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Pathogenesis and genetics of the polydipsia-polyuria defect in the SWV strain of house mice Virgo, Naomi Sheila 1972

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PATHOGENESIS AND GENETICS OF THE POLYDIPSIA-POLYURIA DEFECT IN THE SWV STRAIN OF HOUSE MICE BY NAOMI SHEILA VIRGO B.S.A. (Honors), University of British Columbia, 1968  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Field of GENETICS We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA DECEMBER 1972  In presenting this thesis in partial  fulfilment of the requirements for  an advanced degree at the University of B r i t i s h Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by h i s representatives.  It  i s understood that copying or publication  of this thesis f o r financial gain shall not be allowed without my written permission.  Department of  Genetics  The University of B r i t i s h Columbia Vancouver 8. Canada  Date  January 30, 1973  ABSTRACT  In the SWV strain of mice, old females have a severe increase in water intake and urine output.  This polydipsia-polyuria defect was  investigated.  The SWV females' urine was hypotonic, contained no glucose or blood, and had no increase in protein or pH. diabetes insipidus.  The defect thus appeared to be some form of  The mice were then tested for their response to water  deprivation, saline ingestion, and exogenous vasopressin (pitressin tannate). The results indicated that the defect was nephrogenic diabetes insipidus. Histological studies showed that there was an adequate amount of neurosecretory material in the posterior pituitaries and that the adrenals were normal.  At 4 months of age, the SWV females' kidneys were normal, but from  6 months of age the kidneys were progressively abnormal until by 17 months the kidneys had severely degenerated.  At 12 months of age the females'  kidneys were abnormally large, hydronephrotic, and had severely dilated and hyperplastic tubules in the cortico-medullary zone.  Some of the dilated  tubules contained eosinophilic casts and there was some focal i n t e r s t i t i a l nephritis.  At 17 months, the females' kidneys had severe i n t e r s t i t i a l neph-  r i t i s and some fibrosis around the tubules, and some hyalinization in the cortex.  The females also had a progressive anaemia but preliminary studies  indicated no large increase in blood urea nitrogen. Thus the SWV females had a nephrogenic diabetes insipidus which first appeared at 4 to 6 months of age.  While the defect resembled human nephro-  nophthisis and hypokalemia in some symptoms, it did appear to be unique. Amyloid kidneys, polycystic kidneys, and diabetes mellitus have been ruled out as the cause of the defect but hypokalemia and hypercalcemia are s t i l l  possibilities. The SWV males had a milder expression of the defect and did not show the polydipsia until 7 to 8 months of age.  They had a milder form of the  polydipsia, the concentrating defect, and the anaemia, but showed no kidney abnormalities.  The defect was sex influenced possibly due to the difference  in steroid hormones. Polydipsic SWV mice were crossed to control C3H mice and F-^, F2, BCgyy, and  BCQ^JJ  progeny in each generation indicated that one or two dominant genes  determined the polydipsic defect in the SWV strain. . There was no sex linkage. A quantitative analysis of mean water intakes for each generation indicated that non-genetic factors influenced the polydipsia multiplicatively.  Some  of these non-genetic factors were sex, age, body weight, type of feed ration, and parity.  iii TABLE OF CONTENTS Page ABSTRACT  1  TABLE OF CONTENTS  1 1 1  LIST OF TABLES  v  i  LIST OF FIGURES  viii  ACKNOWLEDGMENTS  xi  INTRODUCTION  1  A.  1  Pathogenesis  B. Genetics  6  MATERIALS & METHODS  8  A. Animals B.  Description of Polydipsia-Polyuria Defect (1) Water Balance (2) Water Intake (a) Influence of Parity in Females (b) Influence of Stress in Males  C.  Differential Diagnosis of Polydipsia-Polyuria Defect (1) Serum Osmolality (2) Response to Dehydration (a) Water Deprivation (b) Saline Ingestion (3) Response to Exogenous Antidiuretic Hormone (4) Similarity to some Kidney Diseases (a) Urinalysis (b) Per Cent Packed Cell Volume (c) Blood Urea Nitrogen (5) Histology (a) Pituitaries (b) Adrenals (c) Kidneys (6) Statistics  D. Genetics of Polydipsia-Polyuria Defect .' (1) Mating Scheme (2) Testing of Progeny. (3) Analysis of Progeny (a) Quantitative Estimate of Number of Loci (b) Qualitative Estimate of Number of Loci (c) Age of Onset  8 9 9 10 11 12 ..  13 13 13 13 14 15 16 16 16 17 17 18 18 18 19 19 19 2 2  ....  23 23 24 5  2  iv RESULTS  27  A.  D e s c r i p t i o n o f P o l y d i p s i a - P o l y u r i a Defect (1) General D e s c r i p t i o n (2) Water B a l a n c e (3) Water Intake (a) Age & Body Weight (b) Dam or S i r e E f f e c t s ..: (c) Feed (d) Strain (e) Sex (f) P a r i t y i n Females (g) S t r e s s i n Males  27 27 29 32 32 34 36 47 50 55 59  B.  D i f f e r e n t i a l Diagnosis of P o l y d i p s i a - P o l y u r i a Defect (1) Serum O s m o l a l i t y (2) Response t o D e h y d r a t i o n (a) Water D e p r i v a t i o n (b) Saline Ingestion (3) Response t o Exogenous A n t i d i u r e t i c Hormone (4) S i m i l a r i t y t o some Kidney D i s e a s e s (a) Urinalysis (b) P e r Cent Packed C e l l Volume (c) Blood Urea N i t r o g e n (5) Histology . (a) Pituitaries (b) Adrenals (c) Kidneys  60 60 62 62 .69 72 80 80 80 82 84 84 84 90  C.  Genetics of P o l y d i p s i a - P o l y u r i a Defect (1) Q u a n t i t a t i v e E s t i m a t e o f Number o f L o c i (a) Females , (b) Males (2) Q u a l i t a t i v e E s t i m a t e o f Number o f L o c i (a) Females (b) Males (3) Age o f Onset  100 100 107 109 110 112 113 115  DISCUSSION A.  D e s c r i p t i o n of P o l y d i p s i a - P o l y u r i a Defect (1) General D e s c r i p t i o n (2) Water B a l a n c e (3) Water Intake  B.  D i f f e r e n t i a l Diagnosis of P o l y d i p s i a - P o l y u r i a Defect (1) Serum O s m o l a l i t y (2) Response t o D e h y d r a t i o n (a) Water D e p r i v a t i o n (b) Saline Ingestion (3) Response to Exogenous A n t i d i u r e t i c Hormone (4) S i m i l a r i t y to some Kidney D i s e a s e s  117 117 117 117 119 ..  121 121 122 122 124 125 130  V  (5)  C.  D.  Histology (a) Pituitaries (b) Adrenals (c) Kidneys  131 131 132 133  '.  Genetics of P o l y d i p s i a - P o l y u r i a Defect  138  (1)  144  Comparison t o o t h e r Syndromes  General  146  Discussion  154  CONCLUSIONS LITERATURE CITED APPENDIX 1.  F o r m u l a t i o n o f Feed R a t i o n s Used  .  156 164  vi LIST OF TABLES Table I.  Page Water balance values + standard error (s-) for the SWV and C3H strains. If N was smaller for the urine osmolality ( U o s m ) , it was given in brackets  30  Means + s- for the water intake, age, and body weight of the SWV and C3H-2 mice used in the multiple linear regression analysis  33  Multiple linear regression analysis of the dependent variable water intake (W.I.) and the independent variables age and body weight (B.W.) in the SWV and C3H-2 mice  33  Analysis of dam and sire effects on water intake (W.I.) in the SWV strain. The water intakes were adjusted for age  35  V.  Daily water intake (ml) + s- in mice fed ration 1  ....  37  VI.  Daily water intake (ml) + s- of SWV mice fed ration 2 . .  38  VII.  Daily water intake (ml) + s^ of C3H-2 mice fed ration 2  39  VIII.  Daily water intake (ml) + s- of C57 mice fed ration 2 . .  40  IX.  Analysis of covariance of the positive regression of water intake on age between the 2 rations in SWV and C3H mice  44  X.  The maximum water intake + s- and the age i t was attained in mice fed ration I  45  XI.  The maximum water intake + s- and the age it was attained in mice tested up tZ 16 months of age and fed ration 2 .  46  Analysis of covariance of the positive regression lines of water intake on age in SWV, C3H-2, and C57 mice fed ration 2. The regression line was calculated up to and including the age at which the peak water intake was reached  51  Covariance analysis of the positive regression lines calculated when water intake was plotted against age in SWV mice fed ration 1. The regression line was calculated up to and including the age at which the peak water intake was reached  53  Serum osmolalities + s- in SWV and control C3H-1 mice . .  61  II.  III.  IV.  XII.  XIII.  XIV.  vii XV.  Effect of water deprivation on body weight (B.W.), urine osmolality (Uosm), serum osmolality (S m), and osmolal excretion in SWV and C3H mice  63  Regressions of urine volume and urine osmolality with time during water deprivation and ad libitium water intake (control)  67  Effect of 0.97= saline on daily ad libitium fluid consumption (ml) and body weight (B.W.) (g) in SWV and C57 mice »  71  Effect of route of administration on increase in urine osmolality ( U ) after 3, daily, injections of % unit pitressin tannate in o i l in control C3H-2 mice  73  Blood urea nitrogen values of the polydipsic SWV and the control C3H-2 strain measured using the azostix reagent strips  83  Relative sizes of the pars nervosa, pars intermedia, and whole pituitary gland in SWV and C57 females  86  OS  XVI.  XVII.  XVIII.  osm  XIX.  XX. XXI.  Estimation of number of l o c i (n) determining polydipsia using Wright's formula. Formula is based on an additive model with semidominance and no interactions. Estimation done using water intake values (ml/24 hrs). The value of n in brackets was calculated using the nontransformed data  XXII.  Frequencies of polydipsic progeny  XXIII.  Average age of onset (mo.) of polydipsia in affected progeny fed ration 2  106 Ill 116  viii LIST OF FIGURES Figure la.  Page Diagram of reciprocal crosses done between the SWV and C3H-2 strains in the first year. F,, F , BC , progeny were produced. The numbers in brackets were the number of mice mated. 0 = female • = male  20  Diagram of reciprocal crosses done in the second year to increase the sample size of each sex to 15 in the reciprocal F-, and BCgyy mice. The B C C 3 H _ 2 mice were also produced. The numbers in brackets were the number of mice mated. 0 = females • = male  21  2  lb.  2a.  SWV  16 month old SWV female showing muscle weakness in hind legs  28  2b.  6 month old control A/WySn female with normal hind legs  3.  The calculated against age in The calculated against age in  4. 5. 6. 7. 8. 9.  10.  11.  12;  ..  regression lines of water intake plotted SWV and C3H mice fed ration 1 regression lines of water intake plotted SWV mice fed ration 2  28 41 42  The calculated regression lines of daily water intake plotted against age in C3H-2 mice fed ration 2  48  The calculated regression lines of daily water intake plotted against age in C57 mice fed ration 2  49  Urine osmolality + s^ during water deprivation and ad 1 ibitium water intake in SWV and C3H mice  65  Urine output + s- during 7 hours of water deprivation and 7 hours of ad libitium water intake in SWV and C3H mice ..  66  Water intake + s- in SWV and C3H mice during a control run and after 3, daily, injections of the vehicle or pitressin tannate  75  Urine output + Sy in SWV and C3H mice during a control run and after 3, daily, injections of the vehicle or pitressin tannate  76  Urine osmolality + S y in SWV and C3H mice during a control run and after 3, daily, injections of the vehicle or pitressin tannate  77  Osmolal excretion + Sy in SWV and C3H mice during a control run and after 3, daily, injections of the vehicle or pitressin tannate  78  ix 13.  Per Cent packed cell volume at different ages in SWV and C3H-2 mice  14a.  SWV female 764, 13% months, pituitary, pars nervosa had abundant aldehyde fuchsin-positive material, mag. X 25.  14b.  C57 female 2119, 13% months, pituitary, pars nervosa stained with aldehyde fuchsin, mag. X 25  85  15.  SWV female 949, 12 months, adrenal, normal except for signs of ageing, mag. X 25  87  16.  C57 female 2208, 11 months, adrenal, mag. X 25  87  17.  SWV female 949, 12 months, adrenal, spindle cells present in cortex and X zone and pigment present in X zone, mag. X 100  88  C57 female 2208, 11 months, adrenal, spindle cells present in cortex and X zone and pigment present in X zone, mag. X 100  88  SWV female 949, 12 months, adrenal, normal zona glomerulosa, mag. X 400  89  ~ 20.  C57 female 2208, 11 months, adrenal, normal zona glomerulosa, mag. X 400  89  21.  Wet kidney weights + s- at various ages in SWV, C3H, and y C57 mice .  91  22.  SWV female 703, 4 months, kidney, normal cortico-medullary zone, mag. X 25  93  23.  C57 female 2063, 13 months, kidney, normal corticomedullary zone, mag. X 25  93  24.  SWV female 703, 4 months, kidney, normal cortico-medullary zone, mag. X 100  94  25.  C57 female 2063, 13 months, kidney, normal corticomedullary zone, mag. X 100  94  26.  SWV female 650, 5% months, kidney, dilated tubules in cortico-medullary zone, mag. X 100  96  27.  SWV female 616, 9 months, kidney, more severely tubules in cortico-medullary zone, mag. X 100  96  23.  SWV female 855, 13% months, kidney, severely dilated tubules and some hyaline casts in cortico-medullary zone, mag. X 25  18.  19.  dilated  81 ..  85  97  X  29.  SWV female 627, 12 months, kidney, hyaline casts and focal i n t e r s t i t i a l nephritis in cortico-medullary zone, mag. X 100  97  SWV female 626, 12 months, kidney, high cuboidal epithelial cells of dilated tubules in cortico-medullary zone, mag. X 400  98  31.  SWV female 626, 12 months, kidney, severe focal inters t i t i a l nephritis in cortico-medullary zone, mag. X 100. ..  98  32.  SWV female 855, 13% months, kidney, distinct blue line on luminal border of proximal convoluted tubules in outer cortex, mag. X 400  99  C57 female 2063, 12 months, kidney, normal proximal convoluted tubules in outer cortex, mag. X 400  99  30.  33. 34.  SWV female 855, 13% months, kidney, glomerulus in outer cortex, mag. X 400  101  SWV female 848, 17 months, kidney, severe dilation and some degeneration of tubules in cortico-medullary zone, mag. X 25  102  SWV female 848, 17 months, kidney, severe i n t e r s t i t i a l fibrosis and nephritis surrounding dilated tubules in cortico-medullary zone, mag. X 100  102  37.  SWV female 848, 17 months, kidney, dilated tubules in cortico-medullary zone, mag. X 400 .1  103  38.  SWV female 848, 17 months, kidney, possible calcification on luminal border of proximal convoluted tubules in outer cortex, mag. X 400  103  SWV female 848, 17 months, kidney, hyalinized glomeruli in cortex, mag. X 100 '.  104  C57 female 2063, 13 months, kidney, normal glomeruli in cortex, mag. X 100  104  SWV male 572, 12 months, kidney, normal cortico-medullary zone, mag. X 100  105  C57 male 2032, 12 months, kidney, normal cortico-medullary zone, mag. X 100  105  35.  36.  39. 40. 41. 42.  ACKNOWLEDGMENTS  I wish to thank my supervisor, Dr. J.R. Miller, and the members of my PhD committee, Drs. D.A. Applegarth, D.S. Lirenman, W.A. Webber, C.W. Roberts, and CO. Person, for their advice and encouragement during this study and the preparation of this manuscript.  I also wish to thank Dr. W.H. Chase for his  advice on the kidney pathology and Professor J. Biely for determining the chemical composition of ration 1.  I am grateful to Elizabeth March for her  technical help with the animals and with the analyses, to Dolores Lauriente for doing most of the statistical analyses on the computer and for preparing the graphs, and to Dr. Robert's students for their programming of the multiple linear regression analyses.  I am also grateful to the other students  in the lab for their interest, advice, and encouragement.  Most of a l l I  wish to thank my husband, Bruce, for his continuous support and encouragement which made this work possible. I was supported by a Medical Research Council Studentship and from the Medical Research Council Research Grant 68-1062.  INTRODUCTION  Laboratory animals which have hereditary diseases similar to ones found in man are becoming increasingly important as investigational models for the understanding of normal and pathological, complex physiological functions.  Environmental manipulation has long been used as a tool to study  physiological processes, but these environmental manipulations often influence many reaction steps or induction systems controlling the final integrated function.  A mutant gene, however, usually alters only one enzyme  step which may, of course, result in a number of final differences  due to  interactions and homeostatic regulation (Thoday, 1967). Polydipsia-polyuria occurs in man and experimental animals due to many different causes.  In this study the polydipsia-polyuria syndrome present in  the SWV strain of mice was investigated to determine: 1.  i f i t is the same c l i n i c a l l y as any of the polydipsia-polyuria syndromes in man or alternatively to determine which disease it resembles the most  2. A.  the genetic mechanism.  Pathogenesis Polydipsia-polyuria is defined as an abnormally large water intake and  a correspondingly large urine output.  A severe polydipsia and an abnormally  large output of dilute, sugar-free, urine is defined as diabetes insipidus. Some authors (for instance, Relkin, 1966) restrict diabetes insipidus to a few specific diseases, whereas Richards & Sloper (1969) consider it a broad syndrome including any diseases with severe polydipsia-polyuria, no glycosuria, and no uraemia due to renal dysfunction.  Thus the term diabetes insipidus  d e s c r i b e s the symptoms, not  and V a l t i n  t i o n of the 1.  (1967).  Primary  behavioural  1961)  but  P a t i e n t s w i t h primary  disturbances.  Primary  been r e p o r t e d strain  ( S z a l a y & M o l l , 1966)  A d i s t u r b a n c e o f the ADH diabetes  insipidus:  cursor; and  failure  Valtin  T h i s has  (1969)  not been r e p o r t e d  Patients with  dehydration,  but  to s y n t h e s i z e v a s o p r e s s i n ;  of the b i o l o g i c a l l y a c t i v e p e p t i d e  can  failure in  from i t s i n a c t i v e  pre-  to s y n t h e s i z e the t r a n s p o r t i n g p r o t e i n ; f a i l u r e o f r e l e a s e ; i n a c t i v e form of v a s o p r e s s i n .  There are many r e p o r t s of hypothalmic  diabetes  gene e r r o r i s not known ( R e l k i n , 1 9 6 6 ;  H e r e d i t a r y hypothalmic  diabetes  i n s i p i d u s has  B r a t t l e b o r o s t r a i n of r a t s ( V a l t i n , absolute defect  they  this  g i v e s 5 p o s s i b l e causes of hypo-  i n s i p i d u s can a l s o be secondary to tumours, i n f e c t i o n s ,  the primary  be  ( S i l v e r s t e i n et a l . ,  s e c r e t o r y mechanism.  failure  p r o d u c t i o n of a b i o l o g i c a l l y  diabetes  of  o f mice.  i n s i p i d u s cannot w i t h s t a n d  respond to exogenous ADH.  (psychogenic  p o l y d i p s i a can a l s o , t h e o r e t i c a l l y ,  i n the STR/N s t r a i n  2.  enzymatic c l e a v a g e  withstand  t h i s d i s e a s e u s u a l l y have a p r i o r h i s t o r y  the TS/A  thalmic diabetes  or combina-  p o l y d i p s i a can  p o l y d i p s i a i s compulsive water d r i n k i n g  and  hypothalmic  one  Sloper  respond to the a n t i d i u r e t i c hormone (ADH), v a s o p r e s s i n .  Patients with  i t has  Richards &  1969).  caused by an o r g a n i c d e f e c t o f the t h i r s t c e n t e r . i n man,  p o s s i b l e causes o f  syndrome can be caused by any  ( R i c h a r d s 6c S l o p e r ,  polydipsia.  cause of primary  polydipsia).  The  following defects  water d e p r i v a t i o n and One  The  i n s i p i d u s have been reviewed by R e l k i n ( 1 9 6 6 ) ,  diabetes (1969),  the p a t h o g e n e s i s .  1967)  i n the s y n t h e s i s o f ADH  i n r e l e a s e o f ADH  i n the h e t e r o z y g o t e s  and  and  trauma.  i n s i p i d u s i n man,  Richards & Sloper,  a l s o been r e p o r t e d  but 1967).  i n the  i s thought to be caused by  (Valtin,  1969)  and  a partial  ( M i l l e r & Moses, 1 9 7 1 ) .  s t r a i n of mice a l s o has v a s o p r e s s i n - r e s p o n s i v e  Hypothalmic  diabetes  The  i n s i p i d u s and  an  defect Ma/J i t is  3 thought to be due 3.  to p i t u i t a r y  l e s i o n s (Hummel, 1960).  The  i n a b i l i t y of the k i d n e y s to respond to c i r c u l a t i n g  Patients with  t h i s nephrogenic or v a s o p r e s s i n - r e s i s t a n t d i a b e t e s  cannot w i t h s t a n d defect the  diabetes  of the m e d u l l a r y  there  of the d i s t a l  is a defect  m e d u l l a r y c y c l i c 3'5' Hereditary  tubule.  i s thought to be  and  the DI +/+  of the m e d u l l a r y  or  i n s i p i d u s has 1969;  been  Stewart &  diabetes  m i l d l y a f f e c t e d mice which have a d e f e c t osmolality;  the V I I 0s/+  1971); the DI +/+  tubules,  i n osmotic d i u r e s i s , and  a reduction  Henle ( K e t t y l e & V a l t i n ,  1972;  Nephrogenic d i a b e t e s diseases, al.,  1972;  to water i n the d i s t a l  s e v e r e l y a f f e c t e d mice which have a d e f e c t i n the d i s t a l  lithium toxicity  osmotic  an  and  i n the ADH-induced  807, r e d u c t i o n of nephrons r e s u l t i n g  i n the l e n g t h of the  Stewart,  tubules;  short  loops  of  1971).  i n s i p i d u s can a l s o be  secondary to c h r o n i c  (Gutman et a l . , 1971;  Lee  Thomsen, 1970), and  build-  s e v e r e l y a f f e c t e d mice which have a  i n the ADH-induced p e r m e a b i l i t y  permeability  i n the  mildly affected  diuresis  the DI 0s/+  reported  Stewart,  807, r e d u c t i o n o f nephrons which r e s u l t s i n an  defect  renal  release.  mice which have an (Stewart,  in  (1972) have suggested  p a r a t h y r o i d hormone s t i m u l a t e d  Naik & V a l t i n ,  interstitial  a defect  In human nephrogenic  There a r e 4 types o f mice w i t h v a s o p r e s s i n - r e s i s t a n t  insipidus:  and  i n the ADH-induced  Fichman & Brooker  production  et a l . , 1964;  tubule,  osmolality.  v a s o p r e s s i n - r e s i s t a n t diabetes  i n mice ( F a l c o n e r 1969).  AMP  of the d i s t a l  interstitial  i n the ADH  insipidus  p o s s i b l e mechanisms f o r t h i s d i s e a s e are a  i n s i p i d u s the b a s i c d e f e c t  permeability  up  Two  i n the ADH-induced p e r m e a b i l i t y  build-up  that  dehydration.  ADH.  aminopterin  toxicity  et a l . , 1971;  renal  Singer  (Bergmann et a l . ,  et  1971;  Rabasa et a l . , 1970). 4.  Resistance  of the osmoreceptors to plasma h y p e r t o n i c i t y .  In  this  4 defect, even i f an adequate stimulus is given, and the hypothalmohypophyseal system is capable of producing ADH,  the osmoreceptors do not recognize the  stimulus and/or cannot signal the hypothalmohypophyseal system to release ADH. 5.  An abnormal inactivation of ADH.  While Hankiss et a l . (1961) and  Richards & Sloper (1969) have reported cases in man and dogs supposedly caused by abnormal inactivation, i t has not yet been proven. 6.  Electrolyte imbalances such as potassium deficiency (Hollander &  Blythe, 1971) or calcium excess (Epstein, 1971).  In both defects the  patients cannot tolerate dehydration or respond to exogenous ADH.  However,  a potassium deficiency can also be a secondary result of prolonged polyuria (Mohring et a l . , 1972a; Thomsen, 1970). Severe polydipsia-polyuria has been reported in the SWR/J strain of mice (Hummel, 1964).  The cause of the polydipsia has not been reported, but  i t does seem responsive to exogenous ADH (Hummel, personal communication 1972).  Dunson & Buss (1968) reported a strain of chickens with hereditary  polydipsia-polyuria which was responsive to ADH.  However, in 1972, Dunson et  al. reported that the diabetes insipidus was nephrogenic; they give i t strongly resembles primary polydipsia.  but from the data  Finally, Jay (1963) has  reported that the JFY strain of hamsters has polyuria or diabetes insipidus, but gives no other information about the disease.  Mild polydipsia-polyuria is caused by a number of diseases including diabetes mellitus, chronic renal disease, polycystic disease of the kidneys, and amyloidosis.  The De/J strain of mice has a mild polydipsia-polyuria  which is secondary to amyloid deposits in the kidneys and adrenals (Chai & Dickie, 1966).  Polydipsia-polyuria is also caused by the kidney diseases  5 n e p h r o n o p h t h i s i s and sidered  to be  the  i n Europe, was  medullary c y s t i c disease.  same e n t i t y a l t h o u g h n e p h r o n o p h t h i s i s was  considered  to be  as m e d u l l a r y c y s t i c d i s e a s e familial, disease  and  had  These d i s e a s e s  was  familial, first  and  had  reported  large medullary cysts  exogenous ADH  i n the  same way  m e d u l l a r y c y s t s ; where-  1971).  react  that  con-  reported  not  considered  Patients with this  to d e h y d r a t i o n  as p a t i e n t s w i t h nephrogenic d i a b e t e s  L y o n & H u l s e (1971) have r e p o r t e d defect with mild  first  i n America, was  (Strauss,  have s t r u c t u r a l l y abnormal k i d n e y s , and  few  are now  and insipidus.  the kd mice have a h e r e d i t a r y  kidney  p o l y d i p s i a - p o l y u r i a which c l o s e l y resembles human nephro-  nophthisis. Thus p o l y d i p s i a - p o l y u r i a can i n man  and  animals.  There are a number of animal models now  which have p o l y d i p s i a - p o l y u r i a due These models are u s e f u l renal physiology vasopressin  and  and  neuroendocrinology,  the drugs c h l o r o t h i a z i d e and  been used to study the  ing  p a t h o l o g i c a l mechanisms i n  the p i t u i t a r y - t h y r o i d a x i s ; i n s i p i d u s and  absence of ADH.  the  diseases.  reported  the one  One  that  neuron - one  the c a u s a l  o f the  models  r a t s have  hormone  hypothesis;  plays  a role in regulati n hypothalmic  the c o n c e n t r a t i o n  (Valtin,  first  mecha-  these DI  DI r a t s are a l s o used as a b i o a s s a y  they are more s e n s i t i v e than normal r a t s  of  insipidus in rats  impaired response to ADH  primary p o l y d i p s i a ; and The  the mode of a c t i o n  i n studying  i s a c o r t i c o - t r o p i c r e l e a s i n g f a c t o r ; i f ADH  diabetes the  following:  studied  o f the above mechanisms.  hypothalmic diabetes  V a l t i n (1967) has  being  diseases  c h l o r p r o p a m i d e which sometimes  i n s i p i d u s ; and  involved hereditary  ( V a l t i n et a l . , 1962).  i n one  i n studying  p o t e n t i a l therapy of the v a r i o u s  to be d e s c r i b e d  i f ADH  to a d e f e c t  i n e l u c i d a t i n g normal and  a c t as a n t i d i u r e t i c s i n d i a b e t e s nisms and  be a symptom of a l a r g e number o f  1967), and  of u r i n e  f o r ADH  since  to study  mode of a c t i o n of c h l o r p r o p a m i d e ( M i l l e r & Moses, 1970a, 1970b) and  in  the  chloro-  6 thiazide (Laycock et a l . , 1972).  Since these rats were only reported 10  years ago, these selected uses illustrate that there is a great demand for well described genetic models. B.  Genetics Genetically determined variation can be either continuous or discon-  tinuous.  Continuous variation usually indicates that there are a large  number of genes involved in the characteristic being studied. is a continuous variable.  Water intake  There is a great range in water intake between  normal individuals and this is determined by polygenic inheritance. there are also extreme deviants which are inherited.  However,  The deviants, when  severe enough to be considered pathological, are usually due to major genie differences  (Thoday, 1967).  These extreme deviants may have occurred spon-  taneously and been selected for in inbred lines, or have been produced by using mutagens.  An example of an extreme deviant in water intake is repre-  sented by the Brattleboro rats which have a hereditary hypothalmic diabetes insipidus defect which is determined by an autosomal recessive gene (Valtin et a l . , 1962).  This recessive gene causes a discontinuous variation with no  overlap between the water intake of normal rats and that of rats with hereditary hypothalmic diabetes insipidus.  However, many common diseases which  occur in later l i f e appear to have a polygenic inheritance.  Cited examples  of polygenic human diseases are schizophrenia, ischemic heart disease, rheumatoid arthritis, and diabetes mellitus (Carter, 1969). The many genes which control water intake each have a specific action. These genes are considered quantitative because of the level of phenotypic classification,  and at a different level of observation they would have a  qualitative action (Spicket et a l . , 1967).  For example, X-zone degeneration  in mice appears to be determined by at least 2 genes (Spicket et a l . , 1967). However, i f the " i n vitro" synthesis of corticosteroids  from a precursor is  used as an indication of adrenal activity, then this characteristic  is a  continuous variable and appears to be inherited in a polygenic manner.  Thus  an increase in the specificity of phenotypic classification decreases the complexity of the genetic system until eventually a character is reached which is inherited in a classical mendelian manner.  8 MATERIALS & METHODS  A„  Animals Three strains of the house mouse, Mus musculus, were used in this study.  The SWV strain was f i r s t obtained from the Defense Research Board, Suffield, Alberta in 1949.  It was maintained as a closed colony at the Central Animal  Depot, U.B.C, until 1959, and then inbred by brother-sister matings. The SWV mice used in this study had reached F^Q to F ^ of inbreeding.  The strain  had been selected for good reproduction and the polydipsia-polyuria was f i r s t noted in 1962 in this laboratory (Miller, 1964). There were 2 inbred strains of mice used as controls for the SWV. The major control was the C3H/M1 strain (henceforth referred to as C3H-1). These animals were obtained by the Central Animal Depot from Rockland Farms in 1956.  They were maintained as a closed colony until 1959 when a brother-  sister mating program was initiated. inbreeding were used in this study.  Mice which had reached F25 to Fg^ of In 1969 the pure strain was lost due to  poor reproduction, but another line which had been derived from outcrossing the C3H/M1 strain to an oel line in 1967, was used instead.  The C3H/sph  line (henceforth referred to as C3H-2) has been inbred since 1967 but is s t i l l only a strain in development.  The mice which were used in this study  ranged from F^ to F-^ of inbreeding.  The term C3H w i l l be used when both  C3H-1 mice and C3H-2 mice were used in the experiment being discussed. The secondary control was the C57B1/6M1 (henceforth referred to as C57) strain which was received from the Jackson Laboratory in 1956 at F ^ of inbreeding.  It has been maintained by brother-sister matings in this labora-  tory and the mice which were used in this study ranged from F23 to ?28'  The mice were maintained in the Zoology Vivarium during this study. They had free access to feed and water ad libitium and were kept under a light regime of about an 8 hour working day.  In 1970 a time clock was in-  stalled, and a light-dark regime of 17 hours and 7 hours respectively was followed.  The temperature was 21-23°C and the relative humidity was 55-65%.  The mice were fed Buckerfields' mouse ration U.B.C. 6-63 (ration 1) until December 1969 when they were switched to Purina Lab Chow (ration 2), because Buckerfields would no longer supply their ration.  The formulations of the  2 rations are given in Appendix 1. B.  Description of Polydipsia-Polyuria Defect (1) Water Balance In order to define the severity of the polydipsia-polyuria defect in  the SWV mice, a water balance study was done on SWV females, SWV males, C3H females, and C3H males of varying ages.  Measurements of water intake, urine  output, feed intake, and urine concentration were recorded for these mice using metabolism cages designed by Wood & Nishimura (1968).  Two minor modi-  fications of these cages were made: (i)  a pint milk bottle with a long glass delivery tube was used for a watering device instead of a 10 ml pipette;  (ii)  in the central chamber the expanded aluminum floor was held in place by a plastic strip riveted to the inner lower margin of the central chamber.  The metabolism cages made i t possible to collect urine which was free of any contamination by feed or feces.  These cages were also used in the water  deprivation and vasopressin experiments to be discussed later. work ration 1 was used.  For a l l this  10 The  f o l l o w i n g r o u t i n e was  animals were p l a c e d  used to determine the water balance.  i n the metabolism cages on F r i d a y  might a c c l i m i t i z e over the weekend. spring-loaded  f e e d e r s was  Four, 24  make.  weight was  The  a f t e r the 24  and  0.1  light,  g.  The  urine  was  a  n  (  during  l feed  feed  they from  f o r the animals The  hour p e r i o d , and  obtained  (UyQjO >  water i n t a k e , u r i n e output  nearest  adjustment  hour runs were done s t a r t i n g on Monday.  measured b e f o r e  that  problem of o b t a i n i n g  the most d i f f i c u l t  marked change i n body weight the data The  i n order  The  the to  a n i m a l s ' body i f t h e r e was  a  the run were e l i m i n a t e d .  i n t a k e were measured to  the  c o l l e c t e d at room temperature, under white,  domestic, p a r a f f i n o i l .  I t was  then c e n t r i f u g e d and  frozen for l a t e r  analysis. The The  u r i n e c o n c e n t r a t i o n was  e x p r e s s e d as  o s m o l a l i t y o f a s o l u t i o n depends upon the number of p a r t i c l e s  i n t h a t s o l u t i o n but physiologically, ability,  not  on the p a r t i c l e ' s s i z e ,  urine osmolality  as mOsm s o l u t e / k g water.  stored  temperature, o  u  l d  The  shaken thoroughly,  and  be w i t h i n the range o f 100  correct dilution, index on a T  g  Osmette model U  (Ogg^)•  dissolved Thus  concentrating  s i n c e the e l e c t r o l y t e s c o n t r i b u t e more to the u r i n e o s m o l a l i t y albumin or u r e a .  w  charge, or shape.  i s a good r e f l e c t i o n o f r e n a l  do m o l e c u l e s such as g l u c o s e ,  ^OSM  the u r i n e o s m o l a l i t y  f r o z e n u r i n e was  2007.  Duplicate, The  0.2  o s m o l a l i t y i s expressed thawed, warmed to room  d i l u t e d w i t h d i s t i l l e d water so t h a t -  the c o n c e n t r a t i o n was  meter.  Urine  ml  1000  mOsm/kg.  estimated  In order  the  to make the  by measuring the  samples were read  o s m o l a l e x c r e t i o n was  than  refractive  on a P r e c i s i o n  c a l c u l a t e d as U _ „ OSM  w  times  V0L* (2)  Water Intake Water balance d e f i n e s  the p o l y d i p s i a - p o l y u r i a d e f e c t v e r y  accurately,  11 but the procedure is time consuming.  The measurement of water intakes alone  is much quicker and therefore more animals can be tested, and/or an animal can be tested at several different ages. both sexes of the SWV,  Water intakes were measured on  C3H, and C57 mice which had been retired from the  breeding colony or from crosses which were being discontinued. takes were measured at irregular intervals.  The water in-  At the time of testing, the age  and body weight of the mice and the type of feed they were on were recorded. The mice were placed into individual pans and allowed to acclimitize for 3 days.  Then 4, consecutive, 24 hours water intakes were measured.  As  in the metabolism cages, the body weight of the animal was measured before and after each 24 hour period, and i f there was a marked change the data for that period were discarded.  The water intake was measured by weighing the  water bottle before and after the 24 hour period.  The feed consumption was  ad libitium and was not measured. (a)  Influence of Parity in Females  Four SWV females which had never produced offspring (nulliparous) were put aside in a pan and aged.  Four SWV females which had been mated to a  single male and whose l i t t e r s had been recorded and discarded at birth (multiparous) were also aged.  The water intakes of these females were  measured at 9, 11, 14, 15, and 16 months of age.  The male had been removed  at 8 months so that the females would not be pregnant during testing. nulliparous C3H-1  Four  females were also aged and their water intakes measured at  8, 10, 11, 12, 14, and 15 months of age.  A l l these females were fed ration 1.  The results from this f i r s t test suggested that the multiparous SWV females drank more than the nulliparous one (p. 55 ).  Therefore a second more  comprehensive test was done, but ration 2 had to be used instead of ration 1. More nulliparous and multiparous SWV females were put aside and aged.  In  order to determine i f there was a difference in water intake between n u l l i parous and multiparous normal females, both types of C3H-2 and C57 females were used.  The water intake was measured once a month starting at 4 months  of age in the nulliparous females and at 6 months of age in the multiparous ones.  The experiment was terminated at 16 months.  The multiparous females  were also compared to lactated females (ie., multiparous females which had nursed their offspring) and whose water intakes had been measured at irregular intervals (p. 11). (b)  Influence of Stress in Males  Males which are housed in groups often fight.  Harris (1969) noted that  KK males which had been fighting never became glucosuric:  ie. did not express  diabetes mellitus. Thus ageing males in groups set up a stressful situation which altered the expressivity of the diabetes mellitus defect in the KK strain of mice.  Most of the SWV and C3H males which were available for study  had been housed singly or with females; whereas the progeny from the genetic testcrosses were housed in groups.  Could the single males' water intakes be  used as standard values against which the grouped male progeny's water intakes could compared, or would a difference in housing affect the water intake.  To determine this, groups of male sibs from the 3 strains were put  aside at weaning and aged.  They were fed ration 2.  measured once a month from 4 to 16 months of age.  Their water intake was At the end of a week in  which the water intakes were measured, the males were placed back in their original groups.  These grouped males were compared to males which had been  used for breeding in the colony, housed individually or with females, and tested at irregular intervals (p. 11).  13 C.  Differential Diagnosis of Polydipsia-Polyuria Defect (1)  Serum Osmolality  The serum osmolality (S ) was measured to determine i f the mice were osnr chronically dehydrated (as reflected by a high S ) or over hydrated (as J  x  Q s m  reflected by a low Sosnr). The Sosm was measured in mice which had had free J  access to water.  Four groups of mice were used:  males, 21 C3H females, and 14 C3H males.  30 SWV females, 21 SWV  Unless otherwise indicated these 4  groups of mice were used for a l l subsequent tests.  Blood was collected from  the suborbital sinus using melting point capillary tubes which contained no anticoagulant.  It was then put into sterile, disposable, polystyrene, 12 x  75 mm tubes, left for serveral hours to clot, and centrifuged in an International centrifuge model HN at 6000 RPM for 20 minutes.  The serum was drawn  off, placed in plastic microfuge tubes, frozen, and stored.  The samples were  then thawed, warmed to room temperature, shaken thoroughly, and diluted 1:1 with d i s t i l l e d water in order to make a 0.2 ml sample.  The osmolality of one  sample was measured twice using a Precision Osmette model 2007. (2)  Response to Dehydration  (a) Water Deprivation The water balance data suggested that the polydipsia-polyuria is severe enough to be diabetes insipidus.  To determine i f the polyuria is  primary or secondary, the animals were dehydrated.  Dehydration increases the  serum osmolality which stimulates the hypothalmo-hypophyseal lease antidiuretic hormone.  system to re-  In the f i r s t test 6 females and 3 males of each  strain were dehydrated by withholding water for 7 hours.  The animals were  placed in the metabolism cages on Friday, and 4, 24 hour water balance runs were done the following week. On the morning of the experiment, each animal's  14 body weight was measured and its water bottle removed, but each animal s t i l l had feed ad libitium.  The cumulative urine volume was recorded at 2, 4, 6,  and 7 hours of dehydration and the urine osmolality and osmolal excretion were measured on the pooled urine from the first 4 hours and the last 3 hours of collection.  The body weight was measured at the end of 7 hours and the  per cent loss of body weight was calculated.  The serum osmolality was meas-  ured at the end of the 7 hour period and compared to the normal value that had been measured 3 to 4 weeks before.  A control experiment using 3 mice from  each of the 4 groups was done in exactly the same way except that the mice had water ad libitium. Three females from each strain were accidently dehydrated for 19 hours. The animals had been given 3, daily, injections of peanut o i l (part of the vasopressin experiment outlined below), but on the third day the water bottles were not put back onto the metabolism cages.  After 19 hours the per cent loss  of body weight, urine volume, feed intake, urine osmolality, and osmolal excretion were measured.  In addition 1 SWV female was accidently dehydrated  for 24 hours because an air lock occurred in the water bottle during a water balance run.  The same parameters were measured at the end of the 24 hours,  but also the gain of body weight over the next 24 hours of hydration was noted. (b)  Saline Ingestion  In another method of dehydration, 4 mice of each sex from the SWV and C57 strains were given physiological saline (0.97«) instead of tap water. Since water intakes alone were measured, this test required much less time than the previous water restriction experiment, and therefore it was possible to observe the effects over a longer period.  Tap water was given for 4, 24  15 hour runs, and then physiological saline was substituted. and body weight were measured daily.  The fluid intake  The design was to k i l l 2 animals from  each group after 6 days of saline and the remaining 2 after 20 days; but i f animals could not tolerate the saline and were losing a large per cent of body weight, those mice plus the appropriate controls would be killed. (3)  Response to Exogenous Antidiuretic Hormone  The dehydration results suggested that the defect was a primary polyuria (p. 69).  The next step was to determine i f the SWV mice could respond  to exogenous antidiuretic hormone (vasopressin).  For this test 7 females and  6 males from the SWV and C3H strains were given pitressin tannate suspended in peanut o i l (Parke Davis).  A l l the animals were put into metabolism cages,  and the f i r s t week 4 control water balance runs were done.  During the second  week 3 mice from each group were tested for their response to the vehicle, peanut o i l .  On day 0 a control water balance run was done and then 3, con-  secutive, daily, intramuscular (i.m.) injections of 0.05 ml peanut o i l were given to each mouse.  The feed intake, water intake, urine volume, urine  osmolality and osmolal excretion were measured for the 24 hours period following each injection. 3-4  weeks.  The mice were then returned to their pans and rested for  In the final week a l l the mice were put into the metabolism  cages on Friday, and on Monday (day 0) a control water balance run Was done. Then 3, consecutive, daily, i.m. injections of \ unit pitressin tannate in o i l were given and the same measurements for each subsequent 24 hour period were taken. A preliminary test had been done on 2 C3H-2 females and C3H-2 males in the same way, except that the vehicle was not tested and that 1 female and 1 male received the pitressin tannate subcutaneously (s.c.) instead of i.m.  16 Since the 2 mice who received the drug i.m. responded more than those who received i t s.c. (p. 72 ), the i.m. route of administration was used in the main experiment.  The procedure agreed with that recommended by Parke Davis  and by Brazeau (1970). (4)  Similarity to some Kidney Diseases  The results from the vasopressin injections suggested that the defect was in the kidney (p. 74); therefore the next step was to determine i f the polyuria was secondary to a kidney disease. (a)  Urinalysis  Combistix test strips (Ames) were used to test for glucose, protein, and a change in pH in the urine of 10 mice from each of the 4 groups.  The  stored frozen urine, which had been collected using the metabolism cages (p. 10) was thawed, warmed to room temperature, and shaken thoroughly. The test strip was dipped in the urine, held for 10 seconds and read  by comparing the  colour of the test strip to that on the colour chart. Hemastix test strips (Ames) were used to test for the presence of occult blood in the urine of 6 mice from each of the 4 groups.  The frozen,  stored urine was again thawed, warmed to room temperature, and shaken vigorously.  The test strip was dipped briefly in the urine, held for 30 seconds,  and read by comparing the colour of the test strip with that on the colour chart. (b)  Per Cent Packed Cell Volume  Cystic disease of the renal medulla often has anaemia as one of i t s symptoms; therefore packed cell volumes were measured in females and males from the SWV and C3H-2.strains. Mice of 6, 8, 12, and 15 months of age were  17 used to determine i f the per cent packed cell volume correlated with the severity of the polydipsia-polyuria defect.  The blood was collected from the  suborbital sinus using microhematocrit tubes.  Duplicate samples for each  animal were centrifuged in an International centrifuge, model HN, with a microcapillary head, model 327, for 15 minutes at 6000 RPM; and then read on an International microcapillary tube reader, model C.R. (c)  Blood Urea Nitrogen  Cystic disease of the renal medulla (nephronophthisis) often also has a high blood urea nitrogen (BUN); therefore the BUN of 10 of each of the following were tested at 13 months of age: females; and C3H-2 males. tested at 15 months.  SWV females; SWV males; C3H-2  Three SWV females and 4 C3H-2 females were also  The BUN was tested by Azostix reagent strips (Ames)  which are designed for screening rather than for precise analytical measurements.  Whole blood was collected from the suborbital sinus using micro-  hematocrit tubes.  The entire reagent area of -the strip was covered by a  large drop of blood.  After 60 seconds, the blood was quickly washed off with  a sharp stream of d i s t i l l e d water for up to 2 seconds.  The reagent strip was  shaken to remove any excess water and read immediately by comparing its colour with that on the colour chart, making sure that any glare was eliminated. (5) Histology Animals about a year old were k i l l e d by cervical dislocation after light ether anaesthesia.  The head was quickly removed, the dorsal cranial  bones overlying the brain were removed, and then the whole head was immersed in calcium formol.  The adrenals were removed and fixed.  Then the kidneys  were removed, weighed, sliced longitudinally in half, and fixed.  At least 2  days later the pituitaries were removed from the brain case and returned to  18 the fixative.  A l l tissues were fixed in calcium formol and embedded in  paraffin using chloroform as the clearing agent. (a)  Pituitaries  The results from the vasopressin experiment suggested that the SWV could not respond to exogenous vasopressin and that the defect was probably in the kidney (p. 74). • To substantiate this the posterior pituitaries were examined to determine i f they had an adequate amount of endogenous vasopressin, which was determined by the presence of neurosecretory material.  Pituitaries  from 4 SWV females and 3 C57 females were serially sectioned at 6 JU and stained with aldehyde fuchsin which stains the neurosecretory material a dark purple (Sloper, 1966).  In preparing the aldehyde fuchsin, the method of  Cameron & Steele (1959) was used since it has the advantage of producing a rather stable staining solution of paraldehyde and basic fuchsin.  The rela-  tive width and height of the posterior lobe, intermediate lobe, and entire pituitary were measured to estimate the size relations of the 3 lobes.  The  area of each lobe was estimated independently using the section with the largest area.  The data were expressed as the ratio of:  width x height of posterior pituitary width x height of pituitary and as the similar ratio for the intermediate pituitary. (b)  Adrenals  The adrenals from 10 SWV females, 4 C57 females, and 5 C3H females were examined.  The adrenals were sectioned at 6 fJ and stained with haema-  toxylin and eosin. (c)  Kidneys  Both kidneys of 10 SWV females,  11 C57 females,  7 SWV males, and 7  19 C57 males between the ages of 11% and 13% months were examined.  In addition  both kidneys of 3 SWV females at 4, 5% to 6, 9, and 17 months of age were examined.  The kidneys were sectioned longitudinally at 7  the tip of the papilla was included in the section. Harris  1  making sure that  They were stained with  haematoxylin and eosin.  In addition to those kidneys examined histologically, wet kidney weights were collected from SWV, C57, and C3H-2 females and males that were autopsied at varying ages.  The mice had no gross signs of tumours or infection at the  time of autopsy. (6)  Statistics  Unless otherwise stated, the following general statistical tests were used:  an analysis of variance with Scheffes multiple comparisons with un-  equal sample sizes, a linear regression analysis, or an analysis of covariance. The data from the saline dehydration experiment, the vasopressin route of administration experiment, and the kidney weights were analyzed using a two tailed _t test. D. Genetics of Polydipsia-Polyuria Defect (1)  Mating Scheme  The SWV strain was crossed reciprocally to the C3H-2 strain as outlined in Figure 1 (a & b).  Some of the matings were done in the first year and  those animals were fed ration 1 (Figure la).  In the second year some of the  matings were repeated to increase the sample size and additional matings were made (Figure lb).  The animals which were used in the second year were fed  ration 2. In the f i r s t year matings were set up to produce reciprocal F^, F2, and BC5^y mice (Figure la).  The F-^ and BCg^y animals became available to me  20  SWV (3) 0  SWV/C3H-2 (2)  SWV  rec iprocal backcross  C3H-2 (1) •  SWV/C3H-2 (2) 0  SWV/C3H-2 (1) •  BC SWV  rec iprocal cross  C3H-2 (1) 0  C3H-2/SWV (2) P  SWV (1)  C3H-2/SWV (1) •  Figure la. Diagram of reciprocal crosses done between the SWV and C3H-2 strains in the f i r s t year. F^, F2, and BCg^ progeny were produced. The numbers in brackets were the number of mice mated. 0 = female, • = male.  21  SWV (1)  C3H-2  1  SWV (1)  SWV/C3H-2  (2)  ?  SWV/C3H-2 (3)  rec iprocal backcross BC  SWV  reciprocal cross  Figure lb.  C3H-2 0  (1)  (2)  C3H-2 • 0  (1)  SWV/C3H-2  •  backcross  backcross  BC  BC  C3H-2  (1)  C3H-2  SWV (1) •  Diagram of reciprocal crosses done in the second year to increase the sample size of each sex to 15 in the reciprocal F-^ and BCgwv mice. The BCc3H-2 m L c e were also produced. The numbers in the brackets were the number of mice mated. 0 = female, • = male.  22 when they were about a year old.  There had been no selection for or against  polydipsia in those F^ and BCgyy mice.  Twenty-four F 2 females and 12 F 2  males from each reciprocal cross were put aside at weaning to be tested.  Only  SWV/C3H-2 hybrids were used in the backcrosses to the SWV and C3H-2 strains^ both in the first and second years. In the second year new matings were set up to increase the sample to 15 of each sex in the reciprocal F^ and BC y animals. At the same time the SW  F^ animals were reciprocally backcrossed to the C3H-2 strain to produce the BC^ou C Jn-  n  2  mice.  Sixteen to 18 progeny of each sex from each reciprocal cross  were put aside at weaning so that a sample size of 15 animals would be available for testing. (2) Testing Progeny The 301 progeny were set aside at weaning to be aged in groups of 4, 5, or 6 nulliparous females and 3, 4, or 5 male sibs. males which were set aside were already multiparous.  A few of the F-^ feThe method of deter-  mining i f an animal was normal or polydipsic-polyuric was to measure their water intake as described on p. 11. In the f i r s t year water intakes were measured at 12 and 18 months of age in the F, progeny; and at 14 to 16% months of age in the F I  progeny.  7  and BC  £  SWV  Those F 2 and BCg^y progeny which were not definitely polydipsic  and which were s t i l l alive at 19 to 20 months of age were retested. In the second year, i t was decided to test an animal several times. The age of onset of the polydipsia varied between animals and sometimes was as late as 17 to 18 months, but since a number of progeny in the first year died before that age, the progeny in the second year were tested periodically after 9 months.  The following procedure was used for the F.., BC  , and  23 BC^ou  o  mice in the second year:  the mice were put aside and aged in the  same manner as in the first year; water intakes were measured at 9, 12, 15, and 16 months of age; and mice which were not definitely polydipsic at 16 months of age were tested once a month until they became polydipsic or were 19 to 20 months old. (3)  Analysis of Progeny  (a)  Quantitative Estimate of Number of Loci  The water intake data were analyzed to estimate the number of loci determining the polydipsic trait using Wright's formulas (Wright, 1968). The means, variances (s ), and coefficients of variation (CV) were calculated for each generation.  These statistics were calculated separately for each sex  and for each ration.  The water intake data were also transformed using  logarithms, and the means and variances were calculated for the transformed data.  These statistics were calculated for each reciprocal cross, and the  reciprocal matings of each generation were tested for heterogeneity.  A _t  test was used for the means and an F test or Bartlett's test for heterogeneity was used for the variances (Sokal & Rohlf, 1969). For each sex the number of loci was estimated from the BC  and  CjH-2  SWV  BC  d a t a  u s i n g  W r i  S  n t  '  s  formula:  n  = (F]_ - ^ i )  2  4 (BC s  2  X  where:  - E s) 2  n = number of l o c i by which F^ differed from P^ F-^ = mean water intake of the F-^ progeny P^ = mean water intake of the parental strain to which the F^ were backcrossed.  2 2 2 • E s. = environmental s which was equal to F^ s or an average of the F^, Pp and ?  2  variances.  24 This formula was only used when a l l the values required were from progeny fed the same ration. female F  In addition the number of loci was estimated using the  data by the formula:  2  - 2 (P - P]_)  _  n  2  '8  where:  (F s 2  E s = \ (P]_ s + P 2  2  2  - E s) 2  s + 2 F 2  2  s) 2  L  Wright's formulas are based on the assumption that the genes are additive, semidominant, and that there is no interaction. the  These assumptions result in  following relations: (1)  Y  = h (P  (2)  BC  (3)  F  (4)  BC^ s = BC  (5)  F  (6)  BC s  l  2  l  = h  = F  l  +  (F + x  s  2  P  1  )  and  BC  2  = % ( ? + p") 1  2  = h (BCi + BC )  x  2  2  2  P2)  >  s = k F 2  2  Fi s  2  s  2  2  9  9  = environmental s + genotypic s  9 l  Only i f these assumptions are met, is a reliable estimate obtained of the number of l o c i determining the trait (Wright, (b)  1 9 6 8 ) .  Qualitative Estimate of Number of Loci  A second estimate of the number of loci determining'the polydipsic trait was made by calculating the frequency of affected progeny in each generation.  Again the data were calculated separately for each sex and for each  ration.  Since water intake is a quantitative trait, an arbitrary division  between normal and abnormal water intakes was chosen.  Since  95.46%  of the  normal population falls below the mean + 2s, this value was chosen as the upper limit of normal water intakes.  Thus any animal which had a water in-  take greater than the mean + 2s of the C3H-2 was considered polydipsic.  This  upper limit was calculated separately for each sex and each age (month) from  25 9 to 20=months of age.  Using these values, an animal could be classified by  comparing its water intake to the upper limit appropriate for its sex, and ration.  age,  An animal which was polydipsic at any age, even i f before or  after that age the water intake was normal, was classified as polydipsic. The frequency of affected progeny was determined for each dam of each generation. In addition, the frequency of polydipsic SWV mice was determined. The heterogeneity G statistic  (Sokal & Rohlf, 1969) was used to test  for heterogeneity between the reciprocal matings of the F^, F2, BC^^, and ^C3H-2 generations.  In addition the frequency of polydipsic male F^ progeny  was tested for heterogeneity between those males fed ration 1 and those fed ration 2.  The frequencies of polydipsic F2 and ^Q2U-2 P r o g e n v  w e r e  tested  for their goodness of f i t to a 1 gene and a 2 gene model using the replicated goodness of fit test (G statistic).  It was possible to test for heterogeneity  between dams and for goodness of f i t on pooled data using this  statistic,  since G„ _ -, = Gpooled 1 + G,heterogeneity . .. v(Sokal & Rohlf,' 1969). total '  The male  progeny fed ration 1 were also tested in this manner except that the expected frequencies were calculated by multiplying the penetrance (penetrance being defined as the per cent of the mice with the polydipsic genotype which have the polydipsic phenotype) in the SWV strain by the theoretical frequency for each generation.  The frequency of affected F 1 and BC  male progeny fed  ration 1 were also tested using a G test with the expected frequency being corrected for reduced penetrance.  The rest of the F^ and B C ^ ^ progeny were  not tested s t a t i s t i c a l l y since none of the statistical goodness of f i t tests can be used i f one of the expected classes is 0 or 1007o. (c)  Age of Onset  The average age of onset of polydipsia was calculated for each genera-  26 tion in those polydipsic progeny fed ration 2. sex were analyzed separately.  Once again the data for each  The earliest age of testing was 9 months of  age; therefore it was not possible to detect an earlier age of onset than 9 months.  In addition the progeny were only tested at 9, 12, 15, and 16 months  of age; therefore over this age period only a rough estimate of the age of onset could be determined.  The same arbitrary division point (mean + 2s of  the C3H-2) was used to classify  the progeny's phenotype.  27 RESULTS  A.  Description of Polydipsia-Polyuria Defect (1)  General Description  The females of the SWV strain weighed about 33 g whereas the females of the control C3H-2 strain weighed about 26 g. The SWV females had a high reproductive success and consequently are used as a production strain. The females were discarded from the breeding colony at 9 to 12 months of age at which point they were drinking a large volume of water (polydipsia) and were starting to appear dehydrated even though they had had free access to water. The dehydrated animals sometimes had a distended abdomen as seen in Figure 2a. It was also noted that as the SWV females became dehydrated, they developed very rapid, shallow breathing.  The polydipsia did not affect the females'  high reproductive success up to 12 months of age, but the females died early, at about 16 months.  The SWV females survived longer i f particular care was  taken that they always had an ample water supply. Some SWV females also showed a muscle weakness in the hind limbs. In an affected SWV female, the hind limbs were extended anteriorly at an odd angle and the toes were claw shaped (Figure 2a), compared to those of a normal albino female (Figure 2b). used for comparative purposes.  An albino female of the A/WySn strain was  The SWV females were not able to bend their  knees and bring the limbs back posteriorly into a normal position.  When the  females moved forward, the hind limbs were dragged, rotated posteriorly until they were at right angles to the hips and then rotated back to the original anterior position.  This 90° rotation occurred with each forward movement.  It was observed 'that this muscle weakness shown by the SWV females was  Figure 2b.  6 month o l d c o n t r o l  A/WySn female w i t h  normal h i n d  legs.  29 different than that shown in muscular dystrophy in which the hind limbs extend posteriorly and cannot be brought forward. The SWV males weighed the same as the SWV females, signs of:  but did not show any  a large increase in water intake, dehydration, muscle weakness, a  shorter l i f e span or rapid shallow breathing.  One 20 month old SWV. male,  which had developed severe diarrehea, showed a mild form of the muscle weakness. (2)  Water Balance  There was no significant difference in any of the 4 groups between the 4 days of control water balance data obtained during the vasopressin experiment (p. 75, 76, 77, & 78). Therefore the 4 days' data from each animal were averaged and the means were used for the analysis of the water balance data (Table I).  There was, however, a tendency for the water intake, urine out-  put, and feed intake to increase while the urine osmolality decreased over the 4 days (p. 75, 76, 77, & 78). The SWV females' water intake of 35 ml/24 hrs was 5 times greater, their urine output of 27 ml/24 hrs was 9 times greater, and their osmolal excretion of 7.8 mOsm/24 hrs was double that of the control females and males; whereas their urine osmolality of 327 mOsm/kg was only 1/7 as concentrated as that of the control males. different.  A l l these parameters were highly  significantly  There was no difference in feed intake between the 4 groups.  The SWV males had a significantly lower water intake, urine output, and osmolal excretion than the SWV females, double those of the controls.  but these values were s t i l l almost  The SWV males' urine osmolality of 1026 mOsm/kg  was only \ as concentrated as that of the control males, and it was significantly lower than those of the control females and males.  TABLE I.  Water balance values + standard error (sy) for the SWV and C3H strains. the urine osmolality (U0sm), it was given in brackets.  Sex  Age (mo.)  SWV  F  10-15.5  17 35.2 + 2.5 A , B ,C  SWV  M  10-15.5  12 12.9+1.7  Strain  N  Water Intake (ml/24 hrs)  Urine Output (ml/24 hrs) 27.0 + 1.9A>B> c 6.0+0.9  Uosra N (mOsm/kg) 327 + 2 5 . 4 A , B , C Q5D 1026+62.0 A > B  If N was smaller for  Osmolal Excretion (mOsm/kg hrs) 7.8+.4 A > B > c  Feed Intake (g/24 hrs) 3.6 + .2  5.4 + . 5  3.5 + . 3  CIO]  C3H  F  10-15.5  13  6.8+0.6  2.5+0.4  1732+ 147.2A El 11  3.6 +.5  2.8 + . 2  C3H-2  M  10-15.5  9  6.6+0.9  2.0+0.3  2324+194.9  4.3 + . 4  3.1 + . 3  C3H-1  F  10-15.5  7  8.3+0.5°  3.3+0.5°  1243+50.2°  4.1 + . 7  3.1 + . 3  C3H-2  F  10-15.5  6  5.0 + 0.5  1.5 + 0.3  2139 + 68.5  3.2 + .6 .  2.4 + .3  C3H-2  F  16-21  11  7.1+0.8  2.8+0.5°  1766+455.6  4.2 + . 8  2.2 + . 2  C3H-2  M  16-21  11  7.4 + 1.0  2.6 + 0.5  1638 + 162.1A  4.0 + . 5  2.6 + . 3  A B C D  = = = =  significantly significantly significantly significantly  different different different different  when when when when  compared compared compared compared  to to to to  C3H-2 C3H SWV C3H-2  males females males females  (10-15.5 (10-15.5 (10-15.5 (10-15.5  mo.) mo.) mo.) mo.)  C53  UQ  p< p< p< p<  0.05 0.05 0.05 0.05  At 10 to 15.5 months the values for the C3H females were the same as those for the C3H-2 males except that the urine osmolality was significantly lower.  The C3H females included both C3H-1 and C3H-2 females, and the C3H-1  females had a significantly higher water intake and urine output and a significantly lower urine osmolality than did the C3H-2 females.  Therefore  it was the C3H-1 females' urine osmolality of 1243 mOsm/kg that pulled down the C3H females' urine osmolality to 1732 mOsm/kg.  When the C3H-1 females  were substituted for the C3H females and the analysis of variance was redone, their urine osmolality was s t i l l significantly different from the C3H-2 males and SWV females,  but not from the SWV males.  The C3H-2 females' urine  osmolality of 2139 mOsm/kg was very similar to the urine osmolality of 2324 mOsm/kg in the C3H-2 males. Finally C3H-2 mice of 16 to 21 months were compared to the C3H-2 mice of 10 to 15.5 months.  In both sexes the urine osmolality had decreased in  the older mice, and in the males the difference was significant.  The sample  size for the old females was low (N=4), and the variance was quite high, which suggested that a larger sample size might show a significant  difference.  The increase in urine output in the old females was significant and there was a slight nonsignificant increase in osmolal excretion. The most important parameter was urine osmolality since the SWV males were significantly different from the SWV females and the controls by this criterion, whereas using water intake the SWV males were not significantly different from the controls. The urine from the 4 groups was clear and became progressively darker with increasing urine concentration. In summary:  the SWV females had a very severe increase in water turn-  over and a very severe decrease in renal concentrating ability.  The SWV  32 males had a milder increase in water turnover and a milder but significant decrease in renal concentrating ability. C3H-1  The results also showed that the  females had a mild but significant decrease in renal concentrating  ability; and therefore as controls they were not as good as the C3H-2 females. (3)  Water Intake  (a)  Age & Body Weight  Water intakes from SWV females, SWV males, C3H-2 females, and C3H-2 males at a number of different ages were analyzed using a multiregressional analysis to determine i f differences in age and/or body weight accounted for some of the variation in water intake.  The water intakes, body weights, and  mean ages of the 4 groups are shown in Table II and the regression equations for the 4 groups are shown in Table III. variable was age.  The most significant independent  There was a significant linear regression of water intake  with age in the SWV females, SWV males, and C3H-2 males, but not in the C3H-2 females. The percentage of variation in water intake accounted for by age was 30% for the SWV males, 23% for the SWV females, and 15% for the C3H-2 males. In the SWV females there was also a significant regression of water intake on age and body weight combined and these 2 variables accounted for 367» of the variation in water intake.  The values in the correlation matrix showed that  age and body weight influenced water intake independently very l i t t l e correlation between age and body weight.  since there was  In the other 3 groups,  however, body weight had no significant influence on water intake.  The water  intake of the C3H-2 control females was not influenced by age or body weight and the regression equation using age was not significant.  TABLE II. Means + s- for the water intake, age, and body weight of SWV and C3H-2 mice used in the ple linea? regression analysis. Age Range (mo.)  Water Intake + s(ml/24 hrs)  Age + s(mo.)  Body Weight + (g)  Sex  N  SWV  F  106  7.5 - 14.0  52.5 + 1.4  10.8 +0.2  32.9 + 0.3  SWV  M  106  7.5 - 14.0  16.1 + 0.4  10.6 + 0.2  33.0 + 0.2  C3H-2  F  39  9.0 - 13.0  6.4 +0.2  11.4 +0.3  26.2 +0.5  C3H-2  M  36  8.0 - 15.5  7.3 + 0.4  11.4 + 0.4  33.6 + 1.0  Strain  TABLE III.  Strain SWV  Y  y  Multiple linear regression analysis of the dependent variable water intake (W.I.) and the independent variables age and body weight (B.W.) in the SWV and C3H-2 mice.  Sex F  2 r Age Age & B.W.  F prob. Age B.W.  .23  .01  .36  .01  Correlation matrix W.I. - Age W.I. - B.W. B.W. - Age .48  .43  .15  Equation Y = C + BX Y = -45.8 + 3.6X + - B.W.1  ag£  8  SWV  M  .30  -  .01  .26  .54  .06  .06  Y= 2.8+ 1.3 X  a g e  C3H-2  F  .02  -  .42  .65  .14  .-.06  .16  Y = 5.2 + 0.1 X  a g e  C3H-2  M  .15  -  .02  .26  .39  -.14  .10  Y = 2.9 + 0.4 X age  34 Since body weight had no influence on water intake in the other 3 groups, its influence on the SWV  females  1  water intake could have been a pathological  part of the polydipsia syndrome.  It would not have been consistent to adjust  the water intake for body weight in the SWV  females and not in the other  groups, therefore the water intake, urine output, and osmolal excretion values were not adjusted in any of the experiments.  In any event the experiments for  which i t was most important to have correct water intake values were the genetic testcrosses in which water intake was used to  determine i f the progeny  were normal or polydipsic. Since the water intakes would be adjusted for body weight only in polydipsic females, the phenotype of the mice would have to be determined f i r s t . polydipsia was not as  After the phenotype was determined the degree of important.  While the water intakes were not adjusted for body weight, mice that were closely matched for age were used whenever possible in a l l the experiments. (b)  Dam or Sire Effects  The same SWV  data were analyzed to see i f dam or sire effects accounted  for any more of the variability in water intake.  The sample size was reduced  since water intakes were not known for a l l of the animals' parents.  The  analysis was done on water intakes which had been adjusted for age using the regression equations in Table III. The water intakes of the dam or sire had no significant influence on the water intake of the progeny (Table IV). was to be expected since the SWV  This  strain had been inbred by brother-sister  matings for 30 - 36 generations and very l i t t l e i f any heterozygosity would be expected in this strain.  TABLE IV. Analysis of dam and sire effects on water intake (W.I.) in the SWV strain. takes were adjusted for age.  Parent's W.I. + s-  The water in-  Comparison  (ml/24 hrs)  Offsprings' W.I. + s o ~y (ml/24 hrs)  Dam vs Daughter  53.1 + 1.3  55.6 + 1.5  .36  .02  Sire vs Son  15.9+0.8  16.8+0.8  .39  .03  Sire vs Daughter  15.8 + 0.7  53.2 + 1.4  .63  .01  Dam vs Son  55.3 + 1.5  16.6 + 0.7  .63  .01  r  F prob.  r2 ^  36 The mice used to analyze the influence of body weight and age on water intake included:  those fed 2 types of rations; nulliparous, multiparous,  and lactated females; and single and grouped males.  In order to obtain the  best reference water intake data for the genetic testcrosses, these factors were examined to determine i f they influenced water intake. Water intake data were obtained from SWV nulliparous, multiparous, and lactated females and from C3H nulliparous and multiparous females which had been fed ration 1 (Table V); and from the 5 groups of mice of the SWV  strain (Table VI), C3H-2  strain (Table VII), and C57 strain (Table VIII) which had been fed ration 2. The data given in Tables V - XIII include a l l the animals used in sections (a) and (b) plus some data collected after the analyses for age, body weight, and dam and sire effects had been done. (c)  Feed  In order to determine i f the ration had a significant influence on water intake, comparisons were made between groups of SWV and C3H mice which had been fed ration 1 and ration 2 (Tables V, VI, 6c VII)..  The-regression of  water intake on age in the SWV females fed either ration shows the same pattern (Figures 3 & 4).  The water intakes increased with age up to a peak  value and then decreased.  A positive regression line was calculated from  the f i r s t month tested up to the age of the highest water intake, and a second negative regression line was calculated from that age of highest water intake to 16 months. Since the water intake data of the mice fed ration 2 (Tables VI 6c VII) covered a larger age range than for those fed ration 1 (Table V), the ration 2 data were truncated so that the same age range (8 - 16 months) was compared. An analysis of covariance on the positive regression lines was done between  37 TABLE V. A.  Daily water intakes (ml) + s- in mice fed ration 1. SWV Strain  Age (mo.)  Nullip arous females  Multiparous females  8.0-9.5  16.8 +  38.7 +  0.9  +  La.ct3.ted females  Single males  4.9  46.5 +  5.4 A  17.0 + 1.4 bC  -  41.7 +  2.4  14.1 + 1.5 BC  58.9  +  9.1  12.7 + 3.3 be  10.0-10.5  32.0 + 6.2  61.9  11.0-11.5  32.8 + 1.1  65.4 + 3.8  12.0-13.0  -  -  57. 8 +  3.2  18.1 + 1.4 C  13.5-14.5  42.5 + 5.4  51.8 + 5.5  52.8 +  4.9  20.1 + 1.6 ABC  15.0-15.5  36.4 + 3.1  37.6 + 9.0  44.2 +  8.1  18.8 +  -  16.0  26.7 + 0  45.4 + 12.1  17.1 +  0.9  -  B. C3H Strain Age (mo.)  Nulliparous females  Multiparous females  8.0-9.5  6.9 + 0.3  -  10.0-10.5  6.6 + 0.3  11.0-11.5  5.5 + 0.3  -  12.0-13.0  5.9 + 0.4  5.2 + 0.3  13.5-14.5  6.2 + 0.5  5.4 +0.1  15.0-15.5  8.0 + 0.9  6.4 + 0.6  16.0  7.4 +  5.3 + 0.6  _  5.3 + 0.2 .  A = significantly different when compared to nulliparous females of the same age and strain p <0.01 B = significantly different when compared to multiparous females of the same age and strain p <0.01 C = significantly different when compared to lactated females of the same age and strain p <0.01 a,b,c, = sames as A, B, C except that p <0.05  TABLE VI.  Daily water intake (ml) + s- of SWV mice fed ration 2.  Age (mo.)  Nulliparous females  Multiparous females  Lactated females  Grouped males  4  9.9 + 1.3  -  -  7.5 + 0.3 a  5  10.7 + 0.8  -  -  9.3 + 1.2  6  19.5 + 2.4  19.6 +  7  27.3 + 3.7  -  8  32.1 + 2.9  36.0 +  9  41.5 + 5.2  10  Single males  10.1 +  -  29.7 + 0.6 ab  10.1 + 1.0 ABC  10.5 + 0.9 abC  34.4 + 1.1  10,9 + 1.0 AC  12.7 + 1.1 AC  5.6  41.5 + 2.2  11.3 + 1.2 ABC  11.1 + 0.6 ABC  46.4 +  6.6  63.6 + 4.9 A  12. 8 + 0.9 ABC  13.5 + 1.0 ABC  53.5 + 7.2  64.2 +  7.9  63.1 + 4.0  15.6 + 1.7 ABC  16.3 + 1.1 ABC  11  51.1 + 3.7  71.2 +  5.3  64.7 + 4.0  16.9  1.1 ABC  16.5 + 1.7 ABC  12  60.9 + 5.0  70.0 +  6.5  66.7 + 2.8  16.0 + 1.2 ABC  19. 8 + 1.1 ABC  13  71.9 + 5.4  66.0 +  5.3  57.0 + 4.2 a  17.4 + 1.9 ABC  19.6 + 2.0 ABC  14  76.4 + 5.9  51.2 +  8.3  63.5 + 5.6  17.0 + .1.6 AC  22.8 + 1.9 AC  15  63.2 + 5.3  48.0 +  6.2  50.4 + 6.4  17.8 + 2.0 AC  20.9 + 1.2 AC  16  67.0 + 5.1  47.1 + 15.0  45.0 + 7.6  15. 8 + 0.9 Ac  24.2 + 2.6 A  1.5  A = significantly different when compared to nulliparous females of the same age B = significantly different when compared to multiparous females of the same age C = significantly different when compared to lactated females of the same age a,b,c = sames as A, B, C except that p <0.05  p <0.01 p <0.01 p <0.01  TABLE VII.  Daily water intakes (ml) + s- of C3H-2 mice fed ration 2.  Age (mo. )  Nulliparous females  Multiparous females  Lactated females  Grouped males  Single males  4  3.8 + 0.1  5  3.9 + 0.1  6  4.3 + 0.2  6.2 + 0.3 A  5.4 + 0.2 a  7  4.0 + 0.1  5.6 + 0.4 A  5.3 + 0.3  8  4.5 + 0.2  5.9 + 0.2 A  5.7 + 0.3 A  9  4.6 + 0.3  7.0 + 0.3 A  4.6 + 0.4 B  10  4.3 + 0.3  6.5 + 0.3 A  6.0 + 0.5 a  11  4.5 + 0.4  6.5 + 0.3 a  6.2 + 0.5 a  12  4.8 + 0.4  6.9 + 0.5  7.3 + 0.5  6.3 + 0.5  9.4 + 1.1 A  13  4.1 + 0.4  7.3 + 0.2 A  7.7 + 0.4 A  8.4 + 0.4 A  7.5 + 0.6 A  14  4.8 + 0.3  6.9 + 0.6  7.3 + 1.1  10.9 + 0.9 Ab  9.3 +  15  5.8 + 0.8  7.4 + 0.6  7.3 + 0.7  10.2 + 0.9  8.9 + 0.1 A  16  7.0 + 0.8  9.1 + 1.6  8.9 + 0.8  11.5 + 0.8  8.2 + 0.7  4.5 + 0.1 A  A = significantly different when compared to nulliparous females of the same age B = significantly different when compared to multiparous females of the same age a,b = same as A, B except that p <0.05  p <0.01 p <0.01  -  TABLE VIII. Age (mo.)  Daily water intakes (ml) + s- of C57 mice fed ration 2. Nulliparous females  Multiparous females  Lactated females  Grouped males  S ingle males  4  5.4 + 0.4  6.5 + 0.3  5  5.5 + 0.3  5.9 + 0.2  6  5.5 + 0.5  5.7 + 0.2  7.1 + 0.6  7  5.4 + 0.3  5.6 + 0.2  6. 8 + 0.4 ab  8  5.1 + 0.2  5.8 + 0.6  7.0 + 0.7  ' 9  5.1 + 0.3  6.0 + 0.5  7.6 + 0.5 A  10  5.2 + 0.3  5.5 + 0.3  9.0 + 0.9 AB  11  5.3 + 0.2  6.3 + 0.5  7.5 + 0.6 a  12  5.3 + 0.2  6.2 + 0.5  6.0 + 0.7  7.7 + 0.7 a  5.6 + 0.2  13  6.1 + 0.2  5.8 + 0.7  6.7 +0.8  7.3 + 0.3  5.1 +0.3  14  6.1 + 0.3  5.8 + 0.4  -  8.7 + 0.3 AB  5.6 + 0.3  15  6. 8 + 0.2  6.1 + 0.3  7.4 + 1.0  8.4 + 0.2  5.2 + 0.5  16  7.0 + 0.0  6.9 + 0.1  6.8 + -  8.3 + 1.1  8.6 + -  A = significantly different when compared to nulliparous females of the same age p <0.01 B = significantly different when compared to multiparous females of the same age p <0.01 p <0.01 D = significantly different when compared to grouped males of the same age a,b,d = same as A, B, D except that p <0.05  41  FIGURE 3. THE CALCULATED REGRESSION LINES DF WATER INTAKE PLOTTED AGAINST AGE IN SWV AND C3H MICE FED RATION 1 100-+  4.  B.  B«  10*  1S»  AGE (MONTHS) ^  POF b BEING ZERO IS < 0*01  14*  IE.  42  FIGURE 4. THE CALCULATED REGRESSION LINES DF THE DAILY WATER INTAKE PLOTTED AGAINST AGE IN SWV MICE FED RATION S 100.+  AGE (MONTHS) 3fc P OF b BEING ZERO IS < 0*01  43 rations for each group of mice (Table IX).  With ration 1 only the SWV n u l l i -  parous females and multiparous females had a significant regression of water intake on age, whereas with ration 2 a l l the groups had a significant regression. In the nulliparous SWV females,  the regression coefficients  (t>) were  the same for both rations, but there was a significantly greater A intercept (from the equation Y = A + B (X - X) ) in those mice fed ration 2.  This in-  dicated when the effect of age was accounted for, the water intakes were significantly higher in the nulliparous SWV females fed ration 2 than in those fed ration 1.  There was no significant difference in b or water in-  takes between the 2 rations in the multiparous SWV females.  An analysis of  covariance was done in the other 4 groups even though there was not a significant regression of water intake on age in those mice fed ration 1.  In  the single males the b values were significantly different, and in the SWV lactated females, C3H nulliparous females, and C3H multiparous females the water intakes were significantly different. When the maximum water intakes for each group fed ration 1 (Table X) were compared to those of each group fed ration 2 (Table XI), there was a significant difference only in the nulliparous SWV females.  When these  females were fed ration 2 they had a maximum water intake of 76 ml/24 hr as opposed to only 42 ml/24 hr when'they were fed ration 1. affect  The ration did not  the age at which each group had the highest water intake. In summary:  the SWV single males, SWV lactated females, and the C3H  females showed a significant regression of water intake on age when fed ration 2 but not when fed ration 1.  In the SWV nulliparous females the b  values of water intake on age were the same, but the water intakes were significantly higher with ration 2 over the range of ages tested.  Therefore  TABLE IX.  Analysis of covariance of the positive regression of water intake on age between the 2 rations in SWV and C3H mice.  p of b  p of A intercepts  0.29  < 0.01  Y -53.2 + 10.5X* Y = -63.5 + 12.4X*  0.61  0.08  8-11.5 8-12.4  Y Y=  -3.8 + 5.2X 10.8 + 4. 9X*  0.92  < 0.01  1 2  8-16.0 8-16.0  Y = Y=  13.4 + 0.4X -0.7 + 1.6X*  <0.01  0.74  Nulliparous females  1 2  8-16.0 8-16.0  Y_ Y=  4.5 + 0.2X 2.1 + 0.2X*  0.52  < 0.01  Multiparous females-  1 2  10-16.0 10-16.0  Y= Y  4.4 + 0.1X 4.0 + 0.3X*  0.30  <0.01  Age (mo.)  Regression Line (Y = C + BX)  Strain  Group  Ration  SWV  Nulliparous females  1 2  8-14.5 8-14.4  Y = -25.9 + 5. IX* Y = -23.0 + 7. IX*  Multiparous females  1 2  8-11.5 8-11.5  Lactated females  1 2  Single males  C3H  * the probability of b being zero is  <0.01  =  45 TABLE X.  Strain  SWV  C3H  The maximum water intake + S y and the age it was attained in mice fed ration 1.  Group  Peak Water Intake + s- (ml/24 hrs) - y  Age (mo. )  Nulliparous females  42.5 + 5.4  14  Multiparous females  65.4  +3.8  11  Lactated females  58.9 + 9.1  11  Single males  20.1 + 1.6 BC  14  Nulliparous females  9.1 + 1.6  15.5  Multiparous females  6.4 + 0.6  15  B = significantly different when compared to multiparous females of the same age p < 0.01 C = significantly different when compared to lactated females of the same age p <0.01  46 TABLE XI.  Strain SWV  C3H-2  Maximum water intake (ml/24 hrs) + s- and the age it was attained in mice tested up to 16 months of age and fed ration 2. Group  Peak Water Intake  Nulliparous females  76.4 + 5.8  14  Multiparous females  71.2 + 5.3  11  Grouped males  17.8 + 2.0 ABC  15  Lactated females  66.7 + 2.8  12  Single males  24.2 + 2.6 ABC  16  Nulliparous females  7.0 + 0.8  16  Multiparous females  9.1 + 1.1  16  11.5 + 0. 8  16  Lactated females  8.9 + 0.8  16  Single males  8.2 + 0.7  16  Nulliparous females  7.0 + 0.0  16  Multiparous females  6.9 + 0.1  16  Grouped males  8.7 + 0.3  14  Lactated females  7.4 + 1.0  15  Single males  5.2 + 0.5 d  15  Grouped males  C57  Age (mo. )  A = significantly different when strain p < 0.01 B = significantly different when strain p < 0.01 C = significantly different when strain p <0.01 D = significantly different when strain p <0.01 a,b,c,d = same as A, B, C, D except  compared to nulliparous females of same compared to multiparous females of same compared to lactated females of same compared to grouped males of same that  p <0.05  47 the water intake data obtained from mice (including progeny from the genetic testcrosses) fed ration 1 were not combined with the data obtained from mice fed ration 2. (d)  Strain  The water balance data demonstrated a significant difference in water intake between the SWV and C3H females but not between the males (p. 29). The water intake data given in Tables VI, VII, and VIII had a larger sample size and a wider range of ages; therefore i t was analyzed to determine i f strain influenced water intake in both sexes.  Comparisons were made between the  SWV, C3H-2 and C57 strains using nulliparous females, multiparous females, and grouped males fed ration 2. The most significant difference was that the SWV females showed a markedly different pattern of water intake with.age than did the C3H-2 or C57 females (Figures 4, 5 & 6).  The SWV females had a significant positive  linear regression of water intake on age up to a peak level and then the curve flattened.  The negative regression lines were calculated from a relatively  small sample and over a very short age range and this may partially explain why the slopes were not significant (Figure 4).  The C3H-2 and C57 females,  on the other hand, had a significant positive regression of water intake on age up to 16 months (Figures 5 & 6).  The b values of the C3H-2 and C57 fe-  males were significantly different from zero, but they were very small (0.23 2 and 0.11) when compared to the'_b of the SWV females (7.08), and the r for the C3H females in Table III was only 0.02 as opposed to 0.23 for the SWV females. When the positive regression lines were compared, the multiparous SWV, C3H-2 and C57 females a l l had significantly different b values and similarly  48 FIGURE 5-  THE CALCULATED REGRESSION L I N E S  OF  WATER I N T A K E P L O T T E D A G A I N S T A G E I N MICE FED RATION 2Q.+  DAILY C3H-2  2«  SINGLE MALES  10-. 4  0..  20-+  LACTATED  ^  4  H  1  +  +  4  d r  FEMALES  10.  0.  20.+  GROUPED M A L E S  10.  0.  20.+  MULTIPAROUS  FEMALES  10.+  -x—*—*—x—x—*—x—*-  0.  20.+  NULLIPAROUS FEMALES  10.+ + — + 0.  H  +  H  H 12-  (MONTHS)  BEING ZERO I S  =r  h-  10. AGE  P OF b  1  1  B.  ^  +  <  0*01  14«  1E<  49  FIGURE G- THE CALCULATED REGRESSION LINES DF DAILY WATER INTAKE PLOTTED AGAINST AGE IN C57 MICE FED RATION E» 20.+ SINGLE MALES 10... 0 -\  4 4 4  3  —I  r  S0.+ LACTATED FEMALES 10-  20*+ GROUPED MALES 10-  _2  SZ  _SZ  S2L  SZ  SZ-  -2—=—v  o.  20 •+ MULTIPAROUS FEMALES 10-+ X 0  H  X  K  X—X  1  *—*  1  X—X  X  *  1  1  1-  *r—*—I  H  -4=—*  20.+ NULLIPAROUS FEMALES 10..  + +  o..  H  4.  B»  +  10*  12*  AGE (MONTHS) % P DF b BEING ZERO IS < 0-01 SAME AS ^f: BUT P < 0«05  14.  16.  2fc  50 the grouped SWV, C3H-2 and C57 males a l l had significantly different b values (Table XII).  The SWV nulliparous females'  b was significantly different from  those of the C3H-2 and C57 nulliparous females;  but the C3H-2 nulliparous  females' _b was the same as that of the C57 nulliparous females, and only their water intakes were significantly different. The water intakes of each group were compared between the 3 strains at each age.  The SWV strain was significantly different from the C3H-2 and C57  strains in a l l cases except for the grouped males at 5 and 6 months of age. The SWV males had not yet started to increase their water intake and had the same intake levels as the C57 males.  The C3H-2 strain was not significantly  different from the C57 strain in any instances. In summary:  the SWV females and males were significantly different  from the control females and males except for the young males.  The SWV mice  had a significantly higher water intake, and age had a greater effect on their water intake.  The 2 control strains differed significantly in the  regression of water intake on age, except for the nulliparous females, and over the 12 month period the C3H-2 strain had a significantly different water intake than the C57 strain even though at each age level there was no significant difference. (e)  Sex  The data were analyzed to determine i f sex influenced the water intake in the C3H-2 and C57 strains, and to investigate the influence demonstrated in the SWV strain (p. 29). In the SWV strain the most obvious difference was that with both rations the males did not show the dramatic increase of water intake and the subsequent decrease with age that the females did (Figures 3 & 4).  The males fed  51 TABLE X I I .  A n a l y s i s o f c o v a r i a n c e o f the p o s i t i v e r e g r e s s i o n l i n e s o f water i n t a k e on age i n SWV, C3H-2, and C57 mice f e d r a t i o n 2. The r e g r e s s i o n l i n e was c a l c u l a t e d up to and i n c l u d i n g t h e age a t which the peak water i n t a k e was reached.  Strain SWV  Regression l i n e Y = C + BX  Group Nulliparous  females  Multiparous  females  Lactated  females  5.1 X*  ) 0.04 Y = -53.2 + 10.5 X*( > 0.02 Y = 1.1 + 5.8 X*'  Grouped males  Y =  5.0 +  0.9 X*v  S i n g l e males  Y =  1.7 +  1.4 X*  All  Y = -17.5 +  7.1 X*  Y =  1.4 X*  females  A l l males  C3H-2  Y = -25.9 +  p. o f b  3.7 +  Nulliparous  females  Y = 2.9 + 0.2 X*  Multiparous  females  Y = 4.4 + 0.2 X*  * **  Females  Y = 3.3 + 0.2 X*  Grouped males  Y = 7.6 + 0.6 X*  Nulliparous  females  Y = 4.5 5 + 0.1 u . l X* X"  Multiparous  females  Y = 5. 1 + 0 . 1  X**  Females  Y = 4.7 + 0.1 X*  Grouped males  Y = 5.8 + 0.2 X*  p. o f b being zero i s <0.01 p. o f b b e i n g zero i s < 0 . 0 5  0.07 0.16  y<0.01  <0.01  ^<0.01  < 0.01  0.35  <0.01  . \<0.0] '  < 0.01  ,42  0.19  0.11  <0.01  \  C57  p. o f A i n Y = A + B(X - X)  '  . \  52 ration 1 did not have a significant regression of water intake on age (Table XIII), and while the males fed ration 2 did have a significant positive regression of water intake on age, their b> was significantly smaller than that of the females (Table XII). When fed ration 1, the males had a significantly lower water intake than the multiparous and lactated females up to 14 months of age (Table V).  The males did not have a significantly lower water intake  than the nulliparous females until 14 months at which point the nulliparous females  had reached their peak water intake.  By 16 months the water intakes  of the nulliparous and lactated females had decreased and were no longer significantly different from the males' water intake. When fed ration 2, the nulliparous females had the same water intake as the grouped males at 4 and 5 months.  From 6 to 13 months a l l the females had  significantly higher water intakes than the males (Table VI).  The n u l l i -  parous females and the lactated females continued to have significantly higher water intakes than a l l the males at 15 months, and than the grouped males at 16 months.  The multiparous females' water intake had decrea-sed  sufficiently by 15 months so that i t was no longer significantly higher than the males'.  .  !  Finally with ration 1, the males had a significantly lower peak water intake than did the multiparous and the lactated females (Table VI), and with ration 2 both groups of males had significantly lower peak water intakes than did the 3 groups of females (Table X). Therefore the SWV males had a significantly lower water intake than the SWV females except at the beginning and end of the age range.  The SWV males'  water intakes were not affected as much by age as were the SWV females' water intakes. In the C3H-2 mice both the females and males had a significant positive  53 TABLE XIII.  Strain SWV  C3H  Covariance analysis of the positive regression lines calculated by plotting water intake against age in the SWV mice fed ration 1. The regression line was calculated up to and including the age at which the peak water intake was reached.  Regression Line Y = C + BX  Group  P-  Nulliparous females  Y = -25.9 +  5.1  X*  Multiparous  Y = -53.2 + 10.5  X*  females  Lactated females  Y =  -3.8 +  A l l females  Y=  9.5+3.3X  Single males  Y = 11.4 + 0.5 X  Nulliparous females  Y = 4.5 + 0.2 X  Multiparous  Y = 4.4 + 0.1 X  females  * p. of b being zero is  <0.01  5.2 X  o f  ^  p. of A intercept  0.04  <0.01  0.37  0.60  0.06  <0.01  .  54 linear regression of water intake on age (Figure 5). nificantly greater b than the females (Table XII).  The males had a sigThe grouped males' water  intake was consistently significantly higher than that of the nulliparous females, but it was only significantly higher than the multiparous females at 9 and 14 months (Table VII).  The single males had a significantly higher  water intake than the nulliparous females but not the multiparous females. There was no significant difference in the peak water intakes between the sexes. In the C57 strain both the females and males had a significant positive linear regression of water intake on age.  There was no significant difference  between the _b values of the 2 sexes, but the A intercepts were significantly different demonstrating that the grouped males had a higher water intake than the females (Table XII).  The grouped males consistently had a signifi-  cantly higher water intake than did the nulliparous females (Table IX), but they only had a significantly higher water intake than the multiparous females at 7, 10, and 14 months of age.  The single males' water intakes were  lower than those of the grouped males and were not significantly different from the females at any age.  There was no significant difference in the  peak water intakes between the sexes (Table X). In summary: and the SWV strain.  sex did influence the water intake in the control strains In the control strains the males had a higher water in-  take than the females.  The C3H-2 males' water intake was also influenced more  by age than was the females' water intake.  In the SWV strain, however, the  females had a severe polydipsia while the males had only a slightly elevated water intake.  Age did influence water intake in the SWV males but much less  than it did in the females.  55 (f)  Parity in Females  Since the SWV  females had a significantly higher water intake than the  SWV males (p. 29 & 52) which suggested an hormonal influence, the data were analyzed to determine i f parity or lactation also affected water intake. Water intakes were determined for a large number of SWV  and C3H-2 females  when they became available from the breeding colony at 9 to 12 months.  Could  these lactated females serve as controls for the nulliparous progeny of the genetic testcrosses? As already mentioned (p. 36) the SWV  females showed a marked peaking  effect in the increase of water intake with age (Figure 3).  In a preliminary  experiment this pattern was more pronounced in the nulliparous and multiparous females in which the positive regression lines were significant (Table XIII).  There was no significant difference between the peak water intakes  reached by these 2 groups, but the multiparous females reached their peak 3 months earlier since they had a significantly greater b than the nulliparous females (Table VI).  Although the multiparous females had a higher water in-  take at each age up to 14 months, the sample size was so small that the difference was not significant (Table V).  At 15 months the water intakes of  both groups had decreased and were the same. Age did not have a significant influence on water intake in the lactated females but the suggestion was s t i l l there (Figure 3).  There was no s i g n i f i -  cant difference in the _b values or the water intakes (A intercept) between the lactated and multiparous females (Table XIII).  There was also no s i g n i f i -  cant difference in the peak water intakes or the ages at which they were reached between the 2 groups (Table VI).  The lactated females started with  a significantly higher water intake than did the nulliparous females at 8 months, but since the lactated females increased their water intake more  56 slowly with age, by 10 months there was no longer a significant difference (Table V).  This test indicated that in the SWV females parity influenced  water intake but lactation did not. In the C3H females age did not influence water intake in either the nulliparous or multiparous females (Table XIII), and there was no significant difference in the peak water intakes between the 2 groups (Table VI).  The  water intakes were significantly different only at 10 months (Table V), but this difference may not be meaningful.  The multiparous group included'only  C3H-2 females, whereas the nulliparous group included only C3H-1 females at 10 months.  There was a significant difference between the 2 C3H sublines in  the water balance (p. 31), therefore the significant difference at 10 months (Table V) may reflect a difference in parity, a difference between the 2 sublines, or an interaction.  If the difference was due to a difference between  the sublines, this effect could have been masked at the other ages because the nulliparous group included both sublines.  This test, therefore,  indi-  cated that parity did not influence water intake in the C3H strain. A second more comprehensive test was done using the 3 groups from the SWV, C57, and C3H-2 strains.  These mice were fed ration 2 instead of ration  1, so the data from the 2 tests could not be combined. In the SWV strain the 3 groups of females showed the same response of water intake with age as in the first test.  The regression lines were cal-  culated in the same way and the positive regression lines were significant in a l l the groups (Table XII). The nulliparous and multiparous females had significantly different t> values (Table XII).  The multiparous females had the same water intake as the  nulliparous ones at 6 months, but increased their water intake much more rapidly with age and thus reached their peak water intake 3 months before the  57 nulliparous females (Table X & Figure 4). There was no significant difference between the 2 groups in the peak water intakes or in the water intake at each month (Table VI). The lactated females started with a significantly higher water intake at 6 months (Table VII) than either of the other 2 groups, but their water intakes increased significantly more slowly with age than did those of the multiparous females (Table XII). Consequently the multiparous and the lactated females reached the same peak water intakes at ab out the same age (Table XII). The b of the lactated females appeared to be similar to that of the nulliparous females, and the water intakes of the latter remained lower until after the lactated females had passed their peak.  By 13 months the  nulliparous females had a significantly higher water intake than did the lactated females. Parity and lactation did not affect the peak water intakes which were reached by the females, but i t did influence how rapidly the females reached their peak.  The nulliparous females took a longer time to reach the peak  than either of the parous groups.  The lactated females started higher but  reached their peak at the same time as the nulliparous females.  The point at  which the 2 parous groups reached their maximum water intake roughly coincided with when they started to appear dehydrated and show muscle weakness (P. 27). In the C3H-2 strain there was a significant positive regression of water intake on age in the nulliparous and multiparous females (Figure 5) but not in the lactated females.  This last group, however, was only tested over  a 5 month age range and i t was beyond the age at which there was a s i g n i f i cant regression in the SWV lactated females. There was no significant difference in the b values but there was in  58 the A intercepts between the nulliparous and multiparous The water intakes of the multiparous  females (Table X i i ) .  females were significantly higher than  those of the nulliparous females from 6-11  months and at 13 months (Table  VIII). There were no significant differences at the older ages, and the maximum water intakes at 16 months were not significantly different (Table X). The lactated females and the multiparous intakes.  females had very similar water  The lactated females had a significantly higher water intake than  the nulliparous females only at 13 months (Table VIII) and there was no significant  difference in the peak water intakes between the lactated  females and the other 2 groups (Table X). Therefore parity did affect the water intake in the C3H-2 strain, but it did not influence the regression of water intake on age.  Lactation did  not seem to influence the water intake between 12 - 16 months of age. In the C57 strain there was a significant, slight increase of water intake with increasing age up to 16 months in the nulliparous and mul.tiparous females but not in the lactated females (probably for the same reason as in the C3H-2 lactated females (p. 57) ).  There were no significant differences  between the nulliparous and multiparous females in the regression of water intake on age (Table XII), in the water intakes (Table IX), or in the peak water intakes (Table X).  There were no significant differences in the water  intakes between the multiparous and lactated females from 12 - 16 months  1  (Table IX) or in the peak water intakes (Table X). In summary:  parity did affect water intake in the SWV  either ration 1 or 2, but in slightly different ways.  females fed  Parity also influenced  water intake in the C3H females fed ration 2; but not those fed ration 1 or the C57 females fed ration 2.  Lactation did not seem as important, but may  have had a slight influence in the SWV  females fed ration 2.  Due to the  59 restricted range of ages tested in the control strains, i t was impossible to rule out lactation having an influence on the regression of water intake on age.  Therefore for the genetic experiments, water intakes of nulliparous,  multiparous, and lactated females could be combined to provide reference data for each age level beyond 13 months in the SWV and C3H strains. (g)  Stress in Males  Since a difference in water intake had been demonstrated between sexes (p. 54) and between multiparous and nulliparous females (p. 56) suggesting an hormonal influence, the data were analyzed to determine i f there was a difference in water intake between single and grouped males.  The grouped  males were stressed by being aged with 3 or 4 males but were not bred, while the single males were bred but not stressed by being with other males. In the genetic experiments, could single SWV and C3H-2 males be used to determine reference water intakes for each age level, when the testcross progeny to be classified were grouped males? Both the SWV grouped and single males had a significant regression of water intake on age up to 16 months (Figure 4), but the single males had a significantly greater _b (Table XII).  At 6 months the water intakes of the  2 groups were the same, but from 7 months on the single males had a slightly higher water intake, although the difference was s t i l l not significant (Table VII). X).  The maximum water intakes were also not significantly different (Table Therefore there was no significant difference in water intake between  the single and grouped males, but the single males had a greater regression of water intake on age. In the C3H-2 and C57 males, there were not enough data to make a good comparison since for the single males there were data only from 12 to 16  60 months.  In both the C 3 H - 2 and the C57 s t r a i n s  t h e r e was  a significant re-  g r e s s i o n o f water i n t a k e on age up to 16 months i n the grouped males but not i n the s i n g l e males ( F i g u r e s 5 & 6 , there was  Table X I I ) .  In the s i n g l e males' data from 12 t o  not even a s u g g e s t i o n of an i n c r e a s e i n water i n t a k e s  16 months ( T a b l e s V I I I 6c I X ) .  The grouped males had h i g h e r water  than the s i n g l e males a t most ages and f o r the C57 s t r a i n  i t was  intakes significantly  h i g h e r a t 14 months and a p p r o a c h i n g s i g n i f i c a n c e a t 12 and 15 months.  The  C57 grouped males a l s o had a h i g h e r maximum water i n t a k e than the s i n g l e males, but the C 3 H - 2 grouped males d i d not ( T a b l e X I ) . In summary: w i t h age, whereas  the c o n t r o l grouped males i n c r e a s e d the s i n g l e males d i d not.  t h e i r water  intake  In the C 3 H - 2 s t r a i n t h e r e was  o n l y a s u g g e s t i o n t h a t the grouped males had a h i g h e r water i n t a k e than the s i n g l e males, but i n the C57 s t r a i n the grouped males had a s i g n i f i c a n t l y h i g h e r water i n t a k e than the s i n g l e males.  The SWV  to s t r e s s than d i d the c o n t r o l C3H.-2 and C57 males. ments, s i n g l e and grouped males c o u l d be combined ence water i n t a k e d a t a a t each age l e v e l  B.  Differential (1)  F o r the g e n e t i c  and C 3 H - 2  experirefer-  strains.  Defect  Serum O s m o l a l i t y  There was SWV  differently  i n order t o o b t a i n  f o r the SWV  Diagnosis of P o l y d i p s i a - P o l y u r i a  males r e a c t e d  females, SWV  was, however,  no s i g n i f i c a n t d i f f e r e n c e  i n the serum o s m o l a l i t y between the  males, C 3 H - 1 females, and C 3 H - 1 males ( T a b l e X I V ) .  a slight  trend  than the females, and a l s o  f o r the males to have a h i g h e r serum o s m o l a l i t y  f o r the SWV  o s m o l a l i t y than the C 3 H - 1 s t r a i n . polydipsia-polyuria,  s t r a i n to have a s l i g h t l y h i g h e r  The SWV  serum  females, which had the severe  had a lower serum o s m o l a l i t y than d i d the SWV  would not have been expected  There  i f the females s u f f e r e d  from c h r o n i c  males which dehydration.  TABLE XIV.  Serum osmolalities + s- in SWV and control C3H-1 mice. Serum Osmolality + s „ (mOsm/kg)  Strain  Sex  Age (mo.)  N  SWV  F  9.0 - 14.5  30  343  +  5.1  SWV  M  9.0 - 14.0  21  351  +  5.6  C3H-1  F  10.5 - 16.0  21  359  +  4.6  C3H-1  M  9.0 - 16.0  14  344  +  3.8  i—  1  62 There was no significant regression of serum osmolality with age in the SWV or C3H-1 strains. (2)  Response to Dehydration  (a)  Water Deprivation  After dehydration the serum osmolality should rise, due to a decrease in serum volume, which in turn should stimulate the release of endogenous ADH.  In this experiment only the C3H females showed a significant  difference  in serum osmolality after 7 hours of dehydration, and the dehydrated serum osmolality was lower (Table XV). obtained from the mice 2 - 3  The control serum osmolality (0 hours) was  weeks before the dehydration run was done.  However, the C3H females, SWV females, and SWV males had a significantly greater per cent loss of body weight after 7 hours of water deprivation than after 7 hours of ad libitium water intake.  The per cent loss of body weight  after the ad libitium control run was unavailable for the C3H males, so that a comparison was not possible.  The C3H mice lost 5% of their body weight  after 7 hours of dehydration indicating that 7 hours was long enough to produce an adequate stress to stimulate the release of endogenous ADH, even though the serum osmolality did not rise (Dies et a l . , 1961). Some SWV and C3H females were accidently dehydrated for 19 hours after being injected with .05 ml peanut o i l .  Since the peanut o i l had no effect on  the water balance of these mice (p. 74), any changes that occurred after 19 hours were caused by the dehydration, although an interaction could not be ruled out. There were very l i t t l e osmolal excretion data obtained during the ad libitium control run because of the low urine output, especially in the C3H strain.  63 TABLE XV.  Effect of water deprivation on body weight (B.W.), urine osmolality ( U ) , serum osmolality ( S ) , and osmolal excretion ( T o t ) in SWV and C3H mice. osm  o s m  osm  Strain SWV  SWV  C3H  C3H  Sex N F  M  F  M  Hours  7o Loss B.W. + s- (g)  U + s (mOsm/kg)  S + s^ (mOsm/kg) 349 + 9.8  o s m  y  o s m  osm ± y (mOsm/24 hrs)  Tot  s  6  0  315 + 25.9  6  4  533 + 37.2  6  7  13.2 + 0.8*  825 + 77.6  3  19  17.0 + 2.7  992 + .147.2  2.4 + 0.5  1  24  23.6  958  4.0  3  0  1041 + 170.0  3  4  1185 + 134.5  3  7  10.0 + 1. 6* 1679 + 265.0  6  0  1472 + 101. 8  6  4  1726 +  6  7  5.2 + 0.5*  2297 + 111.1  3  19  9.4 + 0.4  2701 + 222.1  3  0  1696 + 199.9  3  4  1660 + 140.5  3  7  5.6 + 2.1  1.1 + 0.3 344 + 5.2  346 + 18.0 1.1 + 0.2 377 + 11.2  1.7 + 0.4  340 + 5.5  88.0  2298 + 216.9  1.5 + 0.1  0.5 + 0.1 315 + 6.6  1.0 + 0.1 1.7 + 0.5  345 + 9.3 0.7 + 0.1 348 + 5.0  1.2 + 0.2  significantly different when compared to same group with ad libitium water intake p <0.05  64 In the SWV and C3H females there was no regression of urine osmolality with time during the 7 hours of ad libitium water intake (Figure 7), but there was a significant regression of urine osmolality with time during the 19 hours of water deprivation.  The data was also analyzed using a Scheffes  analysis of variance and with this test there was no significant  increase  after the first 4 hours of dehydration, but the SWV and C3H females significantly raised their urine osmolality by 292 mOsm/kg and 571 mOsm/kg, respectively, during the next 3 hours (Table XV).  Between 7 and 19 hours there  was no further significant increase in urine osmolality.  The 1 SWV female  which was accidently dehydrated for 24 hours showed no further increase of urine osmolality.  An analysis of covariance showed that the regression co-  efficient (b_) was significantly greater in the C3H females than in the SWV females demonstrating that the C3H females could respond better to the dehydration treatment. The SWV females did not concentrate their urine as much as the C3H females did, but they lost a significantly greater per cent of their body weight after 7 hours (13.0% as opposed to 5.0%) and after 19 hours (17.0% as opposed to 9.4%).  The 1 SWV female dehydrated for 24 hours lost 24.0% of  its body weight, but after 24 hours on ad libitium water consumption it had regained a l l its lost weight.  It had also been noted in the colony that i f  mice were accidently dehydrated for over 24 hours, SWV females tended to die, whereas control females did not. There was a significant regression of urine volume with time during the dehydration run and the ad libitium control run with both the SWV and C3H females (Figure 8 and Table XVI).  When an analysis of covariance was  done, the C3H females' regression coefficients of the 2 treatments were the same, but the urine output was significantly greater during the dehydration  FIGURE 7. URINE OSMOLALITY ± Sy DURING WATER DEPRIVATION AND AD LIBITIUM WATER INTAKE IN SWV AND C3H MICE 3000..L  AD LIBITIUM WATER INTAKE • WATER DEPRIVATION H 2500... TO  | 2000... O M 1500..  1000...  500... ft  0 4 7  HOURS  •  0 4 7 19  SWV F  0 4 7  0 4 7  SWV M  0 4 7  0 4 7 1 3  C3H F  0 4 7  C3H  FIGURE B» URINE •LJTPUT ± S DURING 7 HOURS OF WATER DEPRIVATION AND 7 HOURS OF AD LIBITIUM WATER INTAKE IN SWV AND C3H MICE y  6.+  AD LIBITIUM WATER INTAKE • WATER DEPRIVATION m 5...  3:.  5 a... 1...  i i rFi  5 4 6 7  HOURS  >  2 4 6 7  SWV F  2 4 6 7  SWV M  2 4 6 7  2 4 6 7  2 4 6 7  C3H F  2 4 6 7  C3H M  2 4 6 7  TABLE XVI.  Regression of urine output and urine osmolality with time during water deprivation and ad libitium water intake (control).  Urine Output Y = C + BX  Urine Osmolality Y = C + BX  Strain  Sex  Treatment  SWV  F  deprivation  Y = 1.3 + 0.1 X * *  Y = 397 + 36.6 X*  control  Y = 0.1 + 0.6 X*  Y = 393 +  deprivation  Y = 0.2 + 0.2 X*  Y=  control  Y = -0.1 + 0.1 X* ' v '  Y = 1190 - 0.7 X  deprivation  Y = 0.1 + 0.03 X*  Y = 1533 +67.3 X*  control  Y = 0.02 X*  Y = 2150 - 28.3 X  deprivation  Y = 0.1 + 0.1 X  Y = 1587 + 81.0 X  control  Y = 0.03 X*  Y = 2933.- 28.3 X  SWV  ' C3H  C3H  *  M  F  M  0.6 X  984 + 83.1 X  p. of b_ being zero is <0.05  **.the remainder error term after linear regression was high, and the line did not approach the origin, therefore equation might be some other function than linear.  68 run.  It had been expected that the _b and urine output would be less during  the dehydration run, but circumstances dictated that the ad libitium control was run several months after the dehydration test and C3H-2 females had to be used, instead of the C3H-1 females which were used for the dehydration one.  Therefore these results should be interpreted with caution.  The C3H-1  females had a significantly greater urine output than did the C3H-2 females in the water,balance data (p. 31), and therefore would be expected to have the higher base line seen at 2 hours (Figure 8). The SWV females had a significantly lower b for the urine output during the dehydration run than during the control run.  The urine output was also  lower and the difference was approaching significance (p.=.07). In the dehydration run there was a large urine output during the first 2 hours and then i t tapered off as the females became dehydrated. was significant,  The regression line  but the remainder error term after linear regression was  s t i l l high and the line did approach the origin (Table XVI), suggesting that the regression was some other function than linear.  This obvious change in  the regression function after dehydration did not occur with any other group. The SWV females' b was significantly greater than the C3H females' b after both treatments,  but it was significantly greater than the SWV males'  _b only after the control run.  Since the sample sizes were small, especially  in the males, the results should be interpreted with caution. The SWV females and males had a significantly greater osmolal excretion than the C3H females and males after 4 hours of dehydration, but there was no significant difference after 7 and 19 hours of dehydration. In the SWV and C3H males there was no significant change in urine osmolality during the ad libitium control run or the dehydration run (Figure 7 6c Table XVI).  Both groups of males tended to increase their urine  69  osmolality by 635 mOsm/kg after 7 hours of dehydration, but the SWV males lost 10.07o of their body weight whereas the C3H males lost only 5.67, of their body weight.  These losses were not significantly different but the  SWV males' loss of 10.0% was significantly greater than the C3H females' loss of 5.27o.  The males had a significant regression of urine output with time during both the water deprivation and the ad libitium control runs.  For the  males there was no significant difference in b between the 2 tests but the urine output and the urine osmolality were significantly greater during the dehydration run.  In both strains different males were used for the 2 tests,  and the males used for the dehydration run seemed to have a higher base line of urine output at 2 hours and of urine osmolality at 0 hours than did those used in the control run.  The SWV males had a significantly greater b than  the C3H males during both runs.  Thus dehydration did not cause the SWV  males to alter their pattern of urine output. In summary:  the SWV females could partially concentrate their urine  and decrease their urine output when dehydrated, but only at the expense of losing a significantly greater per cent of their body weight than the control females.  The SWV males could not significantly increase their urine con-  centration or decrease their urine output, but they s t i l l lost a large per cent of their body weight. (b)  Saline  Ingestion  The SWV females could not tolerate physiological saline as their only source of water.  After 24 hours 1 female was dead after having drunk only  10 ml compared to its average of 81 ml/day of tap water.  Two more females  were very weak and near death after each had drunk about 7 ml of saline and had lost 25.0% and 31.0% of their body weights, respectively.  The fourth  70 female had drunk 45 ml of saline and had lost only 8.0% of its body weight (Table XVII).  After 48 hours this female had only drunk another 21 ml of  saline and had lost another 20.07o of its body weight.  Since the female was  weakening it was put back onto tap water, however over the next 24 hours it drank only 2 ml of water and lost another 5.6% of its body weight. point the female was extremely weak and was therefore k i l l e d .  By this  After the  female had been on saline for 48 hours, it would not drink out of the water bottle even after the saline had been replaced by tap water, but it did drink a small amount of water out of a dish put into its pan. The SWV males tolerated the saline better, but became quite polydipsic. After 24 hours the males had significantly increased their fluid intake by 39.0% and lost only 3.0% of their body weight (Table XVII).  The males  con-  tinued to increase their fluid intake up to 6 days, at which point i t was twice the normal water intake, and then the fluid intake remained constant. The intake of saline was significantly higher than that of water on a l l 20 days, but there were no significant differences in saline intake between the various days.  The males continued to lose weight for 3 days at which point  i t was significantly different than normal..  After 6 days the males were  starting to regain weight although it was s t i l l significantly lower, and after 20 days the males' weight was back to normal. The C57 females were able to tolerate the saline.  After 1 day their  saline intake was significantly increased 49.07, over their water intake, but there was no change in body weight.  By the second day the saline intake was  s t i l l significantly higher than their water intake, and their body weight had increased significantly by 11.0%.  The water intake remained approximately  the same although it dropped slightly on day 3, but the body weight returned to normal.  There was only 1 female tested from day 4 on, so no comparisons  TABLE XVII.  Fluid  Days  Effect of 0.9% saline on daily ad libitium fluid consumption (ml) and body weight (B.W.) (g) in SWV and C57 mice.  SWV males  SWV females fluid intake B.W.  fluid intake  tap water aver. 75.7 + 2. 5 38.7 + 0.4 22.3 + 1.3 saline 0.9% NaCl  B.W. 32.9 + 0.3  C57 males fluid intake B.W.  C57 females fluid intake B.W. 7.9 + 0.3  28.0 + 0.5  6.5 + 0.2  1  45.4+  -  33.0 +  -  31.2 + 3.3* 32.0 + 0.4  11. 8 + 0.8* 27.8 + 1.3  2  20.7 +  -  27.0 +  -  38.4 + 4.6* 31.6 + 0.9  11.3 + 1.0* 31.2 + 0.8* 7.3 + 0.9  28.1 + 0.6  8.6 + 0. 8* 28.4 + 1.2 28.3 + 1.1  3  44.6 + 5.6* 30.7 + 0.9*  6  40.7 + 5.1* 31.1 + 1.1* 10.3 +  -  30.2 +  -  7.4 + 0.5  28.1 + 1.2  9.1 + 0.0  30.4 + 0. 8 7.5 + 0. 8  27.8 + 1.2  13  43.5 + 1.7* 31.8 + 1.5  11.2 +  -  28. 8 +  -  8.0 + 0.6  29.0 + 1.0  20  38.9 + 4.0* 32.9 + 2.1  10.2 +  -  28.9 +  -  7.4 + 0.3  29.4 + 1.4  * significantly different when compared to average tap water intake  p <0.05  72 were possible. The C57 males seemed to tolerate the saline even better than the C57 females.  There was a transient, significant, 30.0%, increase in fluid in-  take on day 1, but by day 2 the saline intake had dropped slightly and was not significantly different.  The body weight remained the same for the  entire 20 days that they were on saline. In summary:  the SWV females died when given saline as their only  source of water, but the SWV males doubled their fluid intake and only i n i t i a l l y lost weight.  The SWV males developed a polydipsia due to increased  sodium consumption that resembled the polydipsia the SWV females had when drinking tap water.  The C57 controls i n i t i a l l y increased their fluid intake  but returned to normal within a few days.  The C57 females also transiently  increased their body weight but the C57 males kept their weight constant. (3)  Response to Exogenous Antidiuretic Hormone  The results of the preliminary experiment testing the routes of administration are shown in Table XVIII.  In the C3H-2 mice given pitressin tannate  i.m., the urine osmolality was significantly higher on days 1 and 2 but i t dropped in 1 of the animals on day 3. tannate s . c , the 3 days.  In the C3H-2 mice given pitressin  the urine osmolality was not increased significantly on any of Therefore the i.m. route of administration was used for the main  experiment. In the main experiment, the water intake, urine output, urine osmolality, and osmolal excretion were analyzed separately.  For each parameter,  the data  for the 4 days were compared within each treatment and within each group; and also the data for each day were compared between the treatments within each group.  73 TABLE XVIII.  Effect of route of administration on increase in urine osmolality (U o s m ) after 3, daily, injections of \ unit pitressin tannate in o i l in control C3H-2 mice. osm ± s y o f m i c e injected i.m. (mOsm/kg) u  osm ± s y of m i c e injected s.c. (mOsm/kg) u  Day  N  0  2  1914 + 43.8  2278 + 32.5  1  2  2418+57.9*  2638+60.8  2  2  2460 + 62.2  2406 + 41.0  3  2  2523 + 178.9  2446 + 123.1  * significantly different when compared to the control U o s m on day 0, p <0.05  74 The vehicle, peanut o i l , had no significant effect on any of the parameters in any of the 4 groups (Figures 9, 10, 11 & 12). In the SWV  females the pitressin tannate transiently lowered the water  intake and urine volume on days 1 and 2, but the differences were not significant (Figures 9 & 10). excretion (Figure 12).  There was also a nonsignificant rise in osmolal  There was a significant increase in urine osmolality  on day 2, but when the urine osmolality on day 2 of the pitressin tannate run was compared to that on day 2 of the control run, there was no significant difference.  This was because the urine osmolality normally tended to increase  slightly over a 4 day run.  The urine osmolality dropped again on day 3 of  the pitressin tannate run.  If the SWV  ADH,  females had responded to exogenous  there would have been a steady increase of urine osmolality, until after  extended therapy the urine osmolality was within control limits. the transient increase in urine osmolality did not indicate a  Therefore  responsiveness  to ADH. The SWV males showed a slight transient decrease of water intake and urine output and a slight transient increase of urine osmolality after being injected with pitressin tannate, but none of the differences was There was no change in osmolal excretion.  significant.  Thus exogenous ADH had no effect  on the SWV males water balance. 1  In the C3H females there was a transient, nonsignificant decrease in the water intake and urine output and a steady nonsignificant increase in osmolal excretion.  The urine osmolality increased steadily until i t was  significantly higher after 3 injections of pitressin tannate. SWV  As with the  females, the urine osmolality on day 3 of the pitressin tannate run was  not significantly different from that on day 3 of the control run.  However  the urine osmolality steadily increased over the 3 days instead of dropping  75  FIGURE 9-  WATER  INTAKE  ± S  IN  y  SWV A N D C 3 H M I C E  DURING  CONTROL RUN AND A F T E R 3 , D A I L Y , I N J E C T I O N S V E H I C L E OR P I T R E S S I N  1/4  1/3  UNIT  ML-  OF  A THE  TANNATE*  PITRESSIN  PEANUT OIL  TANNATE  IN  OIL  I'M*  UNTREATED  •  DAY  1  =  DAY E  •  DAY 3  SZ  I>M*  40. 30-  T  UNTREATED  •  DAY  1  =  DAY 3  •  DAY 3  ^  BO10v  IS  o. CONTROL 40.  RUN  ri 4  30v  UNTREATED  •  SO10. 0O  1  B  3  0  1  B  3  nnnn 0  1  B  3  A Ann 0  1 2  3  DAYS SWV  F  SWV  M  C3H  F  C3H-2  M  76  FIGURE  10.  URINE  OUTPUT  ± Sy  IN  SWV A N D C 3 H M I C E  DURING  CONTROL RUN AND A F T E R 3, DAILY, I N J E C T I O N S V E H I C L E OR P I T R E S S I N  1/4  UNIT  OF  A THE  TANNATE-  PITRESSIN  TANNATE  IN  OIL  UNTREATED DAY  5  •  I-MDAY  m  1  DAY 3  =  C  LflD.  1/2  ML-  PEANUT OIL  I-MUNTREATED  •  DAY  1  =  DAY 2  B  OAY 3  V  i  CONTROL  RUN  m  40UNTREATED  30-  •  2010O-  0  1 E  3  0  1  S  3  0  H 3  1  O  1 S  DAYS SWV  F  SWV  M  C3H  F  C3H-2  M  3  77  FIGURE  11.  URINE  OSMOLALITY  ± Sy  IN  SWV A N D C 3 H M I C E  A CONTROL RUN AND AFTER 3, DAILY, THE V E H I C L E OR P I T R E S S I N  1/A  UNIT PITRESSIN  UNTREATED  2000*1 ~  D A Y  •  0*.  3  TANNATE  =  T  v  A  IN  OIL  I*M>  rT,liHS  nSflgl 1/E  ML* PEANUT O I L  I*M*  3000*+ UNTREATED  E  2000*1 ^  W  OF  TANNATE*  1000*.  < I  DAY 1  •  2  INJECTIONS  DURING  DAY 2  •  DAY 1  =  •  DAY 3  V  IT  T  1000*1 0  CONTROL R U N §  3000.+ UNTREATED  •  EOOO*..  1000*..  nrinn 0  1  S  3  0  1 5  3  0  1 5  3  0  1 5  3  DAYS SWV #  F  SIGNIFICANTLY RUN P <  0*05  SWV M DIFFERENT  C3H  F  C3H-3  WHEN C O M P A R E D T D D A Y 0  M OF SAME  78  FIGURE 12-  OSMOLAL E X C R E T I O N  ± Sy  IN  SWV AND C 3 H M I C E  DURING  A C O N T R O L RUN A N D A F T E R 3 . D A I L Y , I N J E C T I O N S O F THE VEHICLE  1/4  OR P I T R E S S I N  TANNATE.  UNIT P I T R E S S I N TANNATE IN  OIL  X»M-  12.  B<  UNTREATED  •  DAY 1  =  DAY 2  •  OAY 3  W  i  4...  fi  O1x2  ML-  PEANUT O I L  I-M-  12-  B-  UNTREATED  •  DAY 1  =  DAY 2  •  DAY 3  O  i  4-  OCONTROL RUN 12'  LNTFEATED  •  8-  as  4...  0-  fi  0  1 S 3  0  1 2  3  0  1 S 3  0  1 S 3  DAYS SWV F SIGNIFICANT  SWV M DIFFERENCE  C3H F  C3H-2  BETWEEN T H E 4 DAYS P <  M 0-05  79  off again on day 3 as i t did in the SWV females, therefore the C3H females did respond to exogenous ADH. This confirms that the pitressin tannate was pharmacologically active and that the dose level was adequate. The C3H-2 males did not, however, respond to the pitressin tannate. Their water intake and urine volume increased slightly over'the 3 days and their urine osmolality increased slightly on day 1, but none of these differences was significant. days.  The osmolal excretion increased steadily over the 3  There was a significant difference when an overall analysis of variance  was done, but when individual days were compared using a Scheffe's analysis of variance with multiple comparisons, there were no significant differences. The osmolal excretion is not an independent parameter but a product of the urine output and the urine osmolality, therefore any differences in the latter 2 were magnified  in the osmolal excretion.  On day 0 of the pitressin tannate run, the C3H females' urine osmolality (1593 + 195 mOsm/kg) was significantly different from that of the C3H-2 males (2302 + 203 mOsm/kg); but by day 3, the C3H females' urine osmolality (2397 + 176 mOsm/kg) was no longer significantly different from that of the C3H-2 males (2702 + 202 mOsm/kg). Since the standard errors were the same, these results suggested that the C3H-2 males did not respond to exogenous ADH because their urine was already maximally concentrated; whereas the C3H females' urine was not. C3H-1  The C3H females included both C3H-1 and C3H-2 females. The  females had a lower urine osmolality on day 0 than did the C3H-2  females, but both groups of females showed a significant increase in urine osmolality on day 3. The increase in urine osmolality after 3 injections of pitressin tannate was compared to that after 7 hours of dehydration. osmolality was:  The increase in urine  SWV females, 510 mOsm/kg after dehydration and only 163  80 mOsm/kg after pitressin tannate; SWV males, 638 mOsm/kg after dehydration and only 293 mOsm/kg after pitressin tannate; C3H females, 825 mOsm/kg after dehydration and 805 mOsm/kg after pitressin tannate; and C3H/sph males, 632 mOsm/kg after dehydration and 467 mOsm/kg after pitressin tannate.  There-  fore the SWV females and males could increase their urine concentration more after dehydration. (4)  Similarity to Some Kidney Diseases  (a)  Urinalysis  There was no glucose in the urine of the SWV females, SWV males, control C3H-2 females, or C3H-2 males.  This eliminated the possibility that  the polydipsia-polyuria was caused by diabetes mellitus.  There was no pro-  tein in the SWV females' urine although there was approximately 30 mg/100 ml of protein in the C3H-2 females' urine, and a trace amount of protein in the SWV and C3H-2 males' urine.  The complete absence of protein in the SWV fe-  males' urine was probably a dilution effect due to the severe polyuria. The urine had a pH of approximately 6 and contained no occult blood in the 4 groups of mice. (b)  Per Cent Packed Cell Volume  The C3H-2 females and males showed no change in per cent packed c e l l volume (PCV) with increasing age (Figure 13). The males had a significantly lower PCV than the females at 8 and 15 months of age. The SWV females and males, on the other hand, had a significant negative linear regression of PCV with age (Figure 13) and the females had a larger j^b than the males (P = .04). The SWV females had the same PCV at 6 months that the C3H-2 females had at 8 months, but the PCV decreased significantly over the next 2 - 3 % months so that by 8 - 9% months the SWV females had a significantly lower PCV. The  AGE (MONTHS)  82 SWV females continued to become progressively more anaemic up to 16 months. The severity of the anaemia correlated with the severity of the polydipsia although the anaemia developed after the animals had already become polydipsic:  i e . , at 6 and 8 months the SWV females had a normal PCV (Figure 13)  whereas they were definitely polydipsic (Table VI, p. 38). The SWV females had the same PCV as the SWV males at 6 and 8 - 9 % months, but by 11% - 12% months they were significantly more anaemic.  How-  ever by 14% - 16 months the males had become more anaemic and were no longer significantly different from the females.  The SWV males had a significantly  lower PCV than the C3H-2 males at 11% - 12% months, but not at 14% - 16 months since the PCV of the C3H-2 males had also dropped.  Thus again the SWV  males were intermediate between the controls and the SWV females. The decreased PCV in the SWV females probably reflected a decrease in red blood c e l l mass rather than an expansion of the plasma volume since the mice looked dehydrated and the serum osmolality was not significantly decreased (p. 60).  The anaemia was not caused by a loss of red blood cells  through the urine (p. 80). (c)  Blood Urea Nitrogen  The blood urea nitrogen (BUN) was slightly elevated in the SWV females especially at 15 months (Table XIX), but since the azostix reagent strips were only able to give an estimate BUN the increase was probably not significant.  While a more accurate method would have to be used to determine i f the  SWV females had a small significant increase in BUN, these results indicated that there was no large increase in BUN.  83 TABLE XIX.  Blood urea n i t o r g e n v a l u e s o f the p o l y d i s p i c SWV and the c o n t r o l C3H-2 s t r a i n measured u s i n g the a z o s t i x reagent s t r i p s .  Age Strain  SWV  C3H-2  Blood u r e a n i t r o g e n  Sex  (mon.)  N  F  13  10  17  4.2  15  3  20  6.3  M  13  10  13  1.1  F  13  10  13  2.3  15  4  15  1.6  13  10  11  0.8  M  (mg/100 ml)  + S.D.  84 (5) His tology (a) Pituitaries The dark purple neurosecretory material clearly demarckated the posterior pituitary (pars nervosa) in the SWV and C57 females.  The intermediate  lobe (pars intermedia) was also visible as a lighter shade of purple.  There  was abundant aldehyde fuchsin-positive material present in the pars nervosa of the SWV The SWV  females (Figure 14a) when compared to the C57 females (Figure 14b).  females' pituitary glands were larger than those of the C57 females  (Table XX), but this may have been because the SWV weight.  females had a greater body  The posterior and intermediate lobes occupied the same percentage of  the pituitary gland in the 2 strains.  However since the sample size was  very small, a more comprehensive study would be required to demonstrate that there was no significant difference in the size relations of the various lobes. (b)  Adrenals  The adrenals of the SWV  females (Figure 15) were normal when compared  to control adrenals of the same age (Figure 16). strains showed the usual signs of ageing:  The adrenals of the 3  a proliferation of connective  tissue cells (spindle cells) in the cortex just below the capsule, a migration of the spindle cells inwards through the cortex, and accumulation of these cells at the cortico-medullary junction (X zone), and an accumulation of pigment in the X zone and medulla (Figures 15, 16, 17, 6c 18). of the zone fasiculata cells of the SWV  The cytoplasm  females' adrenals seemed slightly  denser than that of the control females which might indicate a slight stress reaction, however the difference was d i f f i c u l t to quantify. There was no difference in the size of the adrenal or in the width of the zone glomerulosa between the SWV and control strains (Figures 19 6c 20).  35  Figure 14a.  Figure 14b.  SWV female 764, 13% months, pituitary, pars nervosa had abundant aldehyde fuchsin-positive material, mag. X 25.  C57 female 2119, 13% months, pituitary, pars nervosa stained with aldehyde fuchsin, mag. X 25.  #  TABLE XX.  Relative sizes of the pars nervosa, pars intermedia and whole pituitary gland in SWV and C57 females.  Strain  Animal #  B.W. (R)  SWV  855  30.0  2.74  1.46  0.45  0.13  764  40.0  2.46 x 1.73  0.28  0.15  879  31.0  2.37  X  1.46  0.35  0.12  872  31.0  2.46  X  1.28  0.55  0.20  average  33.0  2.50  X  1.50  0.40  0.15  2181  24.7  2.37  X  1.46  0.27  0.10  2119  .27.8  2.74  X  1.10.  0.42  0.13  2179  25.1  2.19  X  1.10  0.33  0.16  25.9  2.44  X  1.22  0.34  0.13  C57  average  Pituitary gland length x height (mm) X  Area of p. intermedia area of pituitary  Area of p. nervosa area of pituitary  87  >•'*."'  '.V.. '  • Jj^-  Figure 15.  SWV female 949, 12 months, adrenal, normal except for signs of ageing, mag. X 25.  Figure 16.  C57 female 2208, 11 months, adrenal, mag. X 25.  Figure 18.  C57 female 2208, 11 months, adrenal, spindle cells present in cortex and X zone and pigment present in X zone, mag. X 100.  89  Figure 19.  SWV female 949, 12 months, adrenal, normal zona glomerulosa, mag. X 400.  Figure 20.  C57 female 2208, 11 months, adrenal, normal zona glomerulosa, mag. X 400.  90 (c) Kidneys The old SWV females had abnormal kidneys. enlarged, and f i l l e d with fluid.  The kidneys were yellowish,  The pelvis was very enlarged and in some  females, approximately 17 months old, the pelvis was so enlarged that very l i t t l e renal tissue remained (hydronephrotic).  The bladder in the SWV females  was also very distended and f i l l e d with clear urine.  However the kidneys  s t i l l had a smooth surface and the capsule was easy to remove. The old SWV females had significantly heavier kidneys (wet weight) than did the SWV males or the controls (Figure 21).  There was no significant  difference in kidney weights between those mice fed ration 1 and 2;  therefore  che kidney weights of a l l mice regardless of their feed were combined.  There  was, however, a significant difference in the kidney weight between SWV nulliparous females and multiparous females at 16 months; therefore these data were analyzed separately.  There was no significant difference between  these 2 groups at 17 months. The SWV females' kidneys were not significantly different from the SWV males' at 4 months, they were significantly lighter at 5 months, and they were significantly heavier from 6 to 21 months.  The SWV females had signifi-  cantly heavier kidneys than the control females and males from 5 to 21 months.  In the SWV females there was a significant increase in kidney weight  between 5 to 6 months and 11 months, but unfortunately no data were collected from 7 to 10 months.  There was a second significant increase in kidney weight  between 14 and 15 months in the multiparous females, and then a significant decrease between 15 and 16 months.  From 16 to 21 months there was no signifi-  cant change in kidney weight. The SWV males had significantly heavier kidneys than the C57 mice, except at 10 months of age.  There was, however, no significant  difference  FIGURE 21 •  WET K I D N E Y WEIGHT  (G)  ± S y A T VARIOUS AGES IN  SWV> C 3 H , A N D C 5 7  T  2-04-  AGE  (MONTHS)  MICE  92 in kidney weight between the SWV males and C3H-2 males.  The SWV and C3H-2  males had body weights of about 33 and 34 g, respectively, whereas the C57 mice had a body weight of about 26 g.  Thus the difference in kidney weight  between the SWV males and C57 mice could have been partially due to the large difference in body weight, but this would not explain the large increase in kidney weight in the SWV females since the females also have a body weight of approximately 33 g. There was no significant difference in kidney weight between the C57 females and males except at 3 months; therefore from 4 to 16 months the data from both sexes were combined (Figure 21).  The C3H-2 males had significantly  heavier kidneys than the C3H-2 females at 16 and 21 months; therefore the data of each sex were kept separate.  Again the difference in kidney weight  between the 2 sexes may be at least partially due to differences  in body  weight since the C3H-2 females had a body weight of only 29 g as opposed to the C3H-2 males' body weight of 34 g. The SWV females' kidneys had a specific lesion when examined microscopically.  The tubules in the inner cortex and cortico-medullary zone were  hypertrophied and the tubular epithelium Was hyperplastic. diffuse and involved both kidneys.  The defect was  There was a steady progression of the  defect from being mild at 6 months to very severe at 17 months. The mouse kidney is divided into 4 zones: outer medulla, and inner medulla.  outer cortex,  inner cortex,  The tubules in the inner cortex and outer  medulla were larger than those in the outer cortex and the epithelial cells were also larger and had more abundant cytoplasm (Figures 22, 23, 24, & 25). The control kidneys had some small focal lymphocytic infiltrations and some of the kidneys had a chronic pyelitis. The kidneys of 4 month old SWV females were normal.(Figures 22 & 24)  93  Figure 23.  C57 female 2063, 13 months, kidney, normal cortico-medullary zone, mag. X 25.  Figure 25.  C57 female 2063, 13 months, kidney, normal cortico-medullary zone, mag. X 100.  when compared to kidneys of 12 month old C57 females (Figures 23 & 25). The tubules in the cortico-medullary zone were not dilated, the glomeruli were normal; and there was no focal chronic i n t e r s t i t i a l nephritis or fibrosis By 5% - 6 months the SWV females' kidneys were abnormal.  The tubules in the  cortico-medullary zone were moderately hypertrophic and dilated (Figure 26) and most of the tubules contained a protein precipitate.  In one of the  females the kidney was affected in patches with apparently normal tubules lying beside dilated tubules.  The kidneys had very l i t t l e focal interstitial  chronic nephritis, no lymphangiectasis,  no hyaline casts, and no v i r a l bodies.  By 9 months the defect was slightly more severe.  The tubules were more  dilated and there was more focal chronic i n t e r s t i t i a l nephritis (Figure 27). The kidneys of 11% - 13% month old SWV females had a marked tubular hypertrophy and dilation (Figures 28 & 29). The tubular epithelium was s t i l l hyperplastic and high cuboidal cells lined the dilated tubules (Figure 30). Some of the tubules were degenerating and some of these contained hyaline casts (Figures 28 & 29). It was unlikely that these casts were caused by protein leakage since they seemed to be restricted to degenerating  tubules.  There was some focal chronic i n t e r s t i t i a l nephritis in a l l of the kidneys examined, but the severity ranged from very mild infiltrations to very severe ones (Figure 31).  There was no fibrosis.  There were a number of  dilated lymphatics which contained a protein precipitate (lymphangiectasis). Also in a few of the proximal convoluted tubules in the outer cortex, a distinct blue line could be seen on the luminal border (Figure 32) which was not present in the C57 kidneys (Figure 33).  This line could represent  calcification, but that would have to be substantiated by staining the kidneys for alkaline phosphatase. This possible calcification could be seen in 50% of the kidneys examined at this age. The glomeruli in the SWV females were  F i g u r e 27.  SWV female 616, 9 months, k i d n e y , more s e v e r e l y t u b u l e s i n c o r t i c o - m e d u l l a r y zone, mag. X 100.  dilated  97  F i g u r e 28.  SWV female 855, 13% months, k i d n e y , s e v e r e l y d i l a t e d t u b u l e s and some h y a l i n e c a s t s i n c o r t i c o - m e d u l l a r y zone, mag. X 25.  F i g u r e 29.  SWV female 627, 12 months, k i d n e y , h y a l i n e c a s t s and f o c a l i n t e r s t i t i a l n e p h r i t i s i n c o r t i c o - m e d u l l a r y zone, mag. X 100.  98  Figure 30.  SW female 626, 12 months, kidney, high cuboidal epithelial cells of dilated tubules in cortico-medullary zone, mag. X 400.  Figure 31.  SWV female 626, 12 months, kidney, severe focal inters t i t i a l nephritis in cortico-medullary zone, mag. X 100.  99  Figure 32.  SWV female 855, 13% months, kidney, distinct blue line on luminal border of proximal convoluted tubules in outer cortex, mag. X 400.  Figure 33.  C57 female 2063, 13 months, kidney, normal proximal convoluted tubules in outer cortex, mag. X 400.  100 normal at this age (Figure 34). Finally in the 17 month old SWV severe.  females the defect had become very  There were a large number of extremely dilated tubules, and many of  them showed signs of degeneration and necrosis (Figures 35, 36 6c 37).  There  was a large amount of chronic i n t e r s t i t i a l nephritis and i n t e r s t i t i a l fibrosis surrounding the dilated tubules in the cortico-medullary zone (Figures 36 6c 39).  Some of the proximal convoluted tubules in the outer cortex had the  same distinct blue line on their luminal border which had been described in the 12 month old kidneys (Figure 38).  In these severely affected kidneys  some of the glomeruli were starting to hyalinize (Figure 39) when compared to control glomeruli (Figure 40), and some fibrosis was present in the outer cortex. The kidneys of the SWV males appeared to be normal (Figure 41) when compared to kidneys of C57 males of the same age (Figure 42).  The SWV males  had some focal chronic i n t e r s t i t i a l nephritis as did the controls.  Since  some focal chronic i n t e r s t i t i a l nephritis was present in the kidneys of the females and males of both strains, i t is probably unrelated to the tubular dilation and hypertrophy in the SWV  females.  However the 17 month old SWV  females' kidneys had such severe focal chronic i n t e r s t i t i a l nephritis and fibrosis, that i t might be related to the kidney defect.  This would have to  be substantiated by examining 17 month old control kidneys. C.  Genetics of Polydipsia-Polyuria Defect (1)  Quantitative Estimate of Number of Loci  The standard deviations (s) of the SWV,  C3H-2, and F-^ males and females  were very different (Table XXI), but the CV's were more alike. the water intake data were transformed using logs.  On this basis  The large difference in  101  102  Figure 36.  SWV female 848, 17 months, kidney, severe i n t e r s t i t i a l fibrosis and nephritis surrounding dilated tubules in cortico-medullary zone, mag. X 100  103  Figure 37.  SWV female 848, 17 months, kidney, dilated tubules in cortico-medullary zone, mag. X 400.  Figure 38.  SWV female 848, 17 months, kidney, possible calcification on luminal border of proximal convoluted tubules in outer cortex, mag. X 400.  104  Figure 39.  SWV female 848, 17 months, kidney, hyalinized glomeruli in cortex, mag. X 100.  Figure 40.  C57 female 2063, 13 months, kidney, normal glomeruli in cortex, mag. X 100.  105  Figure 41.  SWV male 572, 12 months, kidney, normal cortico-medullary zone, mag. X 100.  Figure 42.  C57 male 2032, 12 months, kidney, normal cortico-medullary zone, mag. X 100.  106 TABLE XXI.  Estimation of number of loci (n) determining polydipsia using Wright's formula. Formula is based on an additive model with semidominance and no interactions. Estimation done using water intake values (ml/24 hrs). The value of n in brackets was calculated using the non-trans formed data.  N  Mean  s  CV  Log Mean  Log s  n  Females - Ration 1 (11--16 mo.) SWV SWV  BC  F  l  F  2  C3H-2  12  41.9  17.4  .42  1.578  .049  29  52.4  20.1  .38  1.684  .033  23  23.8  13.4  .57  1.292  .088  41  17.8  10.4  .59  1.185  .075  12  6.4  1.5  .23  0.798  .010  -  . 4.7  Females - Ration 2 (16 mo.)  (B)  SWV F  l  BC  C3H-2  C3H-2  13  57.2  17.6  .31  1.735  .023  16  21.4  5.4  .25  1.319  .014  34  8.3  2.7  .32  0.893  .016  19  8.4  2.3  .28  0.908  .012  17.6  Males - Ration 2 (16 mo.)  (C)  SWV SWV  BC  F  l  BC  C3H-2  C3H-2  9  19.5  5.6  .29  1.276  .014  19  15.9  5.5  .35  1.180  .019  15  11.0  2.5  .23  1.030  .011  31  12.3  2.9  .23  •1.081  .010  7  9.6  2.2  .23  0.973  .012  2.2  (1.1)  (0.2)  107 s but not in CV indicated that the non-genetic factors, at least, tended to act multiplicatively. The variances of the reciprocal matings of the male BC^.^ 2 P r o § e n y were significantly heterogeneous;  but the means were not significantly  different when an approximate test of equality of means for heterogeneous variances was done (Sokal & Rohlf, 1969).  Nevertheless the data for the re-  ciprocal matings were pooled since any variance that was present should be included in the estimation of the number of l o c i .  The only other case of  heterogeneity between reciprocal matings was in the F£ females.  The F 2  females from SWV/C3H-2 dams had a significantly higher mean water intake than did those females from C3H-2/SWV dams.. Again the data from both reciprocal F 2 matings were pooled, as were the data from a l l other reciprocal matings. Nevertheless this reciprocal difference in the  females suggested that  water intake was influenced by the maternal physiology. (a)  Females  The water intake data for the various generations are given in Table XXI(A) for females fed ration 1 and in Table XXI(B) for 2.  females fed ration  These data did not meet a l l the assumptions required to use Wright's  formulas (p. 24).  The F-^ was lower than expected in an additive model when  the females were fed ration 2 (21.4 ml/24 hrs as opposed to an expected 32.8 ml/24 hrs); suggesting that the gene or genes were not semidominant, but rather dominant for a normal water intake.  The F-^ for females fed ration 1  (23.8 ml/24 hrs) was very close to the expected 24.2 ml/24 hrs.  The B C g w v  was much higher than that expected in an additive model (52.4 ml/24 hrs as opposed to 33.0 ml/24 hrs), in fact i t was higher than the SWV of 41.9 ml/24 hrs.  Conversely on the other ration, the BC£<JH_2 was much lower than expected  108 (3.3 ml/24 hrs as opposed to 20.6 ml/24 hrs) and was in fact the same as the C3H-2 of 8.4 ml/24 hrs.  The transformed data did not fit the expected re-  lationships much better except that the F^ of the females fed ration 2 was now the expected value of 1.322.  Thus in the females' water intake data  there were strong interactions occurring in the BC generations in that the BC resembled or surpassed the parental mean even though it should only have 75% of the parental genotype in an additive model. The variances also deviated from their expected relations (p. 24). The F^ s  was very high for both females fed ration 1 (13.45) and for those The F^ s 2 was assumed to represent the non-genetic s2  fed ration 2 (5.4).  since a l l F-^ progeny should have the same genotype unless there was hetero2  zygosity in one of the parental strains. Since the F^ s was high, in fact 2 2 higher than the F2 s and the BCgyy s , it made any estimation of the number of l o c i using Wright's formula very unreliable or impossible. Wright assumed that the F2 s  2  and BC s  2  was made up of the non-genetic s  2  and the genetic  2  s , thus some interaction must have occurred since it was impossible that the 2 9 2 2 I5^C3H-2 s and F2 s had no genetic s and less non-genetic s than the F-^. The  BC^2JJ_2  s  2w  a s  slightly higher than the  2 2 averaging the SWV s , F^ s ,  2 and C3H-2 s .  E  s  2  estimate obtained by  The estimate of n, however, was  very high (n = 17.6) and probably very unreliable due to the strong interactions and dominance.  The F£ was lower than the F^ (17.8 ml/24 hrs as 2  opposed to 23.8 ml/24 hrs) and the F2 s was smaller, instead of being larger, 2 than the F s (10.41 as opposed to 13.45). After being transformed, the F2 (1.185) was close to the expected F^ (1.188); but the F2 s 2 was s t i l l too low when compared to the F^ s2 although 2  it was now twice the BC O T I „ s . Thus while the F 0 data showed less interaction SWV 2 than the BC data, the estimate of n = 4.7 was s t i l l unreliable. The 2  109 estimates were quite different confirming that 1 or both were unreliable, (b) Males The water intake data for the male progeny fed ration 2 are given in Table XXI(C).  The data for the males, as well as the females,  a l l the required assumptions for an additive model.  did not meet  The F^ (11.0 ml/24 hrs)  was lower than expected (14.6 ml/24 hrs) suggesting that the normal genotype was again dominant.  The BCg^y was also lower than expected (15.9 ml/24 hrs  as opposed to 17.0 ml/24 hrs) suggesting some interaction. however, was close to the expected value of 12.1 ml/24 hrs.  The  ^Q^E-I'  The transformed  F-^ was s t i l l too low although the transformed BCgWy was closer to the expected value. o  The male F^ s  was relatively large (2.5) but not as large as the F^  2  females' s  (5.4).  This suggested that although there was less non-genetic  2 s s  than with the females, 2  should equal the  it s t i l l did not f i t an additive model.  BCQ.JH_2 S  2  and be \ the 2  2  s .  The c 3 H - 2 BC  s  2w  The BC  SWV  a  s  approximately the same as the F-^ s , reflecting some interaction; and therefore the BCc3H-2 ^  a t a  Save  a  very small n (0.2).  The BCg^ data gave an  estimate of n = 1.1 which was probably more reliable since there was not as much apparent interaction in the BCgWV data. The transformed B C Q - J J ^ S ^ was 2 less than the transformed F^ s ; therefore no estimate of n could be calcu2  lated.  The transformed BCg^y s 9  the transformed F^ s  was no longer significantly different from 9  or BC^-^^ s , but the estimate of n was higher (2.2).  The F 2 male progeny were fed ration 1; therefore the  statistics  were not compared to the other generations since the F-^ statistics of females fed ration 1 were quite different from those fed ration 2. In summary:  the water intake data of both the female and male progeny  110 did not meet the required assumptions for an additive model.  Therefore any  estimates of n calculated from Wright's formulas were unreliable.  This was  emphasized by the large descrepancy in n when estimates were calculated from ^C3H-2  B  anc  * ^2 ^  a t a  ' There seemed to be less interaction among the males;  therefore the estimate of 1 or 2 loci might be correct. There appeared to be strong interactions between the gene or genes causing polydipsia and the other genes controlling water intake.  This data also emphasized the impor-  tance of non-genetic variability in polydipsia.  This had already been  demonstrated since water intake was influenced significantly by the type of ration, age, sex, and parity in the SWV females. s  2  In addition the SWV females'  2 was much larger than the C3H-2 females' s and after 25 generations of  inbreeding there should be l i t t l e heterozygosity in the SWV strain. (2)  Qualitative Estimate of Number of Loci  The observed frequencies of polydipsia in each generation are given in Table XXII.  The frequencies were calculated separately for each sex.  Since  the type of ration did not alter the penetrance of polydipsia in the SWV females (1007,), the data for a l l females, regardless of their feed, were combined.  A l l the SWV males fed ration 2 were polydipsic, whereas only 797,  of the SWV males fed ration 1 were polydipsic. Therefore, since the expected frequencies for male progeny fed ration 1 would be .79 times the theoretical frequency, the expected frequencies for each generation were calculated separately for each ration.  The theoretical frequencies expected from 1, 2,  or 3 genes are given in Table XXII(A). There were no significant differences in the frequency of polydipsia between reciprocal matings in the F-^, F2, BCgyy, or BCc3H-2 generations. This ruled out any sex linkage or maternal transmission.  There was, however,  Ill TABLE XXII.  (A)  Frequencies of polydipsic progeny.  Theoretical frequencies expected from 1 to 3 dominant genes.  No. genes  penetrance  SWV  1  100  100  (B)  SWV  F]_  F2  100  100  75  50  BC  BC  C3H  78.5  78.5  78.5  78.5  59  39  50  50  50  50  38  25  2  100  100  100  100  56  25  3  100  100  100  100  42  13  100.0  100.0  94.9  70.8  26.5  Males (Rat. 1)  78.6  80.0  21.4  53.8  (Rat. 2)  100.0  100.0  65.0  Observed frequencies. Females  -  61.3  112  significant heterogeneity in frequencies between progeny of different dams in the  BCQ-JH-2  progeny.  §eneratiori-  This occurred both in the female and male  This heterogeneity must be caused either by an environmental in-  fluence on penetrance, or by some heterozygosity in the parental strains. The C3H-2 strain was only in the Fg generation of inbreeding following some minimal outcrossing to another line; therefore some heterozygosity in the C3H-2 strain was possible. (a)  Females  The frequency of polydipsia in the SWV and B C g ^ females was 100%. The W  frequency of polydipsia in the F-^ females fed ration 2 was also 100%; but in the F^ females fed ration 1 it was 95%, (N = 23).  This reduced penetrance  could be due to the ration or some other environmental difference between the 2 years, or the limited testing of progeny in the first year.  The 2  normal F-^ females were only tested at 11 months and might have developed polydipsia at an older age.  In any event, the polydipsia was dominant with  1007, penetrance in the SWV strain. In the F2 generation, 7 1 % of the females were polydipsic. a very close fit to a 1 dominant gene model.  This was  The data were significantly  different from that expected in a 2 dominant gene model (0.025 > p > 0.05) when a G pooled test was done; but when a G total test was done there was sufficient,  but not significant, heterogeneity that the data could just f i t  a 2 dominant gene model (0.10 > p > 0.05). of the females were polydipsic.  In the BC^,^ 2 § e n e r a t : i - o n > 27%  This frequency fitted a 2 dominant gene  model (p > 0.05); but it did not fit a 1 dominant gene model (p < 0.001). The  BC -j}T_2 c  females were tested more often than the F2 females; there-  fore the BCrj3 _2 data should give a better estimate. H  The F2 frequency might  113 be lower than it should be due to animals not being tested after they had become polydipsic.  If the  frequency was too low, this would strongly  support a 1 gene model rather than a 2 gene model. Conversely there were strong interactions occurring in the B C data. This might have caused a lower than expected  BCQ-^J^  frequency since the  K"C3H-2 resembled the C3H-2 more than was expected in an additive model. Thus the polydipsia might in fact be determined by 1 dominant gene and some interaction at the  ^CQ2R-2  level caused a reduced penetrance.  The F 2 and F 2  2 s  suggested that there was less interaction in this generation, and there-  fore the 71% polydipsia could be an accurate estimate of the gene frequency and be due to 1 dominant gene. (b) Males A l l the SWV males fed ration 2 were polydipsic; but only 79% of the SWV males fed ration 1 were polydipsic.  Thus the theoretical  frequencies  for each generation were corrected for 79%, penetrance in those males fed ration 1.  In the BCgWy generation, 100% of the males fed ration 2 were poly-  dipsic and 80% of the males fed ration 1 were polydipsic. was a close f i t to the expected 79%.  The 80%, frequency  The frequency of polydipsia in the F^  generation was much lower than expected - being 657» of males fed ration 2 and 21%, of males fed ration 1.  The 21% polydipsia was significantly  different  from the theoretical frequency of 79% (p < 0.005) expected for dominance with a 79% penetrance.  Thus polydipsia was dominant in the males but there  was reduced penetrance in the males fed ration 1 and in the F-^ generation. The reduction in polydipsia in the SWV, BCgyy and Fi males could be due to either "reduced penetrance,  interactions with the background genotype for  water intake, or heterozygosity.  Since the SWV strain had been inbred for  114 30 to 36 generations and showed no significant dam - progeny or sire progeny regression,  it was unlikely that there was much heterozygosity.  Thus  there was reduced penetrance of the polydipsia genotype which was caused, partially at least, by environmental factors such as the type of ration. F-^ and F^ s  The  had indicated that interactions were occurring and that normal  water intake was dominant over polydipsia.  The low frequency of polydipsia  in the F-^ males might be due to interactions with those genes determining normal water intake. In the F2 generation, 547, of the males were polydipsic.  The frequency  was not significantly different from that expected in a 1 dominant gene model with 807, penetrance (p >0.05).  However, the 547, polydipsia also fitted a 2  dominant gene model with 807, penetrance (p > 0.1). 617, of the males were polydipsic. penetrance was expected. (p > 0.05)  In the B C ^ j ^  generation,  Since these males were fed ration 2, 1007,  The 617, polydipsia fitted a 1 dominant gene model  but not a 2 dominant gene model (p < 0.005).  There was less interaction in the males' water intakes than in the females', but there was s t i l l some interaction in the The  BCrj3jj_2  was low in comparison to the F^  only a limited range of phenotypes present.  BCQ3^_2  generation.  suggesting that there was  The amount of interaction  present in the F^ generation males was not known, but as mentioned there was less testing of the F2 progeny than the B C ^ ^ ^ progeny. 657, polydipsia in the F^ males might be too low.  This meant that the  In addition since the  males were fed ration 1 and there was a marked reduction in penetrance in the males fed ration 1, it is possible that there was also reduced penetrance in the F2 males.  A reduction of penetrance in the F2 males would strongly  support the 1 dominant gene model as opposed to the 2 dominant gene model. In summary:  polydipsia was determined in both sexes by 1 or 2  115 autosomal  dominant genes i n t h i s  the males fed r a t i o n 1, fed r a t i o n  (3)  strain.  There was  reduced p e n e t r a n c e  i n the F-^ males f e d r a t i o n 2,  and  Age  generations  o f Onset  fed r a t i o n 2 a r e shown i n T a b l e X X I I I .  were a n a l y z e d s e p a r a t e l y  f o r each sex.  There was  p o l y d i p s i c by 4 to 5 months o f age, 13 months o f age,  and  t o 18 months.  great v a r i a t i o n The SWV  intakes u n t i l  i n t a k e s t e a d i l y up  i n that  water  i n t a k e , and  ( o n l y 2 m i c e ) , BCswv males,  average age o f onset o f 9 months.  so  reached a peak  and F-^ females had  females had a l a t e r average age o f onset 19 months) than the F-^ females.  average age o f onset  (16  (15  ^> Q2R-2 C  M  A  ^  Similarly  i n t e r m e d i a t e between those o f the Fi  E  S  '  The  BCQ2R-2  months) and a much wider the F-^ males had a  months) and a wider range  However the  an  However the a c t u a l age might have been  s i n c e they were not t e s t e d p r i o r t o 9 months o f age.  males.  The  then stopped b e i n g p o l y d i p s i c .  The BCgyy females  BCgyy  to  death  some mice became p o l y d i p s i c and remained  20 months o f age; whereas o t h e r mice became p o l y d i p s i c ,  -  their  to 20 months o f age.  until  (9  i n t a k e by 11  males were p o l y d i p s i c by 5 to 7 months o f age,  The SWV  but they i n c r e a s e d t h e i r water  earlier  i n the age  females were  reached t h e i r peak water  then had p r o g r e s s i v e l y lower  t e s t c r o s s progeny v a r i e d  o f the v a r i o u s  Once a g a i n the d a t a  of onset both between and w i t h i n each g e n e r a t i o n .  16  females  1.  The average age o f onset f o r the p o l y d i p s i c progeny  at  i n the F^  in a l l  (9  -  males.  later  20 months) than the  average age o f onset  and BC  range  (13  months)  was  In g e n e r a l the males  oW V  had a l a t e r average age o f onset than the females the per c e n t o f the SWV  genotype  p o l y d i p s i c mice became l a t e r .  decreased,  f o r each g e n e r a t i o n ; and  the average age o f onset  i n the  as  116  I  TABLE XXIII.  Average age of onset (mo.) of polydipsia in affected progeny fed ration 2.  range  N  Males mean  range  4  4-5  7  7  5-7  2  9  9  23  9  9-15  10  9  9-12  13  16  9-20  9  15  9-19  19  13  9-18  Group  N  SWV  5  SWV  BC  Fl BC  C3H-2  Females mean  117 DISCUSSION  A.  Description of Polydipsia-Polyuria Defect (1)  General Description  The SWV  strain had a large body weight compared to most inbred strains  (Bernstein, 1966), but this could have occurred inadvertantly during selection for good reproductive performance.  The severely affected SWV  females  weighed the same as the males which only had a mild concentrating defect. The polydipsic chickens (Dunson 6c Buss, 1968) defect similar to human nephronophthisis, body weights.  and the kd mice, which had a  (Lyon 6c Hulse, 1971) had normal  In contrast the homozygous DI rats had a lower body weight  than their heterozygous littermates (Valtin et a l . , 1965), while the DI and DI +/+  mice had heavier body weights than their VII +/+  or VII  0s/+  0s/+  littermates (Naik 6c Valtin, 1969). The polydipsic SWV  females had excellent v i a b i l i t y and f e r t i l i t y as  did the polydipsic chickens (Dunson 6c Buss, 1968) ; whereas the DI rats had inviability and s t e r i l i t y associated with the diabetes insipidus (Valtin, 1967).  Some old polydipsic SWV  females showed muscle weakness.  This also  occurred in some patients with human nephronophthisis (Goldman et a l . , 1966), but was not reported in the kd mice which had a similar disease (Lyon 6c Hulse, 1971). (2)  Water Balance  The SWV  females had as severe a polydipsia-polyuria as the following:  the DI rats (Valtin, 1967) and Ma/J mice (Hummel, 1960) which had a deficiency in ADH production; the DI 0s/+ mice which had vasopressin-resistant diabetes insipidus (Falconer et a l . , 1964; Naik 6c Valtin, 1969); the STR/N  118 (Silverstein et a l . , 1961) and T S / A (Szalay 6c Moll, 1966) mice which had primary polydipsia; the De/J mice which had gross hypertrophy of the adrenal gland (Chai & Dickie, 1966); the SWR/J mice (Hummel, 1964; and personal communication 1972); and, finally, primary polydipsia, vasopressin-resistant and vasopressin-responsive  diabetes insipidus in man (Relkin, 1966).  In. the  following the polydipsia-polyuria was much less severe than that of the SWV females; the polydipsic chickens (Dunson 6c Buss, 1968); human nephronophthisis (Giselson et a l . , 1970); the kd mice which had a defect resembling human nephronophthisis  (Lyon & Hulse, 1971; and gross observation in our laboratory);  and other human, chronic renal diseases (Relkin, 1966). The difference between water intake and urine output was 4.3 ml/day in the C3H females and 8.2 ml/day in the SWV females.  Since the SWV females  appeared dehydrated rather than edematous, the extra 4 ml of water must have been lost in some manner; for example, evaporation due to increased respiration.  This increased loss also occurred in the DI rats (Valtin et a l . , 1965)  and in rats with induced diabetes insipidus (Friedman & Friedman, 1965) in which the difference was 10 ml/day, as opposed to 5 ml/day in normal rats. The SWV males had a normal water intake (Table I, p. 30) but a mild concentrating defect (urine osmolality was 1026 mOsm/kg).  This was slightly  more severe than that of the heterozygous DI rats (Valtin et a l . , 1965) and mild DI +/+ mice (Naik & Valtin, 1969) which had urine osmolalities of 1400 mOsm/kg and 1500 mOsm/kg, respectively, and no polydipsia. The urine osmolality was an important parameter since i t made i t possible to detect the mild concentrating defect in the SWV males. The SWV females' urine osmolality of 327 mOsm/kg was the same as the STR/N's (Silverstein et a l . , 1961) but higher than the DI rats', 190 mOsm/kg, (Valtin et a l . , 1965) or that of the DI 0s/+ mice, 262 mOsm/kg, (Naik 6c  119 Valtin, 1969).  The osmolal excretion was significantly increased in the DI  rats due to increased food intake (Friedman 6c Friedman, 1965; Harrington 6c Valtin, 1968); whereas in the SWV females the osmolal excretion was also increased but the food intake was not.  There was no significant increase in  osmolal excretion in the DI 0s/+ mice (Naik 6c Valtin, 1969). (3)  Water Intake  In the SWV strain, water intake was affected by age, body weight, diet, sex, and parity.  In most strains of mice, age has l i t t l e effect on water  intake (Bernstein, 1966), but i t had a positive effect on the polydipsia in the SWR/J strain (Hummel, personal communication 1972), Ma/J strain (Bernstein, 1966), TS/A strain (Szalay 6c Moll, 1966), STR/N strain (Silverstein et a l . , 1961), and the DI 0s/+ and DI +/+ strains of mice (Falconer et al., 1964). In fact the pattern of water intake up to 12 months of age in the DI 0s/+ and DI +/+ 4, p. 42).  females was very similar to that seen in the SWV females (Figure Conversely Naik (1972) stated that his DI 0s/+ mice had stabilized  their water turnover by 4 months of age.  The polydipsic chickens, on the  other hand, showed an amelioration of their polydipsia with increasing age (Dunson 6c Buss, 1968). Body weight had a significant effect on the severity of the polydipsia in the SWV females but not for the other groups (Table III, p. 33). Body weight also had a large effect on water intake in the DI 0s/+ but not in the DI +/+ mice (Falconer et al., 1964).  However since the water intake was not  simply proportional to body weight, Falconer et a l . adjusted the water intakes using calculated regression coefficients.  The DI 0s/+ females had a  larger b than the SWV females and the DI 0s/+ males (2.63 as opposed to 1.84 and 1.10).  The DI +/+ females had a much smaller b (0.41) but Falconer et a l .  120 did not indicate i f the regression was significant. Naik (1972), Valtin et a l . (1962, 1964, 1967,  Naik & Valtin (1969),  1965), and Valtin (1966a, 1966b,  1969) have expressed their data for diabetes insipidus in mice and  rats as a simple proportion; ie., ml/100 g body weight. The SWV  females had a greater water intake when fed ration 2 which had  a higher fat and a higher sodium content than ration.1. the STR/N mice was also influenced by diet.  The polydipsia of  When the STR/N mice were fed a  high caloric density, high fat content diet their water intake decreased more than could be accounted for by the increased metabolic water (Silverstein et a l . , 1961).  The DI 0s/+ mice decreased their water intake and  increased their urine osmolality when fed a low sodium diet, instead of Purina Lab Chow (ration 2), for 6 to 8 weeks (Naik, 1970a).  This is similar  to human nephrogenic diabetes insipidus in which a decreased solute load slightly ameliorated the water intake and urine osmolality (Relkin, 1966). The SWV  females had a severe polydipsia whereas the SWV males had only  a slight increase in water intake.  The same sex difference was seen in the  TS/A strain (Szalay 6c Moll, 1966), SWR/J strain (Hummel, personal communication 1972), and Ma/J and Ma/J  strain of mice (Hummel, 1960).  In addition the TS/A  strains had a greater water intake in multiparous  nulliparous females, as was the case with the SWV parity has not been reported in SWR/J females).  females than in  females (the effect of The polydipsic chickens  showed an increased severity in polydipsia in females (especially egg-laying females) although the males were polydipsic (Dunson et a l . , 1972). 0s/+ and DI +/+  The DI  female mice also had a greater water intake than the males  (Falconer et a l . , 1964) but there was no sex difference in the DI rats (Valtin et al., 1965).  Human nephrogenic diabetes insipidus, which is  considered to be an X-linked recessive, is usually restricted to males; but  121 a few  cases  o f symptomatic  1969;  B u r s t e i n 6c Chen, 1970).  concentration  Therefore  and  female c a r r i e r s  (Bode 6c Crawford,  show a d e f e c t  comparable to the SWV  d u r i n g pregnancy but d i m i n i s h e s  can  be c o r r e c t e d by a d m i n i s t e r i n g e s t r o g e n s  ( N o c e n t i 6c C i z e k ,  a f t e r meno-  has  been suggested  1970).  the o t h e r hand, food d e p r i v a t i o n p o l y d i p s i a occurs  hamsters and  in renal  males) which becomes  some m o d u l a t i o n of the d e f e c t by e s t r o g e n  (Bode 6c M i e t t i n e n , On  The  (which i s p r o b a b l y  more marked a f t e r puberty pause.  females have been r e p o r t e d  1967).  d i p s i a only before  Female hamsters showed the  puberty,  d i p s i a responded to ADH  a f t e r ageing,  and  only  or sodium c h l o r i d e  food-deprivation  or a f t e r c a s t r a t i o n .  the s u g g e s t i o n was  i n male  The  made t h a t e s t r o g e n  poly-  poly-  enhances  vasopressin secretion. Thus i n some manner sex on  (presumably the s t e r o i d hormones) has  the s e v e r i t y of the d i a b e t e s  diabetes  i n s i p i d u s syndromes.  i n s i p i d u s syndrome i n the SWV Estrogens  i n sodium and water r e t e n t i o n , a t l e a s t Dignam et a l . , 1956).  i n c r e a s e s the water turnover  order  to s u b s t a n t i a t e the e f f e c t  immature females s h o u l d treated with estrogen,  B.  Differential (1)  transiently  i n the SWV  Diagnosis  other  increase 1959;  mechanism  females i s not known.  In  of the sex hormones on the p o l y d i p s i a , some  be c a s t r a t e d and and  (Dance et a l . ,  or some o t h e r  effect  mice and  have been shown to cause an  Whether i t i s t h i s e f f e c t  that  an  some immature males should  be  t h e i r water i n t a k e s measured over a p e r i o d of time.  of P o l y d i p s i a - P o l y u r i a Defect  Serum O s m o l a l i t y  While the SWV compared to the C3H s t r a i n , which had  females had females,  a s l i g h t l y elevated  i t was  not  serum o s m o l a l i t y as  significantly different.  primary p o l y d i p s i a , had  The  a normal plasma o s m o l a l i t y  STR/N  (Silverstein et a l . , 1961); whereas the Brattleboro rats, which had hypothalmic diabetes insipidus, had a significantly elevated serum osmolality (Valtin & Schroeder, 1964).  The DI 0s/+ mice, which had nephrogenic diabetes insipi-  dus, tended to have a slightly elevated serum osmolality, but it was not significant (Naik 6c Valtin, 1969). lalities:  There was a wide range of control serum osmo-  C3H females - 339 +3.8  mOsm/kg, VII +/+ mice - 309 + 3.0 mOsm/kg  (Naik 6c Valtin, 1969), and the STR/N controls - 327 + 3.0 mOsm/kg (Silverstein et a l . , 1961).  Naik 6c Valtin and Silverstein et a l . obtained similar s- for  the serum osmolalities of their control and affected mice as was obtained in the SWV and C3H strains in this study.  Whether the variation was due to  experimental procedures or reflected strain differences (2) (a)  is not known.  Response to Dehydration Water Deprivation  The SWV females raised their urine osmolality to 825 mOsm/kg after 7 hours of water deprivation but lost 13% of their body weight.  The STR/N  mice (primary polydipsia) significantly raised their urine specific gravity to within normal limits after 7 hours of dehydration and only lost 5%, of their body weight (Silverstein et a l . , 1961).  However the DI 0s/+ mice  (nephrogenic diabetes insipidus) tolerated dehydration even less than the SWV females in that they lost 12% of their body weight after 6 hours and could only raise their urine osmolality to 397 mOsm/kg (Naik 6c Valtin, 1969). After 19 hours, 2 DI Os/+ mice were dead and 4 more had lost 27% of their body weight.  After 6 hours of dehydration, 2 Brattleboro rats (hypothalmic  diabetes insipidus) had lost 8% of their body weight and their urine osmolality was 250 mOsm/kg (Valtin 6c Schroeder, 1964).  After 12 hours of dehydration,  the DI rats' urine osmolality was 390 mOsm/kg; after 24 hours, it was 805 mOsm/kg; and after 48 hours it was 1,155 mOsm/kg.  By this point the rats had  123 also lost 227» of their body weight, had a serum osmolality of 400 mOsm/kg (ad libitium was 325 mOsm/kg), and had a fivefold increase in BUN (Valtin, 1966a).  Therefore, the DI rats were not as dehydrated as the SWV females  after 12 hours i f the loss of body weight was used as the criterion for the severity of dehydration (Jakubczak, 227c  1970).  By the time the DI rats had lost  of their body weight, they had achieved a urine osmolality of 1,155  mOsm/kg even though they had no endogenous ADH (Moses & Miller, 1970). Therefore Valtin (1967) postulated that the rats concentrated their urine by decreasing their glomerular f i l t r a t i o n rate and going into severe renal failure.  Although the BUN was not determined,  the SWV females were also  probably in renal failure by the time they had lost 13 - 177, of their body weight, so that the increase of their urine osmolality to 825 mOsm/kg was not necessarily due to an increased secretion of endogenous ADH. Valtin (1967) speculated that a decreased glomerular f i l t r a t i o n rate would result in a decreased flow of fluid through the distal nephron and. the increased reabsorption of a small amount of free water in the distal tubule so that an iso-osmotic fluid would be delivered to the collecting ducts and further reabsorption of water could occur in those ducts. tonic urine.  This would result in hyper-  Both the DI rats and the SWV females showed a rapid recovery  from dehydration when returned to ad libitium drinking. The significance of the serum osmolality not rising significantly after 7 hours of dehydration is not known.  No comparable measurements were made  for the DI 0s/+ mice (Naik & Valtin, 1969)' or the STR/N mice (Silverstein et a l . , 1961).  Heterozygous DI rats raised their serum osmolality by 12 mOsm/kg  (just significantly higher than the controls) after 48 hours of dehydration and a weight loss of 137,.  Thus possibly after 19 or 24 hours of dehydration,  124 there would have been a significant rise in the serum osmolality of the SWV females. (b)  Saline Ingestion  The SWV females could tolerate water deprivation better than the DI 0s/+ mice (Naik & Valtin, 1969), but the DI 0s/+ mice could tolerate saline as their only source of water much better than the SWV females (Falconer et a l . , 1964; Naik, 1970b).  At 2 - 3 months of age the DI 0s/+ females in-  creased their intake from 20 ml of tap water to 30 ml of saline, and the DI 0s/+ males increased their intake from 15 ml of tap water to 28 ml of saline after they had been on saline for 7 days (Falconer et a l . , 1964). lost weight to the same extent as the SWV males. to saline in the same manner as the C57 mice.  They both  The DI +/+ mice responded  Thus in this instance the DI  0s/+ mice, not the DI +/+ mice, behaved like the SWV males.  At 3 - 5 months  of age, the DI 0s/+ mice increased their saline intake 3 times over their normal water intake, whereas the controls drank less (Naik, 1970b).- This was a greater increase than that of the SWV males, but i t took longer to occur since after 3 days the DI 0s/+ mice were drinking less saline than tap water. No report was made of any changes in body weight.  Thus the SWV males and 2 -  3 month old DI 0s/+ mice (and possibly older ones as well) could not eliminate the excess solutes without excreting more water than was ingested.  In  rats, controls preferred saline whereas rats that had experimentally produced diabetes insipidus preferred tap water (Palmieri & Taleisnik, 1969). When the diabetes insipidus rats were fed a low sodium diet their water intake increased; but when they were given 1% saline as their only source of water, 257, of the rats died.  There was an average body weight loss of 20 g and the  rats drank 7 times less saline than their normal water intake.  Palmieri &  125 Taleisnik (1969) postulated that the rats rejected the saline and died of dehydration.  Yet rats with a milder form of diabetes insipidus, which was  also produced experimentally, could drink 1% saline for 3 days without any change in intake (Friedman et a l . , 1962). Palmieri & Taleisnik suggested 3 factors which could be responsible for the rejection of saline by the rats: (a)  a lack of hypophyseal hormones, because vasopressin causes an increased renal excretion of sodium and chloride (Brooks & Pickford, 1958)  (b)  a lesion in the hypothalmic area that may control sodium intake  (c)  an alteration of the intracellular osmotic relationships.  They postulated an increase in the intracellular content of sodium which was not completely matched by water.  This had been demonstrated in rats with  mild diabetes insipidus (Friedman et a l . , 1962).  In mild cases of imbalance  the animal would try to compensate by increasing its water consumption; for example, SWV males, DI 0s/+ mice, and rats with mild diabetes insipidus.  In  severe cases of imbalance; "for example, SWV females and rats with severe diabetes insipidus, the animal would reject the saline solution.  The rats  also rejected other electrolyte solutions with an equal osmolality;  therefore  the intracellular deficit of water was more important than the excess of sodium (Palmieri & Taleisnik, 1969).  The reason why the SWV females could  not tolerate and/or rejected the saline might be a combination of these factors, especially a reduction in their response to vasopressin and a resulting alteration of the intracellular osmotic relationships. (3)  Response to Exogenous Antidiuretic Hormone  The SWV females and males did not respond to exogenous vasopressin.  126 The STR/N strain, which had primary polydipsia, raised their urine specific gravity significantly for 7 to 9 hours and decreased their water intake by \ for several days after being injected once with % unit pitressin tannate (Silverstein et a l . , 1961).  The SWR/J strain also decreased their water in-  take substantially after being given \ unit of pitressin tannate (Hummel, personal communication 1972).  The DI 0s/+ mice, which had nephrogenic  diabetes insipidus, did not respond to exogenous vasopressin when tested in the same manner as the SWV, p. 15, (Naik 6c Valtin, 1969).  Their urine  osmolality increased slightly on day 1 but decreased again and was actually lower on day 3 than on day 0.  Even though the urine of the DI 0s/+ mice  never became hypertonic, while the SWV females' urine osmolality on day 2 was 427 mOsm/kg, their pattern of response over the 3 days was very similar to that of the SWV females. The mildly affected DI +/+ mice showed a response to exogenous vasopressin which was very similar to the SWV males' response (Figure 11, p. 77); whereas the older severely affected DI +/+ animals could not increase their urine osmolality at a l l .  The VII +/+ controls increased their urine osmo-  l a l i t y steadily over the 3 days, but not to a significant extent (_t test); therefore the C3H females responded more to vasopressin than the VII +/+ controls.  Naik 6c Valtin (1969) felt that their controls did not respond  significantly to vasopressin because their urine was already at a maximum concentration on day 0, (2800 mOsm/kg).  The C3H females' urine osmolality  was lower on day 0 (1593 mOsm/kg) and they were able to significantly concentrate their urine when given vasopressin; whereas the C3H males had a urine osmolality of 2302 mOsm/kg on day 0, and responded to vasopressin in the same manner as the VII +/+  controls.  Another cause of unresponsiveness to vasopressin by the C3H males and  127 VII +/+  controls could be that % unit was not a supramaximal dose. However,  Silverstein et a l .  (1961) felt that \ unit was a supramaximal i.m. dose,  because i t was 3000 times greater than the minimum effective intravenous dose (Heller & Blackmore, 1952), and that this would be greater than any difference caused by the route of administration.  A larger dose of 1 unit  s t i l l had no affect on the urine osmolality in the DI 0s/+ mice (Falconer et a l . , 1964) which substantiated the idea that the % unit was supramaximal. In the DI rats, there was no difference in response to %, 1, or 2\ units of pitressin tannate (Harrington & Valtin, 1968). maximal, and since a rat weighs 9-12  Therefore % unit was  supra-  times more than a mouse, \ unit in a  mouse must be supramaximal. The SWV and C3H females responded more to vasopressin than did the males which had higher urine osmolalities on day 0.  While a sex difference  in response to vasopressin was not reported in the DI 0s/+ mice, the females did.have a greater water turnover (Falconer et a l . , 1964).  The VII  +/+  controls and DI 0s/+ males had a higher vasopressor activity than the females (Naik, 1972); consequently  the males would be expected to have a higher urine  osmolality on day 0 and respond less to exogenous vasopressin.  The SWV  and  C3H males might also have a higher vasopressor activity and this might explain their relative lack of response to vasopressin.  The DI rats did not show any  sex difference in water turnover or vasopressor activity (Valtin et a l . , 1965). The DI rats, which had no endogenous ADH,  decreased their water intake  to 1/7 their usual level after receiving \\ units of vasopressin (Mohring et al., 1972a) and also decreased their osmolal excretion to the control level (Harrington & Valtin, 1968); in contrast the SWV  females showed no decrease  in water intake and a nonsignificant increase in osmolal excretion.  The DI  128 rats showed a marked increase in urine osmolality after 3, daily, vasopressin injections although their urine did not reach a normal concentration. The urine osmolality values for the DI rats obtained by Harrington & Valtin (1968) were day 0 - 125 mOsm/kg, day 2 - 860 mOsm/kg; and those obtained by Lee & Williams (1972) were day 0 - 160 mOsm/kg, day 1 - 800 mOsm/kg, day 2 1000 mOsm/kg, and day 3 - 1160 mOsm/kg. Thus the DI rats had a much larger and steadier increase in urine osmolality than the SWV females (Figure 11, p. 77), but there was s t i l l an impaired urinary concentration after 3 idays which was not fully corrected until the DI rats had had 5 weeks of vasopressin treatment (Harrington & Valtin, 1968).  This i n i t i a l impaired response to ADH  which can be corrected by prolonged treatment is also seen in human hypothalmic diabetes insipidus (Harrington & Valtin, 1968; Rado & Szende, 1968; Rado et a l . , 1970), primary polydipsia (Rado et a l . , 1970), and in normal patients who have been water loaded (de Wardener & Herxheimer, 1957).  In the  DI rats, this was due to a washout of the renal medullary hypertonicity and a destruction of the countercurrent mechanism of concentration.  The extended  therapy corrected this by a gradual accumulation of urea in the papi-llary tissues (Harrington & Valtin, 1968; Rado et al., 1970; Schnermann et'fal. , 1969).  However Lee & Williams (1972) stated that the defect was in the de-  creased concentration of papillary sodium and was caused by a defective nephron. It is unlikely that the SWV females' lack of response to vasopressin was due to a washout of the renal medullary osmolality because the urine concentration did not increase steadily over the 3 days of treatment. However to rule out this possibility, vasopressin would have to be given to dehydrated SWV females.  The degree of response to a supramaximal dose of ADH  depends on the osmolality of the renal medulla.  Therefore i f the patient is  129 dehydrated until the urine osmolality levels off, any additional increase after an injection of vasopressin indicates that there is insufficient endogenous vasopressin present (Miller et a l . , 1960).  It is possible to  diagnose primary polydipsia, nephrogenic diabetes insipidus, partial and complete hypothalmic diabetes insipidus using this procedure. However this technique is d i f f i c u l t to do in mice since frequent determinations of urine osmolality are required to determine when i t has levelled off.  The volume of urine produced in the control mice was small; therefore  microtechniques and catheterization would have to be used.  However dehydra-  tion should stimulate the release of sufficient endogenous ADH to produce a maximum effect on the renal tubular permeability; hence animals should have a higher urine osmolality after dehydration than after exogenous vasopressin unless the animals have a defect in the production and/or release of ADH. man,  In  the controls did have a higher (or at least as high) osmolality after  48 hours of dehydration than they did after receiving 5 units of vasopressin (Jones & de Wardener, 1956).  This also occurred in the C3H mice.  The  SWV  mice could also concentrate their urine more after 7 hours of dehydration; therefore their production of endogenous ADH  in the hypothalmo-hypophyseal  system was normal and the defect was nephrogenic.  The SWV  females' kidneys  might be only partially defective in their response to ADH since when compared to the DI 0s/+ mice, the SWV  females:  (i)  had a higher urine osmolality under ad libitium water intake and  (ii)  could concentrate their urine more after dehydration and after 3 daily injections of vasopressin.  The DI 0s/+ kidneys might also be able to respond slightly to endogenous ADH since their urine osmolality decreased to 100 mOsm/kg after bilateral electrolytic destruction of the supraoptic nuclei (Naik, 1972).  130 To determine i f the defective response to ADH was caused by a defect in the vasopressin-induced  permeability of the distal tubule or alterna-  tively by a defect in the build-up of papillary osmolality, ADH should be given after a water load (Naik & Valtin, 1969; Stewart & Stewart, 1969). During water loading there is an inhibition of endogenous ADH which results in impermeability and urine dilution.  If ADH is given after a water load,  there should be a large increase in urine concentration unless there is a defect in vasopressin-induced membrane permeability. (4)  Similarity to some Kidney Diseases  The SWV females had a progressive decrease in per cent packed cell volume (PCV) but did not have a large increase in blood urea nitrogen (BUN). The DI rats had an elevated serum osmolality, an elevated extracellular fluid volume (Friedman & Friedman, 1965), but a normal PCV (Valtin & Schroeder, 1964).  The increased extracellular fluid volume was caused by a progressive  increase of serum sodium.  The SWV females had a normal serum osmolality;  hence there should not be as high a serum sodium concentration; and consequently less expansion of the extracellular fluid volume.  Thus i f the DI  rats did not have a great enough expansion of their extracellular fluid volume to reduce their PCV, the decreased PCV in the SWV probably was due to anaemia rather than plasma expansion. Hardy (1967) reported a large variation in mean PCV between various strains of mice but not between various ages within a strain.  There was  also a trend for the males to have a higher PCV than the females.  Therefore  the steady decrease in PCV with age in the SWV mice was significant pathologically.  In the DI 0s/+ mice there was a significant increase in BUN due to  an 807o reduction of nephrons, but no mention was made of the PCV (Naik &  131 Valtin, 1969). In man, almost a l l patients with nephronophthisis have an insidious progressive anaemia which is normocytic and normochromic (Strauss, 1971). The anaemia is caused by the uremia of renal insufficiency and is invariably associated with a high BUN.(Erslev & Shapiro, 1971).  Thus the SWV seemed to  differ in that they did not develop a high BUN. Patients with nephronophthisis usually have polyuria as their first symptom, however they retain the ability to dilute their urine after water loading (dilution was not tested in the SWV females).  They often become salt wasters and in severe cases re-  quire a large sodium intake to compensate for the renal loss (Strauss, 1971). This is opposite to the SWV females which died when they were given saline drinking water. The kd mice started to have an increased BUN and decreased PCV at 4 7 months and by 8 - 11 months the changes were quite severe (Lyon & Hulse, 1971).  Thus the kd mice seemed to resemble human nephronophthisis more than  the SWV females did.  At necropsy the kd tissues were pale and anaemic-  looking similar to those of the SWV females. (5) (a)  Histology Pituitaries  The SWV females seemed to have an adequate amount of neurosecretory material present in the pars nervosa.  This was also true for the following:  STR/N strain of mice (Silverstein et a l . , 1961), which had primary polydipsia; DI chickens (Dunson et a l . , 1972), which had either primary polydipsia or nephrogenic diabetes insipidus; the DI 0s/+ mice (Naik & Valtin, 1969; Naik & Sokol, 1970), which had nephrogenic diabetes insipidus; and 1 human (Campbell, 1961) and 1 dog (Sloper et a l . , 1967), which had nephrogenic  132 diabetes insipidus.  There have been no autopsy reports for human primary  polydipsia (Sloper et a l . , 1967).  Rats (Valtin, 1967), dogs (Richards &  Sloper, 1969), and humans (Blotner, 1958; Green et a l . , 1967) with hypothalmic diabetes insipidus showed an absence of neurosecretory material in the pars nervosa.  The DI 0s/+ mice (Naik & Sokol, 1970) and DI rats (Sokol &  Valtin, 1965) showed hypertrophy of the hypothalmo-hypophyseal system; whereas human hypothalmic diabetes insipidus (Blotner, 1958; Braverman et a l . , 1965; Green et a l . , 1967), canine hypothalmic diabetes insipidus (Richards & Sloper, 1969), and human nephrogenic diabetes insipidus (Campbell, 1961) showed atrophy of the system. Thus the SW females could only have a deficiency of endogenous ADH i f there was a defect in the release of the hormone from the pars nervosa or i f the hormone released was biologically inactive.  However the defective  response to exogenous vasopressin and the adequate amount of aldehyde fuchsinpositive material in the pars nervosa strongly suggest that the defect in the SWV females was a faulty response to ADH by the kidneys. (b)  Adrenals  The adrenals of the SWV females were normal except for the normal ageing processes; that is, an accumulation of connective tissue in the cortex and X zone, and an accumulation of pigment in the X zone and medulla (Samorajaki & Ordy, 1967).  These connective tissue cells were spindle shaped  and resembled those of the ovarian stroma (Jayne, 1963).  Jayne also reported  strain difference in the susceptibility to the proliferation of these spindle cells. The normal adrenals ruled out the possibility that the polydipsiapolyuria in the SWV females was secondary to amyloidosis as in the De/J  133 mice (Chai & Dickie, 1966).  Patients with human nephronophthisis (Strauss,  1971) and the DI 0s/+ mice had an hypertrophy of the adrenal glands, especially the adrenal cortex, and a prominant zona glomerulosa which Naik 6c Sokol (1970) attributed to the stress of chronic dehydration. Shire & Spickett (1967) reported a large variation in the extent of the zona glomerulosa between inbred strains and species of mice.  They related  the size of the zona glomerulosa to the efficiency of sodium regulation, and thought that an enlarged zone might indicate poor sodium regulation.  Thus  the hypertrophy of the adrenal cortex in the DI 0s/+ mice might be due to poor sodium regulation and chronic stress.  If the SWV females had a defec-  tive sodium regulation, they must have somehow compensated for i t since there was no enlargement of the zona glomerulosa when compared to the C3H and C57 mice. (c)  Kidneys  The old SWV females had enlarged smooth kidneys with no gross indication of cysts; this eliminated polycystic kidneys as the cause of the polydipsia-polyuria defect. They also had a distended bladder and an enlarged pelvis which in some cases was so severe that hardly any renal parenchyma remained.  The SWV males did not have even mild hydronephrosis.  Hydro-  nephrosis has been reported in the old male polydipsic STR/N mice (Silverstein et a l . , 1961), in some severely affected, old DI/+ and DI 0s/+ mice with nephrogenic diabetes insipidus (Falconer et a l . , 1964), in the old rats with hypothalmic diabetes insipidus (Sawyer & Valtin, 1967), and in adults with human nephrogenic diabetes insipidus (Silverstein 6c Tobian, 1961) or human hypothalmic diabetes insipidus in which the patients had not been treated (Friedland et a l . , 1971).  Silverstein 6c Tobian (1961) speculated that the  134 hydronephrosis was caused by the bladder failing to enlarge sufficiently to contain, at normal pressures, the volume of urine excreted between periods of voiding. In the SWV  females, the hydronephrosis was also probably caused  by the enormous water turnover throughout the animal's l i f e . The SWV  females had significantly heavier kidneys than the controls  after 5 months of age.  Schlager (1968) demonstrated.a  kidney weight between different strains of mice.  genetic difference in  The kidney weight had a  high heritability, but the kidney weight which was adjusted for body weight had an even higher heritability.  Therefore the differences between the SWV  and C57 males was partially, but not completely, due to differences in body weight.  Schlager reported that the C57BL/10J males had a wet kidney weight  of .395 g at 9 - 12 months of age and that the SWR/J males had a wet kidney weight of .439 g at the same age.  The C57 males which were used in this study  were derived from the C57BL/10J strain about 16 years ago, nevertheless the kidney weights were s t i l l very similar (.42 g compared to .40 g).  The SWR/J  males had slightly lighter kidneys than the SWV males (.44 g as opposed to .58 g).  The DI rats had lighter kidneys; however, since they were runty  their relative kidney weight was heavier than the controls (Valtin, 1966b). On the other hand, the DI 0s/+ mice had significantly smaller kidneys since the Os gene caused an 807» reduction of nephrons.  The kidneys were contracted  in cases of human (Strauss, 1971) and murine (Lyon & Hulse, 1971) nephronophthisis, but no weights were given.  The most similar case to the SWV  females was that in which mice fed a potassium deficient diet developed enlarged kidneys (Hollander 6c Blythe, 1971). The SWV  females' kidneys were abnormal by 6 months of age.  The kidneys  of the DI rats (Valtin 6c Schroeder, 1964) and polydipsic STR/N mice (Silverstein et a l . , 1961) were normal, confirming that the cause of the diabetes  135 insipidus  i n the SWV mice was nephrogenic.  ported  i n human nephrogenic  niques  (Campbell,  Normal k i d n e y s have been r e -  d i a b e t e s i n s i p i d u s u s i n g l i g h t microscopy  1961; Abelson,  1968; Macdonald,  tech-  1955; S o r e l et a l . , 1968).  However Macdonald a l s o c l a i m e d t h a t t h e r e was some t u b u l a r d i l a t i o n and some e o s i n o p h i l i c casts i n a kidney betes  insipidus.  from a 2 year o l d boy w i t h nephrogenic  U s i n g e l e c t r o n microscopy,  some g l o m e r u l a r d e f e c t s and A b e l s o n defects seen  i n the t u b u l e s .  i n the SWV  (1968) demonstrated  crystalloid  some m i t o c h o n d r i a l t o those  females.  scarring,  structures  reduction i n glomeruli,  interstitial  p r o t e i n c a s t s i n some t u b u l e s , and some cone-shaped i n the d i s t a l  tubules.  Consequently  t h e DI 0s/+  w i t h s e v e r e d i a b e t e s i n s i p i d u s a l s o showed these k i d n e y l e s i o n s Valtin,  demonstrated  However no one has r e p o r t e d d e f e c t s s i m i l a r  The Os gene i n mice caused an 80% inflammation,  S o r e l a t a l . (1968)  dia-  1969) which were d i f f e r e n t  from those o f the SWV  mice  (Naik 6c  females.  However  some young DI +/+ mice (which had a m i l d c o n c e n t r a t i n g d e f e c t , showed some slight  d i l a t i o n o f t u b u l e s i n the medulla  was c o r r e l a t e d w i t h the p o l y u r i a ; old  severely affected Nephrogenic  (Schou,  1958).  and the s e v e r i t y o f the d i l a t i o n  but Naik 6c V a l t i n  DI +/+ mice had normal k i d n e y s .  d i a b e t e s i n s i p i d u s can be produced  by an excess o f l i t h i u m  At a h i g h dose t h e d i s e a s e became i r r e v e r s i b l e and i n the  k i d n e y d e g e n e r a t i v e l e s i o n s o f the p r o x i m a l quite different  (1969) r e p o r t e d that the  from the t u b u l a r h y p e r t r o p h y  t u b u l e s developed. seen  i n the SWV  These were  females.  A s e v e r e p o l y d i p s i a - p o l y u r i a which was caused by the d e p o s i t i o n o f amyloid  i n the k i d n e y s and a d r e n a l s was r e p o r t e d i n the De/J mice ( C h a i 6c  Dickie,  1966).  kidneys  (Cornelius,  stantiated  The k i d n e y s o f the SWV  females  d i d not resemble  murine amyloid  1970), but the absence o f amyloid would have to be sub-  by s t a i n i n g  the k i d n e y s w i t h congo r e d or t h i o f l a v i n T.  136 Diabetes In  SWV);  SWV  by h y p e r c a l c e m i a  d i l a t i o n of  i n the o u t e r c o r t e x (as opposed to the c o r t i c o - m e d u l l a r y zone i n the  and d e p o s i t i o n o f c a l c i u m i n the c o l l e c t i n g  the medulla. females,  Hypercalcemia  a l s o produced  t u b u l e s and  interstitium  g l y c o s u r i a and azotemia.  some p o s s i b l e c a l c i f i c a t i o n o c c u r r e d i n the p r o x i m a l  the o u t e r c o r t e x ; however, t h i s o c c u r s , not t i s s u e damaged by p o i s o n i n g or n e p h r i t i s unlikely  t h a t h y p e r c a l c e m i a was  i n hypercalcemia,  (Strauss, 1971).  but  pletely eliminate this Diabetes  in renal  Therefore i t i s  the p r i m a r y d e f e c t i n the SWV  females;  The k i d n e y  from those seen  i n man,  lesions  not seen  i n t e n s e h y p e r p l a s i a and  l e s i o n s are described.  i n the SWV  old  SWV  stitial  females. nephritis,  In  o c c u r s mostly females  rodents are  One  There  tubule c e l l s  i n the m e d u l l a r y  i s comparable to t h a t seen  and  s c a r r i n g around  and  and  i s also  an  i n the o u t e r  i n a t u b u l a r d i l a t i o n p r o x i m a l t o the  hyper-  zone a f t e r 4 weeks i n k i d n e y s o f 6 "month  these l e s i o n s ;  c o m p l i c a t i o n ( O l i v e r et a l . , 1 9 5 7 ) .  i n the medulla  diff-  major l e s i o n i s  p o t a s s i u m d e p l e t i o n t h e r e i s a l a r g e amount o f  fibrosis,  p r o b a b l y a secondary  SWV  females' k i d n e y s ) .  The amount o f t u b u l a r d i l a t i o n  on a p o t a s s i u m d e f i c i e n t d i e t  (Hollander  c y t o p l a s m o f the i n n e r medulla  s w e l l i n g ot the c o l l e c t i n g  medulla which sometimes r e s u l t s plasia.  by p o t a s s i u m d e f i c i e n c y  i n potassium-depeleted  an a c c u m u l a t i o n o f g r a n u l e s i n the c e l l ( t h i s was  to com-  a l t h o u g h the c o n c e n t r a t i n g d e f e c t i s s i m i l a r ;  t h e r e f o r e o n l y the murine k i d n e y  papilla  never-  possibility.  i n s i p i d u s can a l s o be caused  6c B l y t h e , 1 9 7 1 ) .  In the  tubules of  t h e l e s s the serum c a l c i u m c o n c e n t r a t i o n would have to be determined  erent  1971).  (Epstein,  s e v e r e cases the f o l l o w i n g k i d n e y l e s i o n s have been seen:  tubules  of  i n s i p i d u s can a l s o be caused  The  inter-  these a r e  tubular d i l a t i o n  the e p i t h e l i u m i s f l a t t e n e d ; whereas i n the  the t u b u l a r d i l a t i o n o c c u r r e d i n the c o r t i c o - m e d u l l a r y zone and  the e p i t h e l ium was  high cuboidal.  Thus the p o t a s s i u m d e f i c i e n t k i d n e y s have  137 some dilated tubules similar to those of the SWV kidneys, but there were differences which suggested that the primary defect in the SWV females was not hypokalemia.  To substantiate this, '  SWV females should be fed a  potassium enriched diet from weaning, to determine i f this would prevent or ameliorate the diabetes insipidus syndrome. MShring et a l . (1972a) have just reported that rats with hereditary diabetes insipidus have a secondary hypokalemia which can be corrected by vasopressin therapy.  Kidneys of 3, 3 month old and 3, 10 month old female  DI rats showed vacuolar degeneration of the proximal and tubular cells (Mohring et a l . , 1972b); this defect is the major characteristic of potassiumdepleted nephropathy in man (Hollander & Blyth, 1971). eration was not evident in the SWV females' kidneys.  This vacuolar degenHowever the SWV females  might have a secondary hypokalemia and/or some other electrolyte  imbalances  due to the chronic nephrogenic diabetes insipidus and these might cause secondary complications to the primary kidney lesion. Finally, a mild diabetes insipidus can be caused by human nephronophthisis (Strauss, 1971).  Histologically the kidneys of patients with  nephronophthisis have a  number of defects in the cortex (small glomeruli,  periglomerular fibrosis,  atrophic cortical tubules, and fibrosis),  amount of diffuse  fibrosis,  a large  some macula densa-like lesions (Sherman et a l . ,  1971), and thickened basement membranes; none of which were seen in the SWV females.  In nephronophthisis there is also a large number of cysts at the  cortico-medullary junction but, contrary to the situation in SWV females, the cysts are lined with flattened epithelium.  There are also some eosinophilic  casts and some focal i n t e r s t i t i a l nephritis which were present in the SWV females' kidneys.  The kidneys of the kd_ mice were very similar to those  kidneys of nephronophthisis patients (Lyon & Hulse, 1971), whereas the kidneys  138 of the SWV  females were quite different except that they had dilated tubules.  In summary:  the SWV  females had a progressive kidney lesion which was  distinct from other renal defects which have been reported.  There were some  similarities to nephronophthisis and to hypokalemia, but the SWV kidneys were unique.  females'  At 4 months the kidneys were normal under the light  microscope although the nulliparous females were already starting to increase their water intake (9 ml/24 hrs as opposed to 4 ml/24 hrs in the controls). By 6 months the kidneys were abnormal and the females were definitely polydipsic (19 ml/24 hrs).  This suggests that the dilated tubules were a second-  ary result of some ultrastructural or biochemical  lesion in the kidneys or  alternatively of a toxicity to some abnormal or elevated chemical which was present from weaning or birth.  The primary lesion and/or toxicity combined  with the secondary increase in water turnover became progressively more severe and eventually resulted in the tubular dilation and other structural alterations seen in the kidneys of 12 and 17 month old SWV C.  females.  Genetics of Polydipsia-Polyuria Defect Water and electrolyte metabolism is a highly complex physiological  system and is determined by a large number of genes (Spickett et a l . , 1967). Water intake depends on water metabolism and is also therefore very complex and determined by polygenic inheritance.  The inheritance of the polydipsia  defect was studied by measuring water intake.  Since water intake is many  steps removed from the basic metabolic error (or errors) causing the polydipsia, the genetic mechanism would be expected to appear quite complex.  The back-  ground genotype determining water intake, diet, and other environmental factors would a l l be expected to interact with the polydipsia gene (or genes) to produce the final water intake.  139 A quantitative estimate of the number of loci determining polydipsia w i l l consider a l l the genetic variation involved in water intake between the 2 strains;  that is, the 17 l o c i which differ between the  and C3H strain  include a l l the polygenes influencing water intake as well as the gene or genes causing polydipsia.  This does not necessarily mean that the estimate of  17 l o c i is reliable as mentioned on p. 108.  Comparison of the means and  variances indicated that the environment had a multiplicative influence on water intake and the severity of polydipsia. onset diseases (Falconer, 1967).  This is quite common in late  The SWV females' water intake s^ was very  high and also increased with age; whereas the C3H-2 mice had a smaller water intake s 2 but i t also tended to increase with age.  This increase in s2 with  age is common in various physiological processes in mice (Storer, 1965) and indicates a breakdown of the homeostatic mechanisms which act as a buffer to keep the water intake at a normal level.  In the SWV males, on the other hand,  2 the s  was less and they had a later onset of a milder polydipsia; therefore  they appeared to have a better homeostatic regulation which broke down less with age than it did in the SWV females. The strong interactions that were present in the BC generations probably indicated an interplay between the major gene and the large number of polygenes present in both parental strains.  Thus the C3H-2 strain may have a  number of polygenes which act against, the major gene (negative modifiers) and therefore the ^ ^ 2 ^ 2  ^ema^-es  c  a  n  regulate the error in water metabolism  sufficiently to have a relatively normal mean water intake.  Conversely the  SWV strain may have positive modifiers which combine with the major gene to cause a very severe form of polydipsia in the BCg^y females.  At the F-^ level  these positive and negative modifiers may balance each other so that the normal water intake is only slightly dominant.  As well as the genie inter-  140 actions, there were probably environmental - genie interactions. If a discontinuous variation can be measured, a simpler genetic mechanism is often demonstrated.  There is a discontinuous variation in water in-  take between polydipsic SWV females and normal C3H-2, but there is not between the SWV males and normal C3H-2.  In the testcross progeny there was a  continuous variation in water intake rather than an obvious discontinuous classification.  Therefore the arbitrary classification of the normal mean  plus 2 s was chosen because 95% of the normal population should be below this level.  However the C3H-2 strain does not represent a "normal" mouse since i t  is inbred and a l l inbred strains have a minimal "normal" genetic variability due to one possible genotype being fixed (Thoday, 1967).  Therefore the  results that were obtained only apply to genetic differences between the SWV and C3H-2 strains and cannot be generalized to differences between the SWV and other strains of mice.  Similarly polydipsia was dominant in this study,  but i f polydipsic SWV were crossed to other strains, the polydipsia would not necessarily remain dominant since i t would depend on the genetic background of the other strains. The genetic system was simpler in the qualitative analysis than in the quantitative one.  However i f the primary gene action was known and could be  measured, the genetic system would presumably behave in a classical mendelian manner. The reduced per cent of polydipsia in the SWV males could be due to reduced penetrance of the polydipsic genotype or to heterozygosity in the SWV strain.  The latter is unlikely because of the 30 to 36 generations of in-  breeding and the lack of a significant regression in water intake between parents and progeny.  However to rule out heterozygosity in the SWV strain  selection for a severe polydipsia line and a mild polydipsia line should be  141 undertaken.  It is more likely that there is reduced penetrance due to the  strong influence of the environment and of other genes.  In addition the SWV  males have a milder expression of polydipsia which is correlated with a lower penetrance in a threshold model.  The reduced frequency of polydipsia in the  F]_ males could be due to reduced penetrance plus some heterozygosity in the C3H-2 strain. Quasi-continuous variation is defined as a physiological discontinuity of phenotypes when there is a continuous range of genotypes (Gruneberg, 1952). He gives the following criteria by which quasi-continuous variables can be recognized: (i)  an inbred strain w i l l have more than 2 stable levels of a character.  (ii)  various normal strains may vary considerably when crossed to the same abnormal strain.  The character may vary from being domi-  nant to being recessive. (iii)  a correlation between penetrance and expressivity.  As the  con-  tinuous distribution of genotypes passes the c r i t i c a l level the f i r s t few abnormals w i l l be mildly affected, whereas those abnormal individuals at the extreme end of the normal distribution w i l l be severely affected. The polydipsic trait does not meet criterion 1, but i t does meet criterion 3.  The BCg^y females were 1007, penetrant as opposed to 957, of the F-^ females  fed the same ration, and these BCgyy females were more severely polydipsic than the F\ females.  Also the BCgyy males which were 1007> penetrant had an  earlier age of onset than the Fi^ males which were 657> penetrant.  Thus in the  Fj_ males the C3H-2 polygenes may make more of the males resistant to the deleterious gene so that they are able to compensate and remain below the  142 c r i t i c a l level longer.  Criterion 2 was not tested in this study.  Continuous and quasi-continuous variables should also meet the following c r i t e r i a : (iv)  sensitivity to environmental influences, including intrauterine and post-natal  factors.  (v)  sensitivity to general genetic effects such as sex.  (vi)  sensitivity to major genes.  The continuous character then forms  the "genetic background" for such genes and enters into a "modifier" relationship with them. Polydipsia is sensitive to many environmental influences including maternal physiology.  The F 2 females from SWV/C3H-2 dams had a significantly higher  mean water intake than did those F 2 females from C3H-2/SWV dams.  This  suggested a sensitivity to maternal physiology, however there was no significant difference between reciprocal F-^ females.  Also the expressivity and,  to a limited extent, the penetrance were influenced by sex.  Therefore in  some ways the polydipsia behaves in a manner consistent with a continuous variable.  Carter (1969) gave the example of diabetes mellitus which was  first considered to be caused by an autosomal recessive gene.  However  clinicians had found i t hard to reconcile a single gene hypothesis to diabetes mellitus since there was no clear division between: (i)  normal and abnormal blood sugar curves  (ii)  normal and abnormal threshold for glucose  (iii)  normal and abnormal in change of blood sugar curve with age  also there was: (iv)  absence of any suggestion of bimodality in sugar-tolerance curves of either controls or first degree relatives  (v)  the obvious importance of environmental factors in late onset  143 curves. While i t has since been determined that diabetes mellitus is polygenic (Carter, 1969), the polydipsia-polyuria in the SWV mice act in a similar manner in that i t is d i f f i c u l t to make a clear cut distinction between normal and abnormal in: (i)  water intake  (ii)  urine concentrating ability - in normal conditions - after dehydration - after vasopressin administration  and there is a: (iii)  continuous range of hematocrits with age  (iv)  continuous range of kidney weights with age  (v)  continuous range of kidney structure with age.  However these continuous ranges can be due to an interaction of the background genotype and the environment with 1 or 2 dominant genes. In summary:  this study has indicated that polydipsia is determined by  1 or 2 autosomal dominant genes with reduced penetrance in some cases. However this has been based only on dominance in the cies in the F2 and BC generations.  generation and frequen-  The only conclusive evidence for or  against this hypothesis, would be obtained by breeding" progeny of the segregating generations (Wright, 1934).  For instance, a l l types of F2 progeny  should be bred and i t should be determined i f the resulting frequency of polydipsia in the F^ generation was the expected 5074 (for normal X normal matings) and 837, (for normal X polydipsic matings) for a 1 gene model; or the expected 397. (for normal X normal matings) and 707, (for normal X polydipsic matings) for a 2 gene model.  Alternatively a l l types of progeny from the  144 first  BCQ3H_2  mating could be backcrossed again to the  C3H-2  strain and i t  should be determined i f the expected frequencies for a 1 or 2 dominant gene model were obtained. It would also be informative to cross the polydipsic SWV to a number of other strains and take them to the F  2  and B C generations.  This formidable  program of crosses would provide information on the degree of dominance of polydipsia relative to other backgrounds and on the various interactions between residual genetic and non-genetic variability (Wright, 1968). (1)  Comparison to other Syndromes  Polydipsia in the SWV mice is inherited in a relatively simple dominant fashion.  This does not necessarily mean that the rest of the syndrome  (vasopressin-resistant diabetes insipidus, anaemia, and kidney anomalies) is inherited as a single unit.  There have been a number of hereditary syndromes  reported in man and animals. Hypothalmic diabetes insipidus can be inherited in man as an autosomal dominant, autosomal dominant with incomplete female penetrance, or as a sex-linked recessive (Relkin, 1966); and in rats as an autosomal recessive (Saul et a l . , 1968).  Nephrogenic diabetes insipidus can  be inherited in man as a sex-linked recessive (Relkin, 1966), and also as a sex-linked dominant (Schoen, 1960), or as an autosomal dominant with 1007  o  penetrance in males and variable expressivity in females (Uttley & Thistlethwaite, 1972). genotypes.  In mice the nephrogenic diabetes insipidus is produced by 3  The DI +/+ genotype (which produces a mild  defect) is determined  by 1 or 2 genes, the 0s/+ genotype (which also produces a mild defect) is determined by a dominant gene which is lethal as a homozygote, and when these 2 genotypes are combined (DI 0s/+) a severe defect is produced (Falconer et a l . , 1964; Naik & Valtin, 1969; Stewart & Stewart, 1969). Nephronophthisis  145 (medullary cystic disease) is inherited in man as an autosomal dominant or autosomal recessive (Strauss, 1971).  In mice, the _kd defect, which seems to  resemble human nephronophthisis, is inherited as an autosomal recessive (Lyon 6c Hulse, 1971) . Inherited syndromes of polydipsia have been reported in animals. Dunson 6c Buss (1968) reported a polydipsia in chickens which was an autosomal recessive, but i t has not yet been definitely established i f the defect is primary polydipsia or nephrogenic diabetes insipidus (Dunson et a l . , 1972). Primary polydipsia has been reported in aged females of the TS/A strain (Szalay 6c Moll, 1966) and SWR/J strain (Hummel, 1964) of mice, but no information on the genetic mechanisms was given.  Primary polydipsia also occurs  in 90% of the old mice of the STR/N strain of mice (Silverstein et a l . , 1961) and genetic testcrosses indicated i t was not inherited as a simple recessive (Silverstein, 1961).  A total of 250 progeny from the F^, F2 and backcrosses  were tested at 1 to 1% months of age.  The F-^ progeny had mean water intakes  closer to the normal strain than the polydipsic STR/N strain.  However since  the STR/N males had a water intake of 9.2 ml/24 hrs as opposed to the controls' water intake of 7.2 ml/24 hrs and the STR/N females had a water intake of 9.4 ml/24 hrs as opposed to the controls' water intake of 4.7 ml/24 hrs, i t is d i f f i c u l t to understand how clear segregation at this young age could be expected.  Silverstein reported no difference between F-^ and F2 progeny, but  the progeny of backcrosses to both parental strains seemed to be polydipsic. While I feel i t is d i f f i c u l t to rule out a major gene mechanism from the limited data published, this syndrome is the only one reported that might be polygenic, or at least be influenced by other genes and/or the environment to the same extent as polydipsia in the SWV strain.  Nephronophthisis, hypo-  thalmic and nephrogenic diabetes insipidus appear to be inherited in a simple  146 manner.  Several different modes of inheritance have been suggested for these  diseases which may mean that some of the families were not large enough to accurately determine the inheritance, or conversely there is genetic heterogeneity (McKusick, 1969).  For example, in hypothalmic diabetes insipidus  there may be 3 different causes for the end phenotype each of which is inherited in a different manner.  These possible causes were mentioned on p. 2.  Thus in these diseases only the end phenotypes are being studied, and there are different enzymatic errors that could occur to bring them about. D.  General Discussion This study has shown that polydipsia seems to be determined by 1 or 2  autosomal dominant genes in the SWV strain of mice.  This does not necessaril  mean that the whole syndrome (polydipsia, an impaired concentrating a b i l i t y , anaemia, and abnormal kidneys) is determined by the same gene or genes; for example, the anaemia may be caused by 1 gene and the rest of the syndrome by another gene.  To determine i f this is a unitary syndrome or a fortuitous  association of separate defects,  F 2 hybrids should be examined to see i f ther  is any independent segregation between the different symptoms.  In this  generation one or more of the defects might be separated and have no correlation with polydipsia suggesting that it has no role in the pathogenesis of the vasopressin-resistant  diabetes insipidus.  Vasopressin-resistant  diabetes insipidus can be caused by a number of  c l i n i c a l syndromes, but they a l l are caused by one or a combination of the following 3 lesions (Kleeman, 1972): 1.  Destruction of renal mass resulting in a decreased number of  functional nephrons that in turn results in an increased osmotic load per nephron and an increased glomerular f i l t r a t i o n rate.  The osmotic diuresis  147 per nephron causes a reduction in the medullary i n t e r s t i t i a l osmolality that in turn impairs the concentrating capacity of the countercurrent  mechanism.  This occurs in a number of chronic renal diseases such as polycystic kidneys, nephronophthisis,  or amyloid disease.  The polyuria produced by this lesion  is not as severe as that produced by the other 2 lesions. 2.  An organic lesion in the distal collecting tubule or collecting  duct which impairs the permeability of the membrane to water, even in the presence of ADH.  This can be caused by a reduction in the amount of the  intermediate, cyclic AMP,  produced or released in the kidney.  Brooker (1972) demonstrated a reduction in cyclic AMP  Fichman &  in human nephrogenic  diabetes insipidus and postulated that i t could be caused by: (i)  a deficiency of renal cortical or medullary adenylate cyclase (the enzyme which produces cyclic AMP  (ii)  from ATP)  inability of the hormones to attach to a hormone-cyclase cell membrane receptor site  (iii)  inability of the renal c e l l to produce or release cyclic  AMP,  even i f adenylate cyclase is present. A defect in ADH-induced permeability could also be caused by a structural defect (primary or secondary) which impairs the ADH action even in the presence of cyclic AMP.  If the decrease in concentrating ability is due to a permea-  b i l i t y defect, the tubular fluid w i l l have a lower osmolality than the surrounding medullary interstitium. 3.  Inability to build-up an effective osmotic gradient in the inter-  stitium from the cortex to the papillary tip of the medulla. of ADH there is a flow of water from the hypotonic  In the presence  fluid in the distal tubule  and collecting duct to the hypertonic medullary interstitium; and i f the medullary i n t e r s t i t i a l osmolality is reduced the maximal urinary osmolality  148 is equally reduced.  The reduced medullary  i n t e r s t i t i a l osmolality can be  caused by: (i)  organic lesions which disrupt the anatomical arrangement of the vasa recta and the loop of Henle.  These structures are a  necessary part of the countercurrent mechanism of concentration. No obvious alteration in the anatomical arrangement of these structures was seen using the light microscope.  Thus i t is un-  likely that this is the cause of the, concentrating defect in the SWV mice. (ii)  a defect in the reabsorption of sodium in the ascending limb of the loop of Henle.  At the bottom of the loop of Henle the  tubular fluid is hypertonic.  As the fluid moves up the ascend-  ing limb sodium is reabsorbed but water is not, even in the presence of ADH, nephron.  resulting in hypotonic fluid entering the distal  It is this sodium transfer that creates the osmotic  gradient in the medullary reabsorbed  interstitium and enables water to be  from the distal nephron.  Thus i f sodium reabsorption  in the ascending limb is impaired, the concentration mechanism is also impaired.  Hollander & Blythe (1971) postulated that this  is the primary defect in hypokalemia.  There would be an increased  reabsorption of sodium in the distal tubule (to compensate for the defective reabsorption in the loop of Henle) and this would result in an increased secretion of potassium.  The result would  be a large volume of hypotonic urine with an increased potassium excretion.  The urine would not be as dilute as i t would be in  the complete absence of ADH. Vasopressin-resistant diabetes insipidus can also be caused by toxicity  149 to an excess of some normal electrolyte (for example, calcium) or to some other compound (for example, aminopterin).  The excess compound can cause an  osmotic diuresis that results in the reduction of the medullary i n t e r s t i t i a l osmolality; or it can cause structural damage to the distal nephron that destroys the ADH-induced permeability to water.  Since there was no increase  in the urinary protein content in the SWV females,  the toxic material would  either have to be non-protein, or only a small amount could be'present i f it was a protein.  There could be a slight increase in urinary protein and the  dilution effect would prevent i t being detected by the method used. The SWV females did not seem to have a mass destruction of renal tissue until after 12 months of age and that destruction was probably a secondary complication of the large back pressure produced by the severe polyuria.  To  determine i f the nephrogenic diabetes insipidus was caused by a defect in permeability or in the build-up of the papillary osmolality, the mice should be given ADH after a water load as described on page 130.  If the tubular  osmolality is less than the adjacent i n t e r s t i t i a l osmolality then there is a defect in the ADH-induced permeability of the tubule to water. The SWV females had a significant increase in osmolal excretion even though the urine was hypotonic.  This could have been due to an increase in  the excretion of sodium, potassium, or both electrolytes.  It was also  observed that the SWV females could not tolerate an increased intake of sodium (ie.,  saline substituted for drinking water).  If the increased  osmolal excretion was sodium i t could be caused by decreased reabsorption. Sodium wasting (increased sodium excretion and a negative sodium balance) has been reported in nephronophthisis and other chronic renal diseases (Strauss, 1971).  However in these patients an increased sodium intake compensates for  the sodium wasting; whereas in the SWV females the substitution of saline  150 for tap water aggravated the symptoms.  The SWV  females died from dehydration  due to a severe excess of sodium or due to their refusal to drink saline. Thus i t seems unlikely that the SWV  females were "salt wasters".  Alternatively Naik (personal communication 1972) suggested that an increased sodium excretion may be caused by an increased absorption of sodium from the gut.  Fowler (1962) demonstrated strain differences in the amount  of protein absorbed from the gut when 2 strains of mice were fed the same diet, and strain differences in the amount of electrolytes absorbed seem just as possible.  Increased sodium absorption from the gut would produce an  osmotic diuresis (relative to another strain on the same diet) that would in turn decrease sodium reabsorption from the ascending limb of the loop of Henle and thus prevent the osmolality build-up in the medullary interstitium. If the sodium intake is increased by giving the mice saline instead of water, the osmotic diuresis would be more severe and the polyuria would be aggravated. Alternatively i f the animals are put on a low sodium diet, the urine volume would be decreased and the urine osmolality increased. It has been noted that the SWV  females fed ration 1 had a lower water intake (and presumably  also a lower urine volume and a higher urine osmolality) than those fed ration 2, and Naik suggested that ration 1 should be analyzed for sodium and compared to the value given for ration 2 by Purina.  As shown in Appendix 1,  ration 1 has a much lower sodium content than ration 2:  these results support  Naik's theory. An increased absorption of sodium from the gut would not necessarily be the primary error.  It could be a secondary factor which is in fortuitous  association with the major defect and potentiates the severity of the diabetes insipidus in an additive or geometric manner.  If the primary error was a  partial defect in the ADH-induced permeability, then an osmotic diuresis  151 would prevent the osmolality build-up of the medullary interstitium and thus magnify the concentrating defect.  Even i f there was an absolute defect  in ADH-induced permeability an osmotic diuresis would further impair the concentrating capacity by decreasing the passive diffusion of water out of the collecting duct.  Passive diffusion is decreased when the flow rate and  the volume of tubular fluid entering the distal nephron are increased. The increased osmole excretion could also be potassium and be due to primary or secondary hypokalemia.  Any factor (such as an increase in  aldosterone) which increases the reabsorption of sodium from the distal tubules also increases the secretion of potassium and results in hypokalemia. Hypokalemia can thus be caused by any chronic impairment of concentrating ability.  Mohring et a l . (1972a) demonstrated that rats with hereditary  hypothalmic diabetes insipidus have a secondary hypokalemia which is corrected by ADH.  Some SWV females showed symptoms of a potassium deficiency;  muscle weakness and a dilation of the intestinal tract. higher potassium content than ration 2 (Appendix 1).  ie.,  Ration 1 had a  Therefore any secondary  hypokalemia would be potentiated in females fed ration 2 (as compared to those fed ration 1) and the increased hypokalemia would potentiate the diabetes insipidus.  An increased polydipsia was observed in those females fed  ration 2. The final possibility is that the increased osmole excretion was due to both sodium and potassium and that there was an increased sodium absorption from the gut and a secondary hypokalemia.  To determine which, i f any, of  these mechanisms is occurring, the urinary sodium and potassium concentrations should be measured. Naik (1972) demonstrated that the water intake and vasopressor activities increased while the urine osmolality decreased up to 12 to 14 weeks of  152 age in the DI Os/+ mice (which had vasopressin-resistant diabetes insipidus). He postulated that as the mice matured the endogenous ADH became progressively less effective until i t stabilized at a low level at 12 to 14 weeks.  The  same progressive increase in water intake, decrease in per cent PCV, and increase in severity of the structural kidney defects occurred in the SWV females.  The concentrating a b i l i t y was only tested in year old mice, but it  would be interesting to determine i f there was a progressive decrease in concentrating a b i l i t y .  It would appear that the SWV females have an i n i t i a l  defect which does not become realized until maturity.  Then a number of  secondary factors (such as increased sodium absorption from the diet, sex hormone levels, or a structural defect in the nephrons due to a chronic toxicity) may act to progressively aggravate the diabetes insipidus until a c r i t i c a l level is reached when the females can no longer compensate.  The  kidneys become destroyed by scarring and hydronephrosis and the mice become dehydrated, lose weight, drink less, and eventually die. A number of suggestions for further work have already been made under the appropriate sections.  In addition there are 3 other experiments that  could be done in order to make the SWV females a more useful model for nephrogenic diabetes insipidus. 1.  Patients with human nephrogenic diabetes insipidus (Relkin, 1966)  and mice with vasopressin-resistant diabetes insipidus (Naik, 1970a) respond to a low sodium diet by reducing their urine output and increasing their urine osmolality.  The low sodium diet produces a sodium deficit in the  extracellular fluid that results in an increased reabsorption of sodium and water from the proximal tubule, and thus reduces the volume of filtrate reaching the distal tubule, and that in turn enables more water to be passively reabsorbed (ADH independent).  In addition the decreased sodium concentration  153 of the extracellular fluid may reduce the thirst stimulus so that there is a reduced water intake.  The SWV females fed ration 1 did drink less water  than those females fed ration 2, but the urine volume and urine osmolality were not determined for mice fed ration 2.  Also there are many other  differences between the 2 rations; therefore a low sodium diet, which is otherwise the same as the normal ration, should be given and the water balance measured. 2.  The thiazide diuretics cause a paradoxical antidiuresis in patients  with hypothalmic or nephrogenic diabetes insipidus (Relkin, 1966), but the antidiuresis is not as marked as that produced by ADH or chlorpropamide in hypothalmic diabetes insipidus.  Earley & Orloff (1964) suggested that  chlorothiazide acts by an i n i t i a l blocking of sodium reabsorption in the distal tubule to produce a marked sodium deficit.  This sodium deficit is more  marked than that caused by a low solute diet but acts to produce the antidiuresis in the same way as described above.  The antidiuresis can be en-  hanced by combining a low solute diet and chlorothiazide.  Thus the SWV  females should be given chlorothiazide and the effect on the severity of the diabetes insipidus determined. 3.  Finally 2 isogenic lines differing only in the gene (or genes)  determining the nephrogenic diabetes insipidus should be produced to make this a more useful genetic model. are affected,  Since a l l the females of the SWV strain  the controls have to be of another inbred strain.  One of the  advantages of the DI 0s/+ mice and DI rats is that they have a built in control in that there are normal animals of the same strain which can be used. Thus the vasopressin-resistant diabetes insipidus defect in the SWV strain should be put onto another inbred strain by repeated backcrossings least 9 generations.  for at  154 CONCLUSIONS  The SWV strain of mice has hereditary nephrogenic diabetes insipidus. The females have a severe progressive form which first manifests i t s e l f at 4 months of age whereas the males have a much milder expression of the defect and do not show any symptoms until 7 to 8 months of age.  The syndrome  present in the SWV females has the following characteristics: (i)  severe polydipsia and a large output of dilute,  sugar-free,  otherwise normal urine; (ii)  a severe concentrating defect - inability to concentrate urine or maintain body weight after severe water deprivation inability to concentrate urine after 3, daily, consecutive, injections of \ unit pitressin tannate;  (iii)  a progressive anaemia;  (iv)  normal adrenals and adequate neurosecretory material in the posterior pituitaries;  (v)  abnormal kidneys - in the cortico-medullary zone there are severely dilated tubules which have hyperplastic epithelium. There is some focal i n t e r s t i t i a l nephritis but no fibrosis up to 13 months.  At 17 months the kidneys are severely affected  with extensive degeneration, severe i n t e r s t i t i a l nephritis and fibrosis surrounding the dilated tubules, and some hyalinization in the cortex; (vi)  inability to tolerate saline drinking water.  The SWV males have a milder expression of symptoms i , i i , i i i , and v i , and  155 they have normal kidneys at 12 months of age.  This defect is unique although  it does resemble nephronophthisis slightly in some symptoms and hypokalemia slightly in other symptoms.  Amyloid kidneys, polycystic kidneys, and  diabetes mellitus have been ruled out as the cause of the defect but hypokalemia and hypercalcemia are s t i l l possibilities,  although unlikely.  The nephrogenic diabetes insipidus is inherited in a relatively simple manner.  There are 1 or 2 autosomal dominant genes involved but there is also  a large multiplicative effect by non-genetic factors. influencing the defect's expression are: ration; and parity.  Some of the factors  sex; age; body weight; type of  The defect is sex influenced in that the males have  only a mild form, but there is no six-linkage.  156 LITERATURE CITED  Abelson, H. 1963. Nephrogenic diabetes insipidus. A study of fine structure of the kidney in a 7 month old male. Pediat. Res. 2: 271-282. Bergmann, F., S. Calderari de Lozano, S.L. Rabasa, 1971. Effect of pitressin tannate on the diabetes insipidus induced by aminopterin in the Acta. Physiol. Latinoam. 21: 192-197. Bernstein, S.E. 1966. Physiological characteristics. In "Biology of the Laboratory Mouse". E.L. Green (Ed.), McGraw H i l l , Toronto, pp. 337350. Blotner, H. 1958. Primary or idiopathic diabetes insipidus; a system disease. Metabolism ]_: 191-200. Bode, H.H. & J.D. Crawford, 1969. Nephrogenic diabetes insipidus in North America - the Hopewell Hypothesis. New Eng. J. Med. 280: 750-754. Bode, H.H. & O.S. Miettinen, 1970. Nephrogenic diabetes insipidus: absence of close linkage with Xg. Am. J. hum. Genet. 22: 221-227. Braverman, L.E., J.P. Mancini, D.M. McGoldrick, 1965. Hereditary idiopathic diabetes insipidus - a case report with autopsy findings. Ann. intern. Med. 63: 503-508. Brazeau, P. 1970. Agents affecting the renal conservation of water. In "The Pharmacological Basis of Therapeutics". L.S. Goodman & A. Gilman (Ed.), MacMillan Co., Toronto. pp. 874-885. Brooks, F.P. & M. Pickford, 1958. The effect of posterior pituitary hormones on the excretion of electrolytes in dogs. J. Physiol., Lond. 142: 468-493. Burstein, P.N. & C.M. Chen, 1970. Diabetes insipidus, nephrogenic type, complicating pregnancy: a case report. Am. J. Obstet. Gynec. 108: 1292-1293. Cameron, M.L. & J.E. Steele, 1959. Simplified aldehyde-fuchsin staining of neurosecretory cells. Stain Techn. 34: 265-266. Campbell, W.G. Jr. 1961. Vasopressin resistant diabetes insipidus associated with cytological changes in the supraoptic and paraventricular nuclei. Lancet _ i i : 522. Carter, C.O. 1969. 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C o n c e n t r a t i n g power o f the k i d n e y a f t e r 5 y e a r s o f p i t r e s s i n - t a n n a t e therapy i n a p a t i e n t w i t h D.I. u n t r e a t e d f o r 13 y e a r s . ( I n v e s t i g a t i o n s d u r i n g i n t r a v e n o u s adminis t r a t i o n of hypertonic sodium c h l o r i d e , l y s i n e - v a s o p r e s s i n , clopamate & furosemide). E n d o k r i n o l o g i e 5_5: 359-365. Rado, J.P. & L. Szende, 1968. Use o f h y p e r t o n i c s a l i n e , clopamide and furosemide f o r e v a l u a t i o n o f the c o n c e n t r a t i n g d e f e c t i n p i t r e s s i n treated diabetes insipidus. Med.. Exp., B a s e l 1_8: 185-190. R e l k i n , R. 1966.  Diabetes  insipidus.  N.Y.  S t . J . Med.  66:  2789-2799.  R i c h a r d s , M.A. & J.C. S l o p e r , 1969. Diabetes i n s i p i d u s - the c o m p l e x i t y the syndrome. A c t a , endocr. Copenh. 6J2: 626-646.  of  S a m o r a j s k i , T. & J.M. Ordy, 1967. 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Neurohypophyseal principles in rats homozygous and heterozygous for hypothalmic diabetes insipidus (Brattleboro strain). Endocrinology 77_: 701-706. Valtin, H. & H.A. Schroeder, 1964. Familial hypothalmic diabetes insipidus in rats (Brattleboro strain). Am. J. Physiol. 206: 425-430. Valtin, H., H.A. Schroeder, K. Benirschke, H.W. Sokol, 1962. Familial hypothalmic diabetes insipidus in rats. Nature 196: 1109-1110. Wright, S. 1934. The results of crosses between inbred strains of guinea pigs, differing in number of digits. Genetics, Princeton 1_9: 537-551. Wright, S. 1968. Genetics of quantitative va r i a b i l i t y . In "Genetics and Biometric Foundations". University of Chicago, Chicago. Vol I, pp. 373-420. Wood, A.J. & T. Nishimura, 1968. Energy and water requirements in the house mouse (Mus musculus). Canad. J. Physiol. Pharmacol. 46: 617-620.  164 APPENDIX 1  Formulation of Feed Rations Used 1.  Ingredients (a)  Buckerfield's Mouse Ration U.B.C. 6-63  Ground wheat, ground barley, wheat bran, wheat germ meal, oat groats, fish meal, soya meal, vitagress, skim milk powder, brewers yeast, irradiated yeast, steamed bone meal, iodized salt, stabalized fat, cane molasses, dry vitamin A. (b)  Purina Lab Chow  Meat and bone meal, dried skimmed milk, wheat germ meal, fish meal, animal liver meal, dried beet pulp, ground extruded corn, oat middlings, soyabean meal, dehydrated alfalfa meal preserved with ethozxyquin, cane molasses, animal fat preserved with BHA, vitamin B^  2  calcium pantothenate,  supplement,  choline chloride, folic acid, riboflavin  supplement, brewer's yeast, thiamin, niacin, vitamin A supplement, D activated plant sterol, vitamin E supplement, dicalcium phosphate, iodized salt ferric ammonium citrate, iron oxide, manganous oxide, cobalt carbonate copper oxide, zinc oxide. 2.  Analysis Analysis of purina lab chow was given by Purina and the analysis of  Buckerfield's was provided courtesy of Professor J. Biely, Dept. of Poultry Science, U.B.C.  165 Buckerfields  Purina  26.81  23.40  3.51  4.30  Fibre, 7,  3.98  5.20  Ash, %  6.32  7.30  Sodium, 7,  0.05  0.49  Chlorides, 7,  1.30  0.51  Calcium, %  0.77  1.30  Potassium, 7.  1.18  0.82  Phosphorous, 7>  0.77  0.94  Manganese, ppm  63.20  51.00  Constituent Protein, °h Fat,  7,  

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