convoluted tubules, proximal and d i s t a l , with t h e i r appropriate vasculature. This f i r s t zone corresponds to Smith's d i v i s i o n "cortex". (See Fig.11). Zone 2 - corticomedullary region, i n which there i s the thick 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 portion containing sections of both thick and t h i n limbs of Henle's loop together with bundles of venae rectae. (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 recta. (Smith's "Medulla - inner zone"). Zones 1, 3 and 4 are more e a s i l y defined i n the rat kidney and received more attention i n the examinations. Zone 3, consisting exclusively of "lower nephron" received most atten t i o n and was found on examination to exhibit the c h a r a c t e r i s t i c pathologic damage seen throughout these experiments. Control 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 structure, a fact which would be expected from observations recorded i n Experiment 1. (See Figure 12). Figure 12 Of the 16 test animals, eight showed minimal to moderate changes i n renal tubules, to be described below; two others had questionable changes. Two animals died several hours before t h e i r kidneys could be f i x e d , thereby exhibiting post-mortem change; these sections could not be considered v a l i d . The remaining four animals showed no h i s t o l o g i c a l evidence of renal tubular damage i n spite of the fact that two had urea nitrogens of over 280 mg %, Typical changes appear i n Figure 13 , most marked i n Zones 2 and 3 , but extending into 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 thick portion of Henle's loop) are i n an early stage of degeneration with granular, vacuolated cytoplasm, swollen, pale and vacuolated nuclei and the occasional pyknotic nucleus. A desquamated e p i t h e l i a l c e l l can be seen i n one lumen. These changes accurred i n an animal putting out 3.4, 8.0 and 15.1 cc. of urine per 24 hours and whose B.U.N, rose to at least 250 mg %. Similar 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 furthest removed from the venae rectae, which appear at the upper and lower margins of the figure. This animal (M. 59) died at 24 hours with a postmortem value for B. U. N. of 440 mg % and a urine volume for 24 hours of 2.5 cc. Figure 13 Figure 14 From this 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 renal tubular damage i n a f a i r proportion (50$ of 16 rats) of otherwise intact male albino r a t s . This damage i s indicated by disordered function of the kidney (elevated blood urea nitrogen, increased volume of d i l u t e urine) and disordered structure as well (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 for renal damage, being i n fact the opposite of o l i g u r i a or anuria, but i t never theless indicates a disorder of renal function. - 99 - Experiment 6: The effect of crush in j u r y i n the presence of i n t r a  venously injected myoglobin i n normally hydrated animals was observed i n t h i s experiment. Water intake of four normal control animals was matched to that of a test group of four animals and a control group of four animals i n which the buffer solution alone, here used as a solvent for 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 analysis of observations. In spite of the fact that animals were allowed free s o l i d s , the normal control group did not eat f r e e l y . The amounts consumed, however, compared favourably with the remain ing animals. Water intakes and weight changes also 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 ligated animals, no s i g n i f i c a n t difference between the myoglobin-injected and the buffer-injected groups being evident. Two control animals did, however, show a pronounced diu 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 high figure i n one con t r o l animal i s unexplained and must be presumed to be an error. Urea nitrogen figures may be low owing to hemodilution which was observed i n many animals during cardiac puncture at 24 hours. This was thought i n early experiments to be the result of TABLE 6 LIGATION PLUS MYOGLOBIN INJECTION IN INTACT RATS RAT TOTAL TOTAL FINAL WEIGHT URINE VOLUME URINE pH B.U.N. FOOD WATER AND CHANGE (GMS) (cc.) (GMS) . 24 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 «ajpc M.62 28 51 220 (-6) 4.6 5.4 20.5 7.0 6.0 6.0 100 70 70 o c 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 6.0 80 80 O £ M.66 4 18 224 (-24) 6 . 0 8.0 5.4 7.0 7.0 120 O C M |Z M.6? 13 75 238 (-2) 23 .4 10 .5 9.7 6.0 7.0 6.0 110 90 100 ' i-l < M.68 12 37 228 (-14) 11.2 8.8 4 .2 6.5 7.5 6 .0 120 90 80 2 & M.69 23 62 215 (-5) 10.2 12.0 4 . 8 6.0 7.5 6 .0 120 80 80 ^ < t- O C M.70 19 58 2-50 (-2) 13.6 6.4 4.2 6.0 6.5 7.0 120 80 80 M l - < c M.71 - - - - - - - - - - - - - H S M.72 21 55 236 (-14) 14 .4 8.5 8.7 6.0 7.0 7.5 120 90 80 TABLE 7 DEHYDRATION, LIGATION AND MYOGLOBIN IN INTACT RATS RAT TOTAL FOOD (GMS) TOTAL WATER' (cc) FINAL WEIGHT AND CHANGE (GMS) URINE VOLUME 24 48 72 URINE pH 24 48 72 B. U. N. (mg. % ) - 24 48 72 Q W EH HH •=xi o Q E H tH fe W O Ex] O o M.73 M.74 M.75 M.76 10 10 10 10 23 27 21 39 185 200 180 214 -29) -30) -16) -26) 2.4 0 . 6 1.2 1.9 1-5 2 .4 1.1 0 . 6 1.5 2.3 1.6 10.1 6.0 6.0 6.0 80 110 60 6.0 6.0 5 .5 90 100 70 6.0 6.0 6.0 60 90 70 6.0 6.0 6.0 80 140 70 fe fe o o M M E H E H rf <t\ « o Q . M > H vA W + Q M.77 M.78 M.79 M.80 10 9 7.5 5.0 —g + HH c5 a o o a * 35 43 44 33 220 224 216 196 - 3 0 ) -20) -42) -40) 1.6 3.4 6.4 3 . 2 7.2 10.4 2.4 4 .0 8.6 2.0 5 .0 9 .0 6.0 6.0 7.0 - 110 90 6.0 7.0 6.0 170 150 80 6.0 6.0 7.5 160 120 80 6.0 6.5 7-5 130 130 90 CxJ M M.81 M.82 M.83 M.84 1.0 0 7.0 2.0 41 34 59 25 224 208 224 218 -44) -42) -28) -46) 2.9 5.0 14 .0 2.3 7.4 15.8 4 .9 9 .8 15.1 3 . 0 7.8 9 .2 6.0 6.0 7.0 29O 150 100 6.0 7.0 7.5 160 220 160 6 .0 6.0 7.0 - 200 110 6.0 6.0 7.0 220 170 120 - 102 - haemorrhage from chewed limbs, but these animals had not attacked t h e i r limbs. The response appears to be one of hemodilution to the shock of limb l i g a t i o n , when hydration i s adequate. Constantinides ( 8 l a ) states that i n the production of severe shock i n rats by pinching exposed intestines i n several places, marked and rapidly 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 Inj 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 carried out on normally hydrated animals, again no l e t h a l renal damage resulted, although a d i u r e t i c effect was noticed. In the present experiment these two factors together (myoglobin and crush injury) also produced no l e t h a l kidney damage but again resulted i n a marked and d e f i n i t e d i u r e s i s . In addition, an apparent hemodilution response,to the trauma was observed. Experiment 7: The three factors, 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 with four additional rats subjected to dehydration and l i g a t i o n alone. The control group of four animals was dehydrated 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 plished as i n Experiments? with a phosphate buffer as the s o l  vent. The dehydrated-ligated group received no i n j e c t i o n of buffer. Observations are i n Table 7« Intakes of solids 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 li g a t e d ani mals, however, did lose more weight with one animal l o s i n g 17$ of i t s o r i g i n a l body weight. Urine volume figures 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 difference between the two li g a t e d groups. Urea nitrogen figures i n ligated animals were raised to uremic levels 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 elevation of the myoglobin- injeeted group over the non-injected one, i t i s necessary because of the wide range of figures and the small 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 figures i n order to reach accurate conclusions. When thi s i s done (see Table 7A) i t i s found that,, at the 24 hour reading the difference 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 9% of cases t h i s difference would not occur by chance. The normal histology of the kidney of animal M. 76 i s shown i n Figure 15, contrasting w e l l with 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 renal 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 simi 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 addition, d i s t a l tubules i n the c o r t i c a l region of th i s kidney also were degenerating (Figure 1 8 ) . Figure 17 Figure 18 Results of th i s experiment ( l i g a t i o n + dehydration + myoglobin) should be compared to those of Experiments 5 ( l i g a t i o n + dehydration) and 6 ( l i g a t i o n + myglobin) as well as with the control animals. There were no deaths and there was very l i t t l e - 105 - elevation of B.U.N, with l i g a t i o n and myoglobin as the stress; with l i g a t i o n and dehydration, uremic l e v e l s were reached i n twelve of sixteen test animals and there were no renal deaths. I t would appear, then, that dehydration i s an ess e n t i a l factor i n the development of acute tubular necrosis from crush i n j u r y and that i n j e c t i o n of myoglobin though not essential for the development of the syndrome, adds s i g n i f i c a n t l y to the tubular damage as indicated by urea nitrogen le v e l s and kidney histology. Experiment 8: The effect of increasing the area of crush was i n v e s t i  gated i n four rats subjected to b i l a t e r a l hind limb l i g a t i o n for four hours af t e r being dehydrated for 24 hours previously. Dehydration was continued f or 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 or f i v e hours. Table 8 c l a s s i f i e s the pertinent 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 intake and to a lesser 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 for urine volumes, though incomplete because of deaths, again show polyuria at 24 hours which i s amplified by the water intake at 72 hours. In addition i t was noted that i n two dehydrated control animals wine-coloured urine was excreted though there was no evidence of external bleeding. - 106 - This was apparently a true hematuria. Urine pH figures again show the general trend towards a l k a l i n i t y with d i u r e s i s . Urea nitrogen figures were among the highest yet recorded. Dehydrated control figures 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 elevation was due to dehydration alone i s shown by the prompt return 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 control group, B. U. N. levels were generally higher and persisted at an elevated l e v e l . for a longer time. Only one animal reached an excessively high l e v e l and that animal died apparently of renal damage. In the b i l a t e r a l l y ligated dehydrated test group, the results 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 with elevated B.U.N's and periods of anuria of from s i x to eight hours. The fourth animal of the group survived but maintained an eleva ted 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 controls i n spite of the hematuria observed i n two of the four animals (Figure 1 9 ) . Typical degenerative changes were seen i n Zone 3 of animals M.89, M.90, M .92, M.94, TABLE 7A STATISTICAL ANALYSIS OF FIGURES IN TABLE 7 DEHYDR. + LIG'N "X" DEHYDR. LIG'N, + MYOGLOBIN II Y» DIFF. FROM MEAN »x". DIFF. FROM MEAN l.y.t x 2 170 290 +17 +67 289 4489 &x. = 17 <*M* =12.06 /UH = 39.48 24  HO UR S 160 130 1=153 160 220 M=223 +7 -23 -63 -3 49 529 £x=867 3969 9 £ ^ = 8467 if = 53.12 6My =37.60 ^ = 70 t - 70/39.48 = 1.77 For s i g n i f i c a n c e at 5% l e v e l , need 2.78 110 150 -18 -35 324 1225 6K - 1 4 . 7 6 ^ =8.53 = 17.74 « o K co 150 120 130 1=128 220 200 170 M=l85 +.22 -8 +2 +35 +15 -15 484 64 4 =R7P 1225 225 225 2900 =26.92 <K =15.56 -PH = 57 = 3 .21 For s i g n i f i c a n c e at 5%, need 2 . 4 5 ; a t 3 . 7 1 . 90 100 +5 -22 25 484 cfx. = 5 <^ x = 2 . 8 9 ^ =13.47 CQ 80 160 - 5 +38 25 1 4 4 4 *y = 22.76 <K= 13.16 i)M = 37 O « CM v . 80 90 1=85 110 120 1=122 -5 +5 , -12 - 2 25 25 ^ =100 . 1 4 4 4 2072 t = 2.75 For s i g n i f i c a n c e at 5^» need 2 . 4 5 ; at 1%, 3 . 7 1 . I TABLE 8 DEHYDRATION AND BILATERAL LIGATION IN INTACT RATS RAT TOTAL FOOD TOTAL WATER FINAL WEIGHT AND CHANGE URINE ec. VOL. URINE pH B. U. mg % N. DIED (HRS) (GMS) (cc) (GMS) 24 48 72 24 48 72 24 48 72 J25 OCO Mi_3 M.85 10 36 222 (-38) 2.2 1.8 3.3 6.0 6.0 6.0 110 110 70 E-tO ©a M.86 M.87 10 10 34 24 244 234 (-28) (-34) 0.5 1.6 0.3 1.0 2.8 3 .4 7-0 6.0 6.0 6.0 6.5 7.0 90 100 110 100 70 90 0: M.88 10 28 216 (-28) 1.3 0.3 6.0 6.0 6.5 130 180 80 O M 1-W EH M.89 M.90 10 6.5 39 36 252 230 (-32) (-30) 4.1 3.6 5.4 7.2 8.2 11.3 6.0 6.0 7.0 7.0 7.0 7.5 140 190 120 140 110 90 M.91 0 - - 2.1 0 6.0 - - 410 - - 36-48 M.92 0 15 190 (-46) 1.5 8.1 9.0 6.0 6.5 7.0 210 150 160 + e H O EH M.93 M.94- 0 0 3.5 232 224 (-28) (-32) 2.3 2.8 0 5.1 13-3 6.0 6.0 6.5 7.5 210 190 220 220 36-48 DE HY DR A M.95 M.96 0 0 220 222 (-30) (-28) 0.7 1-9 : - 6.0 6.0 : : 290 260 - - 20 24-26 - 1 0 9 - M .95 and M .96 . (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 addition, the medulla of animal M .95 showed numerous hyaline casts i n Henle's loop; no hematuria was noted i n t h i s animal Figure 21 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 injury coupled with the essential dehydration i s capable of producing a more f a t a l renal 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 point of view -- i . e . , reduce the renal reserve of the animals. Subsequent experiments accomplish th 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 fact that dehydrated control animals r e t a i n water, when i t i s supplied, much better than do those animals whose limbs had been l i g a t e d . Experiment 9t The effect of reducing renal reserve i s studied i n Experiments 9A and 9B, which deal with male, right-nephrect- omied rats subjected to 72 hours dehydration and fi v e hours l i g a t i o n . In 9A, twelve animals were nephrectomied one week pr 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 ligated as well as dehydrated. In 9B, nephrectomy was done three days p r i o r to dehydration, but the control group of four and test 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). - I l l - In Experiment 9A, a l l animals appeared f u l l y recovered following nephrectomy, having regained or exceeded t h e i r pre operative weights. However, a l l were noticeably more sensitive to nembutal sedation. Food, water and weight change figures were not unusual. 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 figures the tendency towards a l k a l i n i t y with d i u r e s i s . A l l figures were higher than comparable figures for dehydrated intact animals. Two dehydrated control animals showed excessively elevated B.U.N, levels and. at autopsy one of these had a granu l a r kidney which showed a chronic i n t e r s t i t i a l inflammation h i s t o l o g i c a l l y . The kidney of the other animal was not examined. Of the remaining eight test animals, s i x reached and held high B.U.N, l e v e l s , two of these as high as 470 mg % and 530 mg %. The remaining two behaved i n a way much l i k e the dehydrated controls. Animals M.105 and M.107 died at 48 and 72 hours respec t i v e l y , as a result of hemopericardium following heart puncture. Animal 1.106 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 for the entire day, was anuric and suffering severe r i g o r s . Animal M.104 was carried 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 with tarry material l i k e old blood. This TABLE 9A LIGATION AND DEHYDRATION IN RIGHT NEPHRECTOMIED RATS RAT FOOD (GMS) WATER (cc) FINAL WEIGHT AND CHANGE URINE VOLUME cc. URINE pH B. U. N. DIED (GMS) 24 4 8 72 24 48 72 24 4 8 72 , (HRS) HH O Q « M .97 M.98 5 5 30 2 7 184 220 ( - 2 6 ) ( - 2 2 ) 2 . 2 1.5 1 . 7 1 . 2 8 . 8 6 . 0 6 . 0 6 . 0 6 . 0 6 . 0 7 . 0 7 . 0 240 110 170 100 1 0 0 90 a M.99 5 30 226 (-14) 1 . 5 0 . 6 7 . 6 6 . 0 6 . 0 7 . 5 180 90 110 DE H'  M .100 5 3 1 2 1 0 ( - 2 0 ) 1 . 2 0 . 8 9 . 8 6 . 0 5 . 5 7 . 0 1 2 0 90 80 TI ON  M .101 5 3 1 2 2 6 (-14) 1 . 8 4 . 3 1 7 . 0 6 . 5 6 . 5 7 . 5 . 260 130 1 0 0 TI ON  M .102 5 3 6 2 2 6 ( - 1 6 ) 2 . 0 1 . 9 9 . 1 6 . 5 7 . 0 7 . 5 230 130 90 ,I GA  M#103 0 4 6 1 9 6 ( - 2 8 ) 1 . 3 2 . 7 2 9 . 3 7 . 0 7 . 0 7 . 5 410 230 140 o M.104 0 9 1 9 2 ( - 2 6 ) 1 . 4 1 . 2 5 . 1 6 . 5 7 . 5 7 . 5 200 240 3 0 0 M.105 0 - 1 8 0 ( - 4 4 ) 1 . 8 7 . 0 - 6 . 0 7 . 0 - 410 230 4 8 IT TO  M .106 0 3 2 1 2 ( - 2 8 ) 1 . 0 0 . 6 - 6 . 0 6 . 5 - 270 220 3 1 0 53 cc; s M .107 0 41 2 2 2 ( - 3 4 ) 2 . 2 7 . 4 2 0 . 8 6 . 0 7 . 0 7 . 5 530 220 1 6 0 72 1 D EH ' M .108 0 23 2 2 0 (-40) 1 . 2 0 . 8 1 6 . 5 6 . 5 6 . 0 7 . 5 220 230 250 TABLE 9B LIGATION AND DEHYDRATION IN RIGHT NEPHRECTOMIED RATS i RAT FOOD WATER (cc) FINAL WEIGHT A T J n HTT A MCI r?. URINE VOL. URINE pH B.U.N. DIED (HR) 'KIDNEY WEIGHT mg per cm.2 24 36 24 36 24 36 ) RA TED  JTR OL M.109 4 - 200 (-34) 1.1 dried 7.0 - 100 80 271.5 - ) RA TED  JTR OL M.110 5 - 238 (-36) 1.9 0.2 6.0 6.0 100 80 269.1 H 0 W O (xl M . l l l 4 - 254 (-40) 1 .6 dried 6.0 - - 140 280.0 Q M.112 4 - 230 (-42) 1.7 dried 7.5 - 110 90 258.9 M.113 0 - 258 (-24) 1.7 0 6.0 - 190 290 32-36 254.9 a O 1 1 M.114 0 - 256 (-32) 2. 2 0 6.0 - 400 - 26 * 276.5 GA T] M.115 0 - 248 (-24) 1.7 - 6.0 - 600 - 304.7 i—t M.116 0 - 242 (-18) 2.9 0.7 6.0 6.0 210 310 293.6 a M.117 0 - 248 (-40) 2.6 1.4 6.0 6 .5 230 270 261.9 a EH M.118 0 - 248 (-38) 2.2 2.3 6.0 6.5 220 200 275.4 :DR A M.119 0 - 264 (-38) 3.1 2.3 6.0 6.0 210 220 - w M.120 0 - 258 (-34) 2 .8 1.0 6.0 6.0 220 260 248.4 - 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 reactions to stress, that of g a s t r o - i n t e s t i n a l haemorrhage. This animal was grossly anemic at post-mortem. In addition, true hematuria was again observed, t h i s time i n a test animal (M.108). Of eight test animals, then, s i x had elevated B.U.N's which were maintained. Six were ac t u a l l y polyuric while one was o l i g u r i c and one anuric for 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); one showed questionable changes; the remaining four did not show histolog i c a l evidence of renal damage when k i l l e d at 72 to 144 hours. Figure 23 Figure 24 An interesting series of observations was made on animal M. 104. This dehydrated ligated animal exhibited o l i g u r i a , progressive uremia and gas t r o - i n t e s t i n a l but not - 115 - bladder haemorrhage. Figures 25 , 2 6 , 27, 28, and 29 show dilated tubules and ischemic glomeruli of the cortex and Zones 3 and 4 (medulla) loaded with hy a l i n , granular and yellowish (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 with 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 of Experiment 9B were e s s e n t i a l l y the same as i n Experiment 9A. One control animal reached a B.U.N, l e v e l of 140 mg % while a l l test animals were over 190 mg. %. Two of eight test animals died 24 to 36 hours following l i g a t i o n , one being anuric for about 20 hours p r i o r to death. AH remain ing animals retained ( i n most cases at higher le 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 ation of 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 eighth i l l u s t r a t e d postmortem changes only. A l l these f i v e had elevated B.U.N.'s and two died i n uremia. Six animals were polyuric and only one was anuric p r i o r to death. Kidney weights at death were recorded for 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 difference between the two groups. Controls averaged 269.8 mg. 2 per cm (range 258.9 to 290.0); test animals averaged 273.6 mg. per cm2 (range 254.9 to 304.7). Comparing these results to those of Experiments 5A and 5B make i t apparent that rats with reduced renal reserve are more susceptible to crush i n j u r y with dehydration as indicated by urea nitrogen l e v e l s , tubular damage and mortality rate. These results compare favorably with those of Experiment 8, i n which the area of crush was increased i n int a c t animals. - 117 - Experiment 10: In an attempt to reduce the number of tests used and to observe the natural course of the syndrome, t h i s experiment was concerned c h i e f l y with the mortality i n right nephrectomized rats subjected to 72 hours dehydration and f i v e hours limb l i g a  t i o n . Because cardiac punctures would i n t e r f e r e with spontan eous deaths, only postmortem B.U.N's were done i n surviving 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 for the remarkable weight loss seen, e s p e c i a l l y i n dehydrated controls, i n which i t i s commonly 30% of body weight. The d i u r e t i c response appears again. B.U.N.'s were not obtained i n the f i r s t two animals nor the three test animals dying spontaneously. Figures for test animals surviving were not elevated. Kidney weights average 319.4 mg. per cm.2 for controls (range 289-5 to 368.8) and 332.7 mg. per cm.2 for test animals (range 307.4 to 348.4) and though 2 t h i s i s a difference 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 control 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 test animals which died, and appeared absent i n the remaining animals, includ 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 also found dead at 24 hours and judging by lack of r i g o r mortis had died 16 to 17 hours following l i g a t i o n removal; M.144 died more than 48 hours after the i n i t i a l stress. These three were f e l t to be " v a l i d " deaths - i . e . from renal f a i l u r e and not from shock - and t h i s mortality rate was taken as a standard for subsequent experi 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 albino rats of the Wistar s t r a i n produced death i n renal f a i l u r e i n three of eight test animals (37%) > Disturbance of renal function as indicated by polyuria and hyposthenuria was seen i n eight of eight (100$) test animals. Experiment 11: Female rats were used at this point i n order to deter mine any sex difference i n the response to the various therapeu t i c agents. In Experiment 11A, twelve uninephrectomied animals were used, s i x as controls and the remaining s i x (alternate animals) were treated with testosterone 5mg. i n o i l and 5 mg. i n saline subcutaneously at the time of l i g a t i o n . A l l animals were subjected to 124 hours p r e - l i g a t i o n dehydration followed by f i v e hours l e f t hind limb l i g a t i o n and 48 hours p o s t - l i g a t i o n dehydration. Again, mortality was the main,factor observed i n order to determine the effect of testosterone i n protecting - 119 - against acute renal f a i l u r e . Observations are recorded i n Table 11A. ' Because i t was f e l t that the testosterone may not have had s u f f i c i e n t time i n which to act, Experiment 11A was repeated (Experiment 11B) giving 5 mg. of testosterone i n o i l 48 and 24 hours p r i o r to l i g a t i o n . The procedure followed was otherwise the same and again mortality was the chief factor observed (Table 11B). In Experiment 11A, figures for urine output and B.U.N, were comparable to previous r e s u l t s . Kidneys of animals receiving testosterone are not s i g n i f i c a n t l y heavier (at the 5$ l e v e l ) than those of control animals (controls averaged 251.2 mg. per cm.2 with a range of 222.8 to 259.95 test animals averaged 266.9 rmg.% cm.2, range 238.8 to 3 0 2 . 5 ) . Five animals i n the control group of s i x , and four i n the, test group of f i v e , died. This increased mortality plagued a l l subsequent therapy experi ments whether i n male or female animals and the problem w i l l be dealt with i n the discussion to follow. 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 stress i s not e f f e c t i v e i n protecting female rats against traumatic uremia and death i n acute renal f a i l u r e . H i s t o l o g i c a l examination of these kidneys revealed, i n test animals, three with t y p i c a l lower nephron degeneration, one with questionable changes, and the f i f t h animal i l l u s t r a t e d post TABLE 10 MORTALITY IN DEHYDRATED, LIGATED. UNINEPHRECTOMIED RATS RAT •"OOD :GM) WATER (ce) WEIGHT AND CHANGE (GM) URINE VOLUME (cc) URINE pH B.U.N. at DIED KIDNEY WEIGHTp. (mg/cm 24 48 72 96 24 48 72 96 96 HRS P M.133 5 16 128 (-66) 1 .6 0 . 5 7.4 4 .8 6.0 6.0 7.0 7.0 - 313.4 E H C K E - Q is S C jij c M.134 M.135 5 5 11 14 126 114 (-64) (-50) 0 .8 1.2 0 . 5 1.0 5.3 6.8 4 .3 2.0 6.5 6.5 6.0 6.0 6.0 6.5 6.5 7.0 100 306.0 368.8 p 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 1T I0  M.138 5 42 130 (-66) 2.0 8 .8 18 .7 7.2 6.0 7.0 7.5 7.5 90 348.4 LI GJ  M.139 0 — 138 (-26) 0 .7 ; 12-24 337.8 + 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 AT K 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 DE HY DR  M.143 0 - 160 (-32) 0.4 - - - .; 7.0 - - - 12-24 345 .5 ' DE HY DR  M.144 0 3 140 (-24) 1.1 1.0 - - L 6.0 6.0 - - - ; 34-36 -TABLE 11A TESTOSTERONE IN CRUSH SYNDROME RAT FOOD (GMS) WATER (cc) WEIGHT AND CHANGE (GMS) URINE VOLUME (cc) 24 48 72 URINE pH 24 48 72 B.U.N, at DEATH DIED (HRS) KIDNEY WEIGHT 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 7.0 170 254.3 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 - - - 56-72 253.3 T M.153 3 50 198 (-24) 1.1 0.6 26.0 6.0 6.5 7.5 90 - 238.8 c M.154 0 0 200 (-26) 0.7 - 6.0 - - 300 20 258.5 T M.155 0 0 152 (-21) drop - - - - 300 17 257.0 C M.156 0 0 188 (-22) 1.0 - 5.5 - - - 26 222.8 - 123 - mortem changes. A feature of the pathology previously men tioned i s here very evident. The fact that the degenerative and pyknotic changes i n Zone 3 tubules are farthest removed from congested venae rectae i s seen i n Figure 30 (low power) and Figure 31 (high power). ... n»t " ' c * • * • - K> If 1 • » « » • * -* V ' / • / V m ' " * *• Figure 30 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 f a t a l  i t y rate i s i l l u s t r a t e d . Eight of the animals were "found dead" 24 hours after l i g a t i o n was applied and so the problem of shock death rather than uremic death i s raised. Four ani mals (two test and two control) died a f t e r t h i s 24 hour period and are taken to be certain uremic deaths; two of these (the test animals) had raised urea nitrogen figures. 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 necrosis (see Figure 32) which were also apparent i n the d i s t a l tubules of the cortex. These kidneys also exhibited the frequently - 124 - observed marked congestion of the medulla (Figure 33). Kidney- weights i n mg. per cm.2 at death again show no predictable plan, Figure 32 Figure 33 average figures being 271.6 mg. per cm.2 for test animals (range 260.5 to 280.0) and 277.2 mg. per cm.2 for controls (range 258.0 to 310.8). In considering results of Experiments 11A and 11B i t must be concluded that testosterone proprionate given i n ade quate doses to l i g a t e d , dehydrated, uninephrectomied female rats does not reduce t h e i r mortality rate. Whether or not the hormone has some p a l l i a t i v e effect as measured by decreased st r u c t u r a l damage or lower urea nitrogen levels i n test 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 pr i o r to l i g a t i o n , as well as at time of l i g a t i o n , i n alternate animals. Table 12 presents the observations. TABLE 11B TESTOSTERONE IN CRUSHED FEMALE RATS RAT FOOD (GMS) WATER (cc) WEIGHT AND CHANGE (GMS) URINE VOLUME (ce) URINE pH B.U.N. at DIED (HRS) KIDNEY WEIGHT 24 4b 72 24 48 72 DEATH Mg/cm2 T M.157 0 0 196 (-18) 0.3 - 7 .0 - - - 24 269.8 C M.158 0 0 190 ( - 2 6 ) ' 0 . 8 - 7.0 - - 24 310.8 T M.159 0 0 202 (-18) 0.2 - - - - - 24 269.4 C M.160 0 0 176 (-24) 0.4 0 - - - - 32-48 286.9 T M . l 6 l 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 (-20) 0.2 - _• 24 258.2 T M.167 0 0 190 (-20) 0.4 - 6.0 24 264.9 C M.168 0 0 170 (-22) 1.2 - - 6 .0 24 267.4 TABLE 12 TESTOSTERONE IN CRUSHED MALE RATS RAT FOOD AND WEIGHT AND CHANGE URINE VOLUME (cc) URINE pH B.U.N. at DIED KIDNEY WEIGHT WATER 24 48 72 24 48 72 DEATH mg/cm2 T C T C M.253 M.254 M.255 M.256 0 0 0 208 (-14) 206 (-20) 192 (-16) 0 - 0.1 - 0 - - - - - 24 24 24 266.4 214.8 203.7 5E C T C M.25.7 M.258 M.259 M.260 0 0 0 0 186 (-16) 200 (-24) 194 (-16) 202 (-20) 0 - - 0.5 - 0.3 - 0.3 - - 24 24- 24 24 230.1 250.6 226.0 230.5 T C T 0 M.261 M.262 M.263 M.264 0 5 gm 60cc 0 210 (-18) 188 (-24) 196 (-22) 0.4 - 0.3 3.4 30.0 0.2 - 5.0 6.0 310 24 24 249.3 279.8 201.8 - 127 - A l l animals appeared; markedly shocked when ligatures were removed after f i v e hours and nine of ten were dead when seen the following morning (24 hours a f t e r ligatures were applied). Two other animals died during the period of l i g a t i o n . These re 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 frequently. Kidney weights averaged 235.5 mg. per cm.2 (range 201.8 to 279.8) for control and 235.1 mg. per cm.2 (range 203.7 to 266.4) for treated animals. One untreated control animal ran a 72 hour course show ing a t y p i c a l c l i n i c a l picture of acute renal 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 , of 310 mg. %. H i s t o l o g i c a l l y , that kidney showed changes taken to be regeneration following tubular damage: areas of flattened, basophilic tubular c e l l s associated with c e l l u l a r debris i n the lumens (see section on "Discussion"). Testosterone /propionate therefore would appear to be in e f f e c t i v e i n maintaining or prolonging l i f e i n male rats exper iencing shock and acute renal f a i l u r e . Experiment 13t The effect of cortisone acetate on mortality i n uni-r nephrectomied, dehydrated and ligated female rats i s considered i n t h i s experiment. Two mgs. of cortisone i n saline were injected 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, alternate - 128 - ones being treated with the test drug. Observations are i n Table 13 . Mortality rate was again high, but most animals l i v e d beyond the 24 hour period. A l l treated animals died i n the 24 to 28 hour period, while two control animals died i n the 32 to 48 hour period. Of eight animals i n which B.U.N, levels were determined at death, a l l were elevated to uremic levels and a l l showed h i s t o l o g i c a l changes of acute tubular necrosis. Representative areas of Zone 3 are i l l u s t r a t e d i n Figures 34 (control) and 35 ( t e s t ) . Figure 34 Figure 35 These animals were also anuric for 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. - range 240.4 to 272.2) to untreated (av. 246.4 mg. per cm.2 - range 235.5 to 2 6 0 . 5 ) . Cortisone acetate would appear to be i n e f f e c t i v e i n - 129 - reducing the mortality from or severity of acute tubular necrosis i n female rats subjected to uninephrectomy, dehydration and crush i n j u r y . Experiment 14: The above experiment with cortisone i s here repeated using, instead, 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 exper iment. Observations are i n Table 14. A l l twelve animals ( s i x treated, s i x untreated) were dead when observed 24 hours after ligatures were applied, so that no observations were made, other than weight loss and 24 hour urine volume. No explanation for t h i s exaggerated increase i n early mortality was apparent, but the problem i s considered i n the section on "Discussion 1 1. Kidney weights show an isolated example of significance 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 effect i n pro tecting male rats from death from shock and/or renal f a i l u r e . I t becomes apparent that the standardized production of acute TABLE 13 CORTISONE IN CRUSHED FEMALE RATS RAT FOOD (GMS) WATER (cc) WEIGHT AND CHANGE (GMS) URINE VOLUME (cc) URINE pH B.U.N. at DEATH mg. % DIED (HRS) KIDNEY WEIGHT Mg/cm2 24 -48 72 24 48 72 T M.169 0 0 180 (-20) 1.4 - - 5.5 - - 190 25 243.7 C M.170 0 ' 0 180 (-20) 1.0 - - 400 26 260.5 T M.171 0 0 180 (-20) 0.5 - 5.5 - - 230 27 240.9 C M.172 0 0 170 (-30) 1.2 0 6.0 - 270 48 235.5 T M.173 0 0 178 (-18) 0.4 0 - 370 27 251.4 C M.174 0 0 184 (-24) 0.6 - - - 12-24 248.6 T M.175 0 0 174 (-28) 1.3 0 6.0 - 290 27 272.2 C M.176 0 0 182 (-30) 0.7 0 6.0 - 32-48 - T M.177 0 0 184 ( -20 ) 0.6 : 0 5.5 - - 500 28' 251.4 C M.178 0 0 210 (-22) 0.6 0 - 5.5 - - - 30 236.8 T M.179 0 0 194 (-34) 0.6 - 6.0 - - - 12-24 244.7 C M.I80 0 0 212 (-28) 1.1 0 6.0 400 27 250.6 TABLE 14 CORTISONE IN CRUSHED MALE RATS RAT FOOD (GMS) WATER (cc) WEIGHT AND CHANGE (GMS) URINE VOLUME (cc) URINE pH B.U.N. at DIED KIDNEY WEIGHT 24 48 72 24 48 72 DEATH (HRS) Mg/cm2 T M.241 0 0 190 (-20) 0.3 - - _ _ _ _ 12-24 248.6 C M.242 0 0 192 (-22) 0.3 - - - - 12-24 209.1 T M.243 0 0 198 (-16) 0 .9 - - - - 12-24 220.4 C M.244 0 0 232 (-16) 0.5 - - - - 12-24 231.1 T M.245 0 0 180 (-16) 0 .9 - " - 12-24 238.0 C M.246 0 0 190 (-16) 0.1 _ - 12-24 191.8 T M.247 0 0 204 (-22) 0.4 - - 12-24 260.3 C M.248 0 0 184 (-14) 0.4 - - 12-24 196.1 T M.249 0 0 • 228 (-22) 0.6 - - 12-24 252.9 C M.250 0 0 190 (-18) 0.2 - -• 12-24 221.6 T M.25I 0 0 224 (-18) • • 0 . 5 - - - - 12-24 241.5 C M.252 0 0 214 (-18) 0.3 - - - 12-24 236.3 - 132 - tubular necrosis and renal f a i l u r e by these methods i s much less possible than was indicated i n e a r l i e r experiments. Experiment 15? A combination of testosterone, with i t s renotropic (333) and/or protein-sparing actions, and cortisone, with i t s co-called life-maintaining f a c t o r , might conceivably be e f f e c t  ive i n cases of acute tubular necrosis, where either one of these agents alone would f a i l . These hormones were therefore used together i n t h i s experiment, with dosages arranged as i n Experiments 11B and 13. Twelve female animals were again used with alternate animals receiving the test drugs. Table 15 presents the observations. These animals were smaller than usual, being 165 to 180 gm. i n weight, which fact may account i n part f or the increased mortality at 24 hours. Only four animals l i v e d beyond the 24 hour period; three of these were untreated control animals and one had been treated with 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 necrosis. Kidney weight figures averaged 284.5 mg. per 2 2 cm. (range 261.0 to 322.9) for test animals, 277.4 mg. per cm (range 251.7 to 287.5) for controls, 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 for an TABLE 15 TESTOSTERONE PLUS CORTISONE IN CRUSHED FEMALE RATS RAT FOOD (GM) WATER (cc) WEIGHT AND CHANGE (GM) URINE VOLUME URINE pH B.U.N. at DEATH Me. t DIED KIDNEY WEIGHT Mg/cm.2 24 48 72 24 48 72 T M.181 0 0 168 (-12) 0.5 - - _ - 12-24 261.0 C M.182 0 0 148 (-20) 0 . 3 - - - - - - 12-24 287.5 T M.183 0 0 152 (-18) 0 . 6 - - - - 12-24 282.1 C M.184 0 0 162 (-20) 0 . 9 - - 6.0 - 410 25 273.3 T M.185 0 0 156 (-18) 1.1 - - 6.0 - - 12-24 265.4 C M.186 0 0 146 (-20) 0 . 6 6 .5 - r - 12-24 283.9 T M.187 0 0 152 (-16) 0 . 6 5.5 - - 12-24 322.9 C M.188 0 0 140 (-14) 0 . 7 - 5.5 " 230 25 251.7 T M.I89 0 0 146 (-20) 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 - - 12-24 278.8 C M.192 0 0 154 (-18) 0 . 6 6.0 - - 12-24 282.6 TABLE.16 TESTOSTERONE AND CORTISONE IN CRUSHED MALE RATS RAT FOOD (GM) WATER (cc) WEIGHT AND CHANGE (GM) URINE VOLUME (cc.) URINE pH B.U.N. at DIED KIDNEY WEIGHT^ Mg/cm. 24 48 72 24 48 72 DEATH Mg.$ (hrs) T M.193 0 0 200 (-28) 0.15 - — — — — 12-24 281.9 C M.194 '•- T M.195 0 11 220 (-34) 0.95 - ^ 5 - - 36-48 261.6 C M.196 0 0 212 (-40) 0.3 - - - - - 12-24 - T M.197 0 0 224 (-34) 0.4 - - 12-24 285.0 C M.198 0 0 210 (-36) 0.5 - - - - - 12-24 269.5 T M.199 0 0 214 (-42) 0.5 - - 12-24 318.4 C M.200 5 55 228 (-38) 1.9 17.2 9.2 5.5 6.5 7.0 180 - 295.9 T M.201 0 0 222 (-38) 0.1 - - - - 12-24 286.4 C M.202 0 7 194 (-36) 0.4 0 5.5 - - 30 281.9 T HI. 203 0 0 208 (-32) 0.5 - - - - - 12-24 296.9 C p i . 204 3 57 200 (-58) 0.55 14.7 20.8 5.5 6.5 7.0 200 - 276.7 - 135 - adequate t e s t , i t would appear that testosterone and cortisone together afford no protection 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 effect of testosterone and cortisone on the mortality i n uninephrectomied, dehydrated and ligated male albino rats of the Wistar s t r a i n . Twelve animals were used, but one control 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 well as 5 mg. at the time of l i g a t i o n ; that of cortisone 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 lessen the stress and pro long l i f e to allow determination of whether or not the animals reached a state of acute renal f a i l u r e . Observations appear i n Table 16 . Again, seven animals died within the 24 hours following l i g a t i o n i n spite of being of adequate body weight; one died during the l i g a t i o n period of massive retroperitoneal haemorrhage following nembutal i n j e c t i o n ; two animals (one test and one control) survived for 24 to 48 hours; two remaining animals - 136 - (both controls) survived for 72 hours and were k i l l e d . These had urea nitrogen levels 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 debris 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 of Zone 3 degeneration and of Zone 4 (medulla) with frequent casts. 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^ for s i x treated animals (range 261.6 to 318.4), 281.0 mg. per cm for four controls (range 269.5 to 295.9). As i n Experiment 15, i t can only be concluded that testosterone and cortisone have no therapeutic value i n male rats with acute tubular necrosis. - 137 - Experiment 17: The efficacy of Compound F i n the treatment of acute tubular necrosis i n uninephrectomied, dehydrated, li g a t e d male rats i s tested i n Experiment 17A. Observations are made on urine output, blood urea nitrogen, kidney histology and mortal i t y rate. Six animals were so tested, with s i x untreated controls. Compound F was given as a d a i l y dose of 2 mgs. i n saline 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 of this substance, 3 mgs. d a i l y s t a r t i n g 72 hours before ligatures were applied. Alternate animals of a group of twelve males were so treated. Observations were made on urine outputs and mortality rate but B.U.N's were determined only postmortem on animals freshly dead. These appear i n Table 17B. By 24 hours following l i g a t i o n , nine animals i n Experi ment 17A had died. The three remaining animals survived for 72 hours to be k i l l e d at that time for kidney histology. These were treated animals. Urine outputs were t y p i c a l of the d i u r e t i c response, and urea nitrogen levels 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 to be healing acute tubular.necrosis (see Figures 38-40 i n Experiment 17B). Kidney TABLE 17A COMPOUND F IN CRUSHED MALE RATS RAT FOOD (MG) WATER (cc) WEIGHT AND CHANGE (GM) URINE VOLUME cc. URINE PH B . U . N . me.#. DIED KIDNEY WEIGHT Mg/cm2. 24 48 72 24 48 72 2 4 48 72 (hrs) T M.205 9 66 172 (-36) 1.5 5-9 2 6 . 4 6 . 0 6.0 6 .5 240 - 170 _ 231.2 C M.206 0 0 224 (-24) 0 . 4 - 12.24 251.2 T M.207 0 0 188 (-28) 0 . 0 - - - - - - 12-24 201.0 C M.208 0 0 172 (-24) 0 . 5 - - 6.0 - - - - 12-24 202.3 T M.209 0 0 174 (-24) 0.75 - - 5.5 - - - - 12-24 197-7 C M.210 0 0 180 (-28) 0 . 3 - - - - - 250 - 130 - 260.5 T M.211 9 63 184 (-44) 1.8 6 .7 19.8 6.5 7.0 7.5 - - 12-24 262.4 C M.212 0 0 188 (-28) 0 . 4 - 12-24 247.2 T M.213 0 0 186 (-30) 0 . 3 - - - - - - 12-24 210.9 C M.214 0 0 170 (-28) 0 . 1 - - - - - . - 12-24 203.4 T M.215 9 40 172 (-36*) i . a 2 . 9 7.o 6.5 6 . 0 6 .5 190 - 150 - 225.4 C M.216 0 0 198 (-24) 0 . 6 12-24 212.5 TABLE 17B COMPOUND E IN CRUSHED MALE RATS RAT FOOD (GM) WATER (cc) WEIGHT AND CHANGE (GM) (GM) URINE VOLUME (cc) URINE pH , . B.U.N. mrt • DIED (hrs) KIDNEY WEIGHT mg/cm.2 24 48 72 24 48 72 T M.229 0 99 218 ( -38) 2.6 16.6 47.4 5.5 6.0 7.5 200 289.9 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 6.0 6.5 7.0 170 - 229.2 C M.232 0 0 . 190 ( -20) 0 . 2 - - 6.0 - - • - 12^24 208.1 T M.233 0 0 226 ( -30) 0.9 0 - 6.0 - - 280 25 218.7 C M.234 0 3 230 ( -38) 1.2 0 • - 6.0 - - 420 26 236.6 T M.235 0 8 220 ( -32) 0.6 0.3 - 6.0 - - - 29-33 222.4 C M.236 0 0 196 ( -22) 0.7 - - 6.0 - - - 12-24 201.0 T M.237 0 0 190 ( -28) 0 . 4 - - - - - 12-24 245.9 C M.238 0 61 224 ( -52) 2.1 8.0 31.0 6.0 6.0 7.5 240 - 246.3 T M.239 0 0 190 ( -24) 0.7 0 - . 6.0 - - 300 25 251.3 C M.240 0 0 184 ( -24) 0 . 2 -. - - - - 12-24 234.8 - 140 - weights were again not s i g n i f i c a n t s treated animals averaged 221.4 mg. per cm.2 (range 197*7 to 262.4), untreated controls 229.5 mg. per cm.2 (range 202.3 to 2 6 0 . 5 ) . On the basis of Experiment 17A with twelve male ani mals, i t can be concluded that Compound F shows some promise i n the treatment of acute tubular necrosis induced by dehydration i n crushed animals. Three of s i x treated animals survived while none of s i x untreated animals survived. 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. It was f e l t t h a t , i f kidney damage was already present, free water intake would not improve the ultimate outlook and so not interfere with comparisons of mortality rate. Kidneys were here and subsequently fixed i n Herlant's solution because of technical d i f f i c u l t i e s with Zenker's f i x a t i v e and results amply j u s t i f i e d the switch. Urine outputs i n those animals surviving 48 hours or more showed a remarkable d i u r e t i c response and indicated that these animals with damaged kidneys could not handle water intake s a t i s f a c t o r i l y . I t appeared that the increased o r a l amount was ra 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 surviving to 72 hours (two treated, - 141 - two untreated), B.U.N, levels remained elevated, indicating continuing renal damage, and renal histology showed a picture of what has been described previously as healing acute tubular necrosis. This healing picture i s shown i n Figure 38 (Zone 2 of animal M. 229) i n which new, low basophilic cuboidal c e l l s are seen appearing i n disorganized or degenerated areas; Figure 39 (Zone 3 of M. 229) showing e p i t h e l i a l debris with nuclei i n tubules; and i n Figure 40 (medulla of M. 229) which again shows Figure 38 Figure 39 Figure 40 the e p i t h e l i a l casts. Two 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 diuresis again i n d i c a t i n g kidney dysfunction. Three other animals (two treated, one untreated) died 25 to 36 hours after ligatures were applied, 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 levels (0 . 7 to 1.2 cc) and h i s t o l o g i c a l acute tubular necrosis. - 142 - A t o t a l of eight animals died spontaneously, four having been treated with Compound F and four untreated; the remaining four survived to 72 hours. Kidney weights averaged 242.9 mg. per cm. (range 218.7 to 289.9) for treated and 221.4 mg. per cm2 (range 201.0 to 246.3) for untreated control animals. Conclusions to be drawn from t h i s experiment include the following: 1) Compound F does not appear to prolong the l i f e of or reduce mortality i n male rats suffering from experi mental acute tubular necrosis. Since t h i s agent did seem to have some p a l l i a t i v e effect i n Experiment 17A, determination of i t s true value i n treatment of the syndrome must await further experimentation. 2) Animals with evidence of renal damage are seen to handle o r a l intake of water i n an i n e f f i c  ient and disadvantageous manner. Experiment 18: A fourth and f i n a l hormonal agent, desoxycoricosterone acetate, was tested i n male rats i n which dehydration and crush in j u r y were used to produce acute tubular necrosis. In particu l a r , mortality rate was noted, but observations on urine output, blood urea nitrogen and kidney histology were also made. Again, twelve animals were used, alternate ones being treated with 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: DESOXYCORTICOSTERONE IN CRUSHED MALE RATS RAT FOOD (GM) WATER (cc) WEIGHT AND CHANGE (GM) URINE VOLUME (cc) URINE pH B.U.N. Mg. % DIED QDNEY »ffiIGHT 2 (Mg./cm~) 24 48 72 24 48 72 24 48 72 T M.217 0 0 236- (-24) 1.9 0 5 . 5 300 32-48 230.7 C M.218 0 0 250 (-12) 0 . 5 - - - - - - 12-24 213.4 T M.219 0 0 228 (-36) 1'° 2.0 - 6.0 6.0 - - - 32-48 214.7 C M.220 5 60 210 (-32) 2.9 2.3 25.9 6.0 5 . 5 7 . 5 280 - 190 - 256.9 T M.22I' 0 0 224 (-26) 2.3 0 .7 - 5 . 5 5 . 5 - 420 - 32-48 198.0 C M.222 0 0 224 (-20) 2.1 0 . 1 - 5 . 5 5 . 5 - 240 - - 32-48 195.6 T M.223 0 0 182 (-16) 1.0 0 - 5 . 5 - - - 31 202.7 C M.224 0 0 194 (-26) 1.2 0 - 6.0 - 290 - 32-48 223.4 T M.225 0 0 188 (-24) 0 . 7 0 - 5 - 5 - - - 32-48 209.2 C M.226 0 0 186 (-12) 0 - - - - 360 - 24 243 .8 T M.227 0 0 174 (-16) 0 . 9 0 - 5 . 5 • - - - 31-32 2 0 0 . 5 C M.228 0 0 178 (-26) 1 . 5 0 - 5 - 5 - 210 - 32-48 186.4 - 144 - Only one animal, M . 220 (a control animal) survived to 72 hours; this animal had an elevated B . U . N , and kidney histology showed f o c i of regeneration i n Zones 2 and 3 as previously described. I t has been pointed out before that this change was seen frequently i n animals known to have suf fered kidney damage but which eventually recovered. A second control animal, M . 226, was observed to die i n convulsions 24 hours a f t e r the ligature was applied and at that time the B . U . N , was 360 mg. % and kidney histology was t y p i c a l of "lower nephron nephrosis" (Figure 41) together with frequent proximal tubule vacuolization. From th i s observation i t becomes apparent that acute tubular necrosis with death i n uremia can indeed be pro duced within 17 to 18 hours following removal of the crushing l i g a t u r e . Figure 41 Five other animals had B . U . N ' s elevated to from 210 to 420 mg. % and died at from 32 to 48 hours following l i g a t i o n . A l l apparently died i n acute renal f a i l u r e with a varying number of hours anuria preceding death. Kidney histology of the two - 145 - test animals was probably r e l i a b l e , though the animals could have been dead for one hour and ten 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 tubular necrosis i n both cases. Kidney weights following f i x a t i o n averaged 209.3 nig. per cm.2 (range 198.0 to 230.7) for treated animals and 219.9 mg. per cm.2 (range 186.4 to 256.9) for untreated. I t can be concluded that DCA, given in^adequate dose, to male rats suffering from acute tubular necrosis, does not decrease t h e i r mortality rate nor prolong l i f e . I t can also be stated that death i n uremia from acute tubular necrosis re 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 following release of l i g a t i o n . DISCUSSION AND CONCLUSIONS From these experiments several conclusions can be drawn which give r i s e to some discussion; but before present ing these points i t i s essential to 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 rats by varying three stresses, 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 of testosterone, cortisone, 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 factors responsible for the production of acute tubular necrosis. Dehydration has been shown to be an essential factor i n the production of traumatic uremia i n the r a t . Even severe dehy dration (Experiment IB), when alone, succeeded i n producing only s l i g h t uremia and o l i g u r i a with almost immediate recovery on re-hydration, without h i s t o l o g i c a l evidence of tubule damage. These results are probably adequately explained by simple hemo- concentration; though prolonged dehydration could conceivably produce shock, such a condition was never observed i n animals (even uninephrectomied ones) dehydrated as long as 72 hours and therefore could play no part i n the urea nitrogen increase and o l i g u r i a . 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 effects i n int a c t animals and i n right nephrectomied animals. This finding that the state of.hydration i s an important factor i n the production of the syndrome i s i n agreement with the works of L a l i c h (202, 203)» Maluf (232) and many others. I t i s also obvious (Experiment 5) that there i s a second essential factor 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 tissue (125, 162, 3 0 , 89) cannot be excluded as the pathogenetic mechanism, shock with renal ischemia was - 147 - probably the chief cause of renal damage. That shock actually was present could only be assumed from the appearance of the ani mals 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. There was a second observation in favour of anoxia — i.e. ischemia as a result of shock — as the damaging agent. It was consistently noted that various degrees of post mortem change could in no way be distinguished from the tubular damage seen in kidneys of animals freshly dead as a result of ligation and dehydration. Since postmortem autolysis must essen ti a l l y be primarily an anoxic change, then i t is probable that the degenerative changes seen in test animals is also anoxic (see Figures 5 1 - 6 2 ) . The method of leg compression was used first by Bywaters and Popjak (74) in order to simulate as closely as possi ble the clinical 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 in dogs and noted the similarity to crush syndrome. Eggleton et al (125,126), using eats and dogs, recorded low blood pressures following elastic rubber tube binding of limbs but was of the opinion that renal dam age resulted from a released nephrotoxic agent. Corcoran and Page (85) also used a method of limb ligation in their studies of the relationship of myoglobin to crush syndrome, and Keele and Slome (191) noted a marked reduction in blood pressure following release of a - 148 - wrapped limb in cats. There would appear to be l i t t l e doubt, therefore, that this method of limb compression can indeed produce shock in experimental animals. It is apparent therefore that the combination of severe dehydration and crush injury is capable of producing renal tubular damage as evidenced by elevated blood urea nitro gen 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 prolonga tion of ligation, bilateral ligation, myoglobin injection and uninephrectomy being merely attempts to produce a more predict able 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 in 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 in 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 in the human (in which i t commonly runs a 7 to 14 day course) i t is apparent that i t runs a fore-shortened course. The corresponding events in the rat appeared to occur within one to three days following trauma, - 149 - those animals surviving for 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 often d i f f i c u l t to obtain s e r i a l observations since affected animals often died within 24 hours; second, i t was often 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 renal f a i l u r e , when i t succumbed within 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 fi v e hour l i g a t i o n period, though animals were sedated they nevertheless behaved vigorously and i n a wide-awake fashion when that sedation subsided. But following removal of the l i g a t u r e they f e l l immediately into a period which we called "shock". They crouched far back i n t h e i r cages, eyes closed and fur r u f f l e d , often developing marked tremor. They remained i n t h i s state for from two to four hours at which time t h e i r condition 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 possible for a rat (animal M.226) to die i n acute renal f a i l u r e with acute tubular necrosis 17^ hours following 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 myo globin i s not essential to the production of acute tubular necrosis from crush Injury i n the rat (Experiments 5 , 6 and 7 ) . This - 1 5 0 - statement i s i n contrast to the o r i g i n a l work of Bywaters and Stead ( 7 5 ) who, though unable to produce renal f a i l u r e by i n j e c t i n g myoglobin alone or by leg compression alone, produced the syndrome by myoglobin i n j e c t i o n following leg compression or following a c i d i f i c a t i o n of the urine to pH 4 . 5 to 6 . 1 with ammonium chloride. In our experiments, i t can be noted that though urine was consistently of pH 5 * 0 to 6 . 5 and animals were dehydrated, myoglobin i n j e c t i o n did not produce detectable renal damage (Experiment 3 ) « I t i s in t e r e s t i n g to note that,, although Bing (31j 3 2 ) found that 80 to 120 gm. of ammonium chloride given to dogs to a c i d i f y urine did i t s e l f produce no renal damage, Govan and Parkes ( 1 6 0 , l 6 l ) found that both ammon ium and calcium chloride produced renal lesions and death i n rabbits. I t would therefore appear that Bywaters' work ( 7 5 ) should be considered only with reservations. Corcoran and Page ( 8 5 , 8 6 ) have also reported that crush syndrome i s reproducible by intravenous i n j e c t i o n of metamyoglobin after release of com pression from one crushed hind limb of r a t s . They reported as well " p a r t i a l l y recoverable renal 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 aciduric dogs. Bing's ( 3 2 ) work, however, contrasts with these observations; he f a i l e d to produce any s i g n i f i c a n t impairment of renal function by i n j e c t i o n of myohaemoglobin into normal or aeidotic dogs. Whether or not myoglobin adds to the damage induced by crush and dehydration should be apparent i n Experiment 7» - 1 5 1 - 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 elevation of urea nitrogen l e v e l s for myoglobin injected ani mals over non-injected ones was found. I t can be stated there fore that intravenously injected myoglobin adds to renal damage induced by dehydration plus crush i n j u r y , although by i t s e l f or coupled with either one of these f a c t o r s , the pigment i s non t o x i c . There was no h i s t o l o g i c a l evidence that the aggravation of renal dysfunction was due to obstructive casts. The p o s s i b i l i t y that these observations of the effect 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 was colored one to two hours af t e r i n j e c t i o n . This change was never seen i n our experiments. Also, since the myoglobin was i n part injected intravenously 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 proportion ( 6 0 to 7 0 $ ) of the myoglobin was c e r t a i n l y dissolved so that the effective dose would at least be at the upper end of the range calculated by Bywaters ( 7 5 ) and used also by Corcoran and Page ( 8 5 ) . And i n myoglobin-injected animals a post-injection polyuria was frequently observed; since the only variable was the presence of the hypertonic solution of myoglobin, t h i s fact 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 tubular f l u i d to r e s u l t i n an increased urine flow. I t i s reasonable to conclude therefore that myoglobin i n adequate - 152 - dosage passed through the kidneys. Because the clinical 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 in the experimental production of the syndrome. It soon became obvious, however, that not only was i t difficult 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 its 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 in normally hydrated animals but was present also in 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 in the literature. Eggleton et al (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 is not observed in the crush syndrome in dogs. Block et al (41) reported that polyuria was a striking feature in dogs following a hypotensive period. These workers suggest an explanation which has long been used in the polyuric phase of chronic glomerulo-nephritis — decreased functioning renal tissue requires that remaining nephrons eliminate the necessary nitro genous 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 filtration, 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. A third possibility may also be considered: hemodilution. Hemodilu tion was observed frequently in ligated animals but was usually associated with haemorrhage from bitten limbs. It was, however, also observed occasionally in animals which showed no sign of external haemorrhage and in those in which gastro-intestinal haemorrhage and hematuria were observed. It has been observed (81A) that in producing severe shock in rats by pinching the intestine in several places for short periods, marked and rapid ly developing hemodilution appeared. - 154- - Perhaps the most probable explanation of the later polyuria is one which accounts for the diuretic phase of human acute tubular necrosis, that of tubular damage to the extent that normal water reabsorption is inhibited. Surely anoxic tubular damage can be such that the normal reabsorptive mechan ism is 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 in rats is a con sistent observation and can be used as a definite indication of renal dysfunction which does not necessarily indicate recovery but is only one stage in the reaction of a damaged kidney. In those animals which showed eyidence of renal dys function either by polyuria, anuria or uremia, histological changes were exclusively in the kidney tubules. The intra capsular granular eosiniphilic granular debris and cubical meta plasia of capsular epithelium were not observed. An occasional glomerulus was observed, however, in which the capsular space appeared to be dilated in such a way as to incorporate the upper extremity of the proximal convoluted tubule, giving the appear ance of cubical metaplasia of the capsular epithelium (Figure 42 and Figure 43, which shows a less obvious case of the same phen omenon). This change occurred in control and test animals alike and i t is interesting to note that a similar though appar ently true cubical metaplasia has been described as an action of DCA ( 3 3 3 ) . V Tubular changes could be recognized at two stages. In animals which died i n obvious acute renal f a i l u r e , tubular c e l l s showed changes ranging from early degeneration to necrosis. Cytoplasm became granular and vacuolated, nuclei swollen, pale and vacuolated and i n severe cases, nuclei progressed to the small, dark pyknotic stage of degeneration. In a l l cases the basement membrane appeared to remain in t a c t (See Figures 3 1 , 32 , 3 4 , e t c . ) . In those animals which showed signs of renal dysfunc t i o n -- polyuria and uremia — but went on to recovery, kidney histology was amazingly normal. However, consistently i n these cases there were seen fo c a l areas of b l u i s h , granular degenera t i o n of tubules with desquamation of these c e l l s to form casts, and evidence of regenerating tubular epithelium. In addition, medullary tubules showed occasional casts of e p i t h e l i a l debris including 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, healing and regenerating phases of the process. (See figures 38 - 40). The l o c a l i z a t i o n of these tubular lesions within the nephron i s in 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 the involved segment, hence Lucke's (213) term Lower Nephron Neph r o s i s . Later reports (146) observed degenerative changes often more advanced i n the proximal tubule and eventually Oliver et a l (271) pointed out that the essential l e s i o n of acute tubular necrosis ("tubulorhexis") could i n fact 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 often develop lesions i n the lower nephron (59> 6 0 , ' 8 8 ) , while those subjected to renal artery occlusion show proxi mal tubule lesions (194, 195) . In the experiments reported herein, i n which rats were subjected to the stress of crush and dehydration, the s i t e of the l e s i o n was consistently the d i s t a l tubule, c h i e f l y i n Zone 3 of the kidney but also (though to a lesser extent) i n the cortex. The presence or absence of the brush border i n proximal tubules can be used as a very fine index of damage to that unit and i n the kidneys examined t h i s structure was consistently present (Figure 44). There was one exception to t h i s statement i n an experi ment which was discarded because of a high incidence of chronic - 157 - Figure 44 kidney disease in 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 in animals used. Hydronephrosis (Figure 47) and chronic inflammatory changes (Figures 48 and 49) were the chief diseases seen. In one case, this last change appeared to - 158 - account for an elevation of the blood urea nitrogen. Almost consistently the hydronephrotic change occurred only i n the right kidney, which was of course removed p r i o r to experimenta t i o n so that i t was f e l t that t h i s did not int e r f e r e with observations. In any case, i t i s u n l i k e l y that chronic disease would interfere with acute experiments such as were carried out. Figure 48 - 1 5 9 - Figure 49 The s i m i l a r i t i e s between early (up to seven hours) postmortem change and acute tubular necrosis due to crush and dehydration have already been referred to. I t was noted that the d i s t a l tubules quickly 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 (Fig 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 , 61 and 6 2 ) . The postmortem d i s t a l tubule degeneration was very noticeable i n the c o r t i c a l region as w e l l (Figure 5 0 ) . Two of the three c l a s s i c a l responses to stress were observed frequently, 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 often 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 in later experiments was a baffling one. Referring to Experiment 10, i t will 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 in Figure 51 Figure 52 Figure 53 - 161 - Figure 57 Figure 58 Figure 59 Figure 60 Figure 61 Figure 62 r- 162 - hormonal experiments. In Experiment 14, not only did 10G % of control animals d i e , hut they did so within 24 hours of the crush in j u r y so that the problem of shock deaths i s ampli f i e d . 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 for t h i s phenomenon. The tension of the s t r i n g l i g a t u r e could not be absolutely standardized and with 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 occlusion could affect the degree of syste i c shock since the mass of tissue damaged and the duration of l i g a t i o n were the same i n a l l cases. The second apparent fac tor involved i s the time of year — i . e . , during the course of these experiments winter had become spring and summer and the environment was noticeably warmer and more humid. This i s a vague but not an unusual ef f e c t i v e factor i n a l t e r i n g experi mental observations and here may have reduced the resistance of the animals by a l t e r i n g t h e i r water balance. Interesting i s the fact that the increase i n mortality was a gradual one. Hamilton, P h i l l i p s and H i l l e r (166) have referred to an increased mortality rate i n dogs subjected to renal artery l i g a t i o n for varying periods when environmental temperature and humidity were increased. I t should be unnecessary to point out that as many - 163 - factors as possible were kept constant throughout these experi ments. Differences i n s t r a i n of r a t , body weight, sex, duration of l i g a t i o n , mass of tissue involved or sedatives used could not account for the change i n mortality rate. 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 tubular necrosis. In the case of Compound F. some promise was shown i n Experiment 17A so that some s l i g h t reservations about t h i s agent are held and further experimentation i s j u s t i f i e d . Testosterone was used i n t h i s work i n the hope that i t s "renotropic" action might lessen the damage induced i n the kidney or hasten i t s recovery; i t s effect on protein metabolism ( a sort of protein-sparing action i n the usual stress breakdown Of body protein) might also 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 tubules. The hormone also appears to afford some protection against mercury bichloride poisoning (333). Although i t has been shown (333) i n the dog that 1G0 mgs. testosterone per day produces a rapid r i s e i n TmD, 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 rate, renal plasma - 164 - flow, Tnip^ or Tm^ Smith also points out that the hormone has been used i n the treatment of chronic nephritis and i n cholera, i n which "better s u r v i v a l " with 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 renal hypertrophy was apparent, especially i n Experiment 11A, but no reduction i n uremia or mortality rate was observed. Disturbed e l e c t r o l y t e balance, which accompanies acute renal f a i l u r e , has frequently 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 arrest (181). For t h i s reason, DCA would seem to be a useful thera peutic agent. As a mineralocorticoid, i t i s known to promote sodium and water retention and potassium excretion so that plasma sodium increases while plasma potassium decreases ( 3 2 5 ) . This action of DCA i s thought to be a d i r e c t action on the renal ( d i s t a l ? ) tubules to promote sodium reabsorption (333) but also on the c a p i l l a r y permeability and tissue a f f i n i t y for water and ele c t r o l y t e s ( 3 2 5 ) . 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 Tm 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 rats i n our experiments, DCA did 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 ureteral l i g a t i o n or b i l a t e r a l nephrectomy) the elevation of serum potassium i s such that cardiac damage i s the cause of death, i n mercuric chloride 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 electrocardiographic 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 role of potassium retention i n deaths i n acute renal f a i l u r e and might also explain the f a i l u r e of DCA to prolong l i f e i n rats suffering from the syndrome. In using cortisone (17 hydroxy - 11 dehydrocortieo- » sterone, Compound E) as a therapeutic agent i n the traumatic anuria syndrome (23) i t was hoped that the hormone would lessen the mortality by counteracting the shock of the early phase of the alarm reaction (325, 326) seen i n response to limb l i g a t i o n . Selye (326) found that " c o r t i n " was highly e f f e c t i v e i n t h i s regard, reporting that DCA alone had l i t t l e e f f e c t . Weil et a l (358) reported s i m i l a r findings i n rabbits. Ingle (185) on the other hand found that neither DCA, cortisone nor adrenal c o r t i c a l extract reduced the mortality rate of rats 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 bio l o g i c properties of cortisone, pointing out that i t can no longer be thought of as a simple glucocorticoid. I t s effect on e l e c t r o l y t e and water balance i s variable but i n acute experi ments i n rats an increased excretion of sodium, chloride and potassium l a s t i n g one to three days has been reported. C o r t i  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; to maintain renal function in adrenalectomy; and to increase the PAH secretion in normal males by up to 35% (186, 324). In high doses and prolonged administration i t produces hyalin- ization 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 in 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 clinically, to be of value where cortisone is ineffect ive. Compound F is in fact very similar to cortisone in chemical composition (325) and in action (287). It is said to be less active in 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 in rats and increases kidney mass (145) Though in Experiment 17A, Compound F appeared to be of some considerable value, this phenomenon was not observed in Experiment 17B. Nevertheless i t is apparent that further experimentation with Compound F would be advisable. - 1 6 7 - In considering the failure of cortisone or Compound F to be of benefit to rats, in acute renal failure i t is perhaps worthy of note that Selye (326) points out that adrenal cortical hormones in shock are more effective given in divided doses, . and that pre-treatment is useless and may be harmful. Pre- treatment may well depress the normal adrenal cortical activity. It is very unlikely that the dosage used in 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 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 p i c t u r e . He described tubular necrotic lesions for which the chemical toxins (mercuric chlor ide, carbon tetrachloride) were responsible, and tubulorhectic lesions which were c h a r a c t e r i s t i c of the shock kidney. These lesions could appear at any l e v e l i n the renal tubule and were characterized by destruction of the basement membrane. Pigment casts were apparent i f intravascular pigment release was assoc iated with the i l l n e s s . The work of P h i l l i p s , Van Slyke and associates (291, 292, 355, 356), 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 s p e c i f i c ex t r i n s i c renal toxins play a major role i n s p e c i f i c cases. The role of hemoglobin appears to be c h i e f l y i n the production of obstructive casts l a t e r i n the course of the disease; these pigments are precipitated 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 rats were studied i n eighteen experi ments concerning crush syndrome. I t was concluded that the most important single factor tending to aggravate the renal effects of crushing i n j u r y i s the antecedent state of dehydra t i o n . Myoglobin i s not an essential factor i n the development of renal damage but tends to aggravate the exi s t i n g uremia. Acute renal f a i l u r e was seen to be a l a t e effect of shock; 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 c i r c u  l a t o r y f a i l u r e . Development of t h i s delicate balanee of factors was aided by reduction of renal 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 persistent d i u r e s i s i n response to the trauma of limb l i g a t i o n . This polyuria 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 reabsorption of water because of tubular damage. It 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 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. - 170 - Although there was no doubt that the syndrome of acute renal failure due to acute tubular necrosis could be produced in large numbers of these relatively inexpensive laboratory animals by dehydration and limb ligation, produc tion 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 is felt 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. 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