Department of Anatomy, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Quebec, Canada

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1 Department f Anatmy, Faculty f Medicine, University f Sherbrke, Sherbrke, Quebec, Canada SALT AND WATER METABLSM AND NEURHYPPHYSEAL VASPRESSR ACTVTY N MCE WTH HEREDTARY NEPHRGENC DABETES NSPDUS By D. V. Naik ABSTRACT Three gentypes f mice, V s/, D / and D s/, with nephrgenic (vaspressin resistant) diabetes insipidus (D) are cmpared t a nrmal V / strain with respect t water intake, urine utput, urine smlality, urine sdium and ptassium, and the extractable vaspressr (ADH) activity frm the neurhypphysis. Frm the results btained, it is cnfirmed that the severely diabetic D s/ mice prduce large vlumes f dilute, hyptnic urine and have a 2\m=1/2\fld vaspressr increase as cmpared t nrmal V / mice, whereas, the mildly diabetic V s/ and D / stand in between the D s/ and V / with intermediate vlumes f hypertnic urine and a vaspressr increase f 1\m=1/3\and 1\m=1/2\fld respectively. Further, a parallelism between the severity f diabetes insipidus and increased vaspressr activity is demnstrated in this study. The ttal excretin f sdium in the urine f V s/ and D s/ is higher than that f the V / and D / mice. This natriuresis is assciated with s gene and furthermre, its pssible hypthalamic invlvement is discussed. The increase f ADH in these hereditary nephrgenic diabetes insipidus mice is a new syndrme and the pssible rle f ther factrs invlved is discussed. This study was supprted by a Medical Research Cuncil f Canada, Grant N. MA-3759.

2 A cmparisn f the mrphlgical and physilgical characteristics f three gentypes (D s/, D / and V s/) f hereditary nephrgenic dia betes insipidus (D) mice, was made with a nrmal strain f (V / ) mice, by Falcner et al. (1964), Naik Sc Valtin (1969) and Stewart Sc Stewart (1969). The diabetic mice drink large quantities f water and excrete cpius amunts f dilute, sugar free urine. This defect is nt crrected by treatment f ex genus vaspressin, and hence is vaspressin resistant. The D s/ and V s/ mice are characterised by ligsyndactyly (s) f the fre limbs and/r hind limbs, accmpanied by unusually small kidneys with fewer nephrns. There is abnrmally high bld urea cncentratin and a higher serum smlality in these tw types f mice as cmpared t D / and V / mice (Naik 8c Valtin 1969). n the D s/ mice, Naik (1966) re prted hypertrphy f the neurhypphysis and the adenhypphysis. Further mre, with different stress experiments, Naik (1970a) demnstrated the rela tinship between the hypertrphy f the hypthalam-hypphysial neursecretry system and the hypertrphy f all the parts f the hypphysis, espe cially the pars intermedia. Naik Sc Skl (1970) made a cmparisn f the hypthalam-hypphysial neursecretry system in the abve 4 gentypes f mice and bserved that there is a parallelism between the severity f the diabetes insipidus and the extent f hypertrphy f the neursecretry system. The hypertrphied neursecretry system is als accmpanied by an increase f xytcin and vaspressin (ADH) in the pituitaries f the D s/ mice as cmpared t thse f V / nrmals (Naik Sc Kbayashi 1971). n certain dmestic as well as labratry animals, the physilgical and pathlgical aspects f diabetes insipidus are better understd (dgs, Slper et al. 1967; Richards Sc Slper 1969; rats, Valtin 1967; Sawyer et al. 1964; mice, Falcner et al. 1964; Naik Sc Valtin 1969; Naik Sc Skl 1970; Naik Sc Kbayashi 1971; Stewart Sc Stewart 1969). But in human diabetes insipidus (hypthalamic r nephrgenic) the exact disrders in the hypthalamic hyp physial neursecretry system are nt well understd (Frssman 1945; Martin 1959; Slper 1969). n the present paper, a cmparisn f the severity f diabetes insipidus, its mde f water turnver, electrlyte balance and the extractable vaspressr activity frm the neurhypphysis in 3 gentypes f diabetic and the nrmal mice is made. MATERAL AND METHDS The 4 gentypes f mice, V /, V s /, D / and D s/ were bred in this labratry fr several generatins by mating males and females f V stck (V / and V s/) and D stck (D / and D s/ ). Detail charac terisatin f these gentypes is described by Falcner et al. (1964) and Naik 8c Valtin

. (1969). Metablism experiments were cnducted in an air-cnditined rm maintained between 23-25 C and under cntrlled humidity and light (12L, 12D).

3 . (1969). Metablism experiments were cnducted in an air-cnditined rm maintained between C and under cntrlled humidity and light (12L, 12D). The animals were fed at all times with Purina Lab chw and tap water ad libitum. The littermates used fr this study were: V stck, 14 nrmal V /, 12 V s/; and D stck, 12 D / and 14D s/ Since water turnver stabilizes after 90 days f age, in all grups, males and females between days f age were used. Water intake, urine flw and urine excretin f sdium, ptassium and smlality f individual muse were measured. The individual muse was kept in the muse metablism cages (Acme Metal Prducts nc., Chicag, 111.), during three 24 hur perids when animals tk fd and water ad libitum. The urine was cllected under mineral il in 50 ml flask and its smlality was measured as it was dne previusly (Naik 8c Valtin 1969). Urine excretin f sdium and ptassium was estimated by direct flame phtmetry (143 Mdel, nstrumentatin Labratry nc., Bstn, Mass.). When the metablism studies were ver, 3-4 animals were kept tgether in each cage and given rest fr 8-10 days. The animals were sacrificed and the neurhypphysis alng with pars intermedia was carefully separated frm each animal and dried individually in acetne fr vaspressr biassay. After several changes f acetne, the tissues were further dried under phsphrus pentxide in a The individual dried tissue was carefully weighed (Mdel M-5 S/A) and the weight was recrded. Previus prcedure was fllwed fr the extractin and biassay f the individual gland (Naik Sc Kbayashi 1971). Vas vacuum desiccatr. n a sensitive Mettler balance pressr activities f the neurhypphysis extracts were estimated with the rat pressr methd (Dekanski 1952), 8-lysin vaspressin (Sandz) being used as the reference standard. The animals used fr vaspressr assay were anaesthetized with urethane and treated with heparin and dibenamine as described by Dekanski (1952). A fur pint assay methd was adpted fr the estimatin f each sample. As the average vaspressr activity in the males and females f the same grup did nt shw much difference, their results were cmbined fr the simplicity f calculatin. Three male D s/ mice shwed lwer water intake, lwer urine utput and in creased urine smlality (Table 1) than the rest f the D s/ animals. These culd be cnsidered as mildly diabetic. ut f these three, 2 were pituitaries used fr vas pressr biassay; pituitary f the third animal and the three hypthalami were fixed in Buin's fr histlgical studies. Sectins were cut at 5 and 7 /<m respectively and stained with Gmri's aldehyde fuchsin fr the neursecretry study as it was dne previusly (Naik 1910a; Naik 8c Skl 1970). RESULTS The data n average fluid balance fr each grup f mice based n the bdy weight, per 24 hurs is given in Table 1. Each grup value represents the mean f three 24 hur cllectins. By cmparing the results f the nrmal V / mice t thse f the diabetic V s/, D / and D s/, it is evident that the severity f the diabetes in D s/ is reflected by a mean increase f water intake 137 ml/100 g bdy weight and in mean urine flw f 91 ml/100 g bdy weight in 24 hurs and by a crrespndingly lw mean urine smlality f 297 msm/kg. Values fr fluid balance in D / and V s/ mice fall between thse fr D s/ and V / nrmals.

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5 .. n D s/ and V s/, the urine flw is higher and the urine sm lality is significantly lwer than thse f nrmals (Table 1). The urine smlalities f D / (1629 msm/kg) and V s/ (1898 msm/kg) are signi ficantly higher than that f D s/ (297 msm/kg) and significantly lwer than that f V / nrmals (3054 msm/kg). The severity f the diabetes in D / and V s/ lies between that f D s/ and V / and hence they can be cnsidered as mildly diabetic. By judging the water intake, urine flw and the urine smlality, the D s/ mice are severely diabetic with urine smlality lwer than their serum smlality (als see Naik Sc Valtin 1969). The three mildly diabetic D s/ mice, described earlier, tk less water and gave mre cncentrated urine as cmpared t the ther D s/ These animals were tested peridically fr mre than 6 weeks. The average results f all the experiments f water intake (80 ml/100 g bdy weight), urine utput (52 ml/100 g bdy weight) and urine smlality (567 msm/kg), are quite different frm thse f D s/ (Table 1). ut f these three, ne animal was used fr histlgy and 2 fr vaspressr biassay. The degree f hyper trphy f the neursecretry system in this mildly diabetic D s/ is much less than that f the severely diabetic D s/, but similar t that f D / mice as described by Naik Sc Skl (1970). The data n average ttal urine excretin f sdium and ptassium in each grup f mice is given in Table 1. The ttal urine excretin f sdium in nrmal V / (16.81 meq.) and D / (16.41 meq./l g bdy weight) mice is almst the same, whereas, there is a significant increase f ttal sdium in the V s / (22.43 meq.). The ttal urine excretin f ptassium values d nt differ significantly in all the 4 grups (Table 1). The dry neurhypphysial weights shw n difference in V / and V s/ mice (0.073 mg), but in the D / and D s/, they shw a significant increase, and mg respectively (Table 2). The dry neurhypphysial weight includes the dry pars intermedia weight als. Mean vaspressr assay values fr each grup f mice are given in Table 2. The highest vaspressr value is in D s/ 208 mu, where the water turnver is maximum. D / and V s/ vaspressr values, 128 mu and 177 mu respectively, are significantly lwer than the D s/ and signi ficantly higher than the V / nrmals, 87 mu. nspite f the fact that the V / and V s/ are littermates, the vaspressr value fr V s/ is l'/e fld higher. D / and D s/ shw vaspressr increase f Vî and 2V2 flds respectively, against the nrmal V / Here again, D / and D s/ are littermates with a difference f l2/s fld in the vaspressr activity. n all these grups, there is a parallelism between the water turnver and the vaspressr activity. The vaspressr values f tw mildly diabetic D s/ mice are markedly lwer (135 and 141 mu) than the

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7 rest f the D s/. Mrever, the water turnver is markedly these 3 mildly diabetic D s/ mice (Table). n all these 4 tempted. Hwever, Naik 8c reduced in gentypes f mice, the xytcin biassay has nt been at Kbayashi (1971), have biassayed the neurhyp physial xytcic activity frm V / and D s/, and fund a great increase f xytcin in the latter grup. Similarly, Stewart Sc Stewart (1969), have biassayed the neurhypphysial hrmnes f D / and D s/ mu mice and irrespective f grups, their activity is equivalent t fr vaspressin and mu fr xytcin. As these results are fr D s/ and D / cmbined, we cannt cmpare these results t ur earlier results (Naik Sc Kbayashi 1971), nr the present nes. Hwever, we can cnclude Stewart (1969) that these values are higher than frm the results f Stewart 8c thse f V / and V s/ mice. DSCUSSN Earlier histlgical studies f the hypthalam-hypphysial neursecretry system in D mice (Naik Sc Skl 1970), the biassays f xytcin and vas pressin (the antidiuretic hrmne-adh) in the D s/ mice (Naik Sc Kbayashi 1971) and the present vaspressr biassay in all the 4 gentypes f mice clearly indicate that the hypertrphy f the neurhypphysis and increase in vaspressr activity g parallel with each ther. The unusual results in 3 male D s/ (cnsidered as mildly D), further give a strng supprt t the fact that the hypertrphy f the hypthalamhypphysial neursecretry system and the vaspressr activity f the pituitary are directly prprtinal t the water turnver, whereas, the urine smlality is inversely prprtinal. There is n dubt that the increased water turnver and the increased vaspressr activity in all these D mice is due t the majr defect in the kidney. n D s/ and V s/ mice, the kidneys are very small with fewer nephrns and cccsinally damaged tubules (Naik 8c Valtin 1969; Stewart Sc Stewart 1969). Besides these mrphlgical defects, they may be sme bichemical defects in the kidney als, since in the D s/ and V s/ mice, similar mrphlgical defects in the kidney may nt give such a great difference in the water turnver and the urine smlality (Table 1), unless there are greater bichemical defects in the kidney tubules f the D s/ mice. n the D / kidneys, n abnrmalities were bserved under light micrscpe, but there may be sme ultrastructural defects and/r sme bichemical defects. S, it is essential t lk in fr details f these diabetic kidneys under electrnmicrscpe. Naik Sc Valtin (1969) have ruled ut the pssibility f the medullary cystic disease, sickle cell disease, amylidsis,

8 . ptassium and magnesium deliciency and prbability f hypercalcaemic renal disease in the D mice. The 0s/ gene in the D 0s/ and V 0s/ is assciated with ttal in be due t the fact crease f urine excretin f sdium (natriuresis). This may that their kidney tubules are shrter and hence sdium reabsrptin by the distal end tubules is reduced resulting in natriuresis. Naik (unpublished) fund that the Juxta Glmerular ndex (JG) f the kidney and the width f the adrenal zna glmerulsa in the D s/ and V s/ are markedly reduced in cmparisn with the nn-s/ gentypes. Lw sdium diet (GB) helped t increase bth f these parameters enrmusly. Frm bth the abve results, therefre, it seems that the salt-renin-aldsterne respnse is nt hampered in the s/ gentypes. f there was mre bdy sdium lss, naturally, the JG and the width f zna glmerulsa wuld have been increased t prevent mre bdy sdium lss. There is n difference f fd intake in all the 4 gentypes (Naik Sc Valtin 1969). Therefre, it culd be pssible that sme defect in the hypthalamus may be respnsible fr mre sdium absrptin, which is lst thrugh the abnrmal kidney tubules. n experimental rats, Drn Sc Prter (1970) and Cvian Sc Antunes-Rdrigues (1963) have demn strated the hypthalamic invlvement in sdium intake and excretin. n all these D gentypes, the kidneys are anatmically and/r bichemically de fective, and influence the degree f water turnver, which increases the serum smlality, which in its turn activates the hypthalamic thirst centre, resulting in the hypertrphy f the neursecretry system and increased vaspressr activity. Naik (1970Z>) bserved that keeping these D s/ mice n lw sdium diet (GB) fr 6-8 weeks, results in marked decrease f water intake and significant increase f urine smlality, accmpanied by decrease in the neursecretry system. Further studies shwed a significant decrease in the neurhypphysial vaspressr activity f these animals (Naik, t be published). This supprts ur abve statement that the degree f water turnver influences the neursecretry system and vaspressr activity. The histlgical bservatins in pstnatal (2 t 12 weeks f age) D mice as cmpared t thse f the nrmals, shw a faster hypertrphy f the neur secretry system. The water intake, urine utput, urine smlality and neur hypphysial vaspressr activity in the pstnatal D mice (Naik, t be pubi.) shw the fllwing results: average urine smlality at 3 weeks f age in D s/ mice is 543 msm/kg and it gradually decreases t its minimum f 297 msm/kg by weeks f age, and later stabilizes. The water intake als gradually starts increasing frm 3 weeks f age nwards and reaches a steady high level by weeks f age. The neurhypphysial extractable vaspressr activity at 3 weeks f age is abut 68 mu/gland in D / and D s/ But, in late pstnatal develpment (4-12 weeks f age), the vas-

9 pressr in the D s/ increases at much faster rate and reaches the plateau f abut 205 mu at 12 weeks f age (Naik, unpublished). Frm the neur hypphysial extractable vaspressr cntent and the water turnver studies in the pstnatal D mice, it can be cncluded that as the D s/ mice reach their maturity, the ADH in the kidney becmes less and less active, and it reaches its lwest by the time the mice reach 12 weeks f age, and then re mains steady (Naik, t be published). S, it appears that there may be sme hereditary enzyme deficiency and/r slw dwn f the prper respnse f ADH actin n the tubular cells f the D s/ mice, when they reach maturity. Similar defect may be present in the ther tw gentypes f D / and V s/ mice in different degrees. S, it appears that in D s/ mice, the syndrme is quite different frm that described by Fancni (1956) and Hankiss et al. (1961), that there is faster destructin f ADH in the liver and kidney in the human diabetes insipidus. n these D mice, it appears that the activity f ADH in the kidney gradually decreases frm 3 weeks f age t 12 weeks f age. Naik (197Gb) bserved the reductin f stained neursecretry material in the dehydrated and stressed D s/ mice. When stressed by frmalin (intracutaneus injectin f 0.4 ml, 4 /) and dehydrated by saline (intraperitneal injectin f 1 ml, 5%), the D s/h- mice shwed a marked reductin f neurhypphysial extractable vaspressr activity after 5 and 15 min respec tively (Naik, t be published). These experiments f dehydratin and stress indicate a much faster rate f release f ADH frm the neursecretry system. But this des nt give any infrmatin abut the nrmal rate f release f ADH in these D mice in resting cnditins. With the technique presently available, it is nt pssible t measure the circulating ADF in the bld f such small animals. The fact that the urine smlality increases a little in the exgenus ADH treated, water laded D mice (Naik Sc Valtin 1969), shws that the ADH is functining t its minimal level in D s/ mice as cmpared t the ther types f mice, Naik (1970c) and Naik (in press) has bserved a marked decrease in the urine smlality (80 t 150 msm/kg) by the destructin f bth supraptic nuclei by electrlytic lesins in the D s/ mice, in cntrast t 297 in cntrl grups, which shws that the endgenus ADH is functining in the D s/ mice t a minimal level as cmpared t D / and V s/ mice. Frm the present biassay studies and the previus hypthalam-hypphysial neursecretry studies (Naik Sc Skl 1970), it can be cncluded that the neur hypphysis f V s/, D / and D s/ mice seems t functin mre actively than that f nrmal mice. Therefre, the elevated cntent f the vas pressr in the neurhypphysis prbably arises as a cnsequence f the in-

10 creased synthesis and strage f the secretins withut a cmmensurate accele ratin f their release. The present results and the evidences f thers (Naik 8c Valtin 1969; Naik Sc Kbayashi 1971; Stewart Sc Stewart 1969), therefre suggest very strngly that the diabetes insipidus in the 3 gentypes f D mice, unlike the familial diabetes insipidus in the Brattlebr rats {Valtin 1967) is nt due t a failure in the synthesis f ADH. Als, Naik Sc Valtin (1969) shwed that the D mice d nt suffer frm primary plydipsia as d STR/N mice described by Silverstein et al. (1961). t is als different frm the syndrme f inapprpriate secretin f ADH, described in human by Bartter Sc Schwartz (1967), and dif ferent frm diabetes insipidus due t increased hrmne inactivatin described by Hankiss et al. (1961) in human. The increase f ADH in these nephrgenic diabetes insipidus mice is a new syndrme. Fr such an increase f ADH, there may be multiple reasns cntrlled by increased water lss, resulting in higher serum smlality, and stimulatin f smreceptrs, variatins in vlume and distributin f the bld and the extracellular fluid as is suggested by Share (1969) in his recent review, and may partly be due t a genetical defect f the central nervus system cntrl f the secretin and release f ADH. ur wrk is currently in prgress in rder t answer sme f these questins. ACKNWLEDGMENTS thank Dr. Maurice Burgain, f the Sandz Labratry, at Drval, Quebec, Canada, fr kindly supplying 8-lysin vaspressin (Sandz) fr this experiment and t Miss Denise Gaudet fr her excellent technical assistance. REFERENCES Bartter F. C. Sc Schwartz W. B.: Amer. J. Med. 42 (1967) 790. Cvian M. R. & Antunes-Rdrigues J.: Amer. J. Physil. 205 (1963) 922. Dekanski J.: Brit. J. Pharmacl. 7 (1952) 567. Drn J. 8c Prter J. C: Endcrinlgy 86 (1970) Falcner D. S., Latyszewski M. Sc saacsn J. H.: Genet. Res. (Camb.) 5 (1964) 473. Fancni G.: Helv. paediat. Acta 11 (1956) 506. Frssman H.: Acta med. scand. Suppl. 159 (1945) 1. Hankiss J., Keszthelyi M. 8- Sir B.: Amer. J. med. Sci. 242 (1961) 125. Martin F.. R.: Quart. J. Med. 28 (1959) 573. Naik D. V.: Amer. Zl. 6 (1966) 518. Naik D. V.: Z. Zellfrsch. 107 (1910a) 317. Naik D. V.: Anat. Ree. 776 (1970è) 353. Naik D. V.: Prc. canad. Fed. bil. Sc. 13 (1910c) 147. Naik D. V. Sc Kbayashi H.: Neurendcrinlgy 7 (1971) 322. Naik D. V. Se Skl H. W.: Gen. cmp. Endcr. 75 (1970) 59. Naik D. V. Sc Valtin H.: Amer. J. Physil. 277 (1969) 1183.

11 Richards M. A. Se Slper J. C: Acta endcr. (Kbh.) 62 (1969) 627. Sawyer W. W., Valtin H. Sc Skl H. W.: Endcrinlgy 74 (1964) 153. Share L. n: Ganng W. F. and Martini L., Eds. Neurendcrinlgy, xfrd Univer sity Press nc., New Yrk, Lndn, Trnt (1969) 183. Silverstein E.. Sklff L., Mickelsen. & Jay Jr. G. E.: Amer. J. Pathl. 38 (1961) 143. Slper J. C: Acta neurl. belg. 69 (1969) 581. Slper J. C, Karim M. A. Sc Richards M. A. n: Stutinsky F., Ed. Neursecretin. Prv. V ntern. Sympsium n Neursecretin, Strasburg (1966), Springer Verlag. Berlin, Heidelberg, New Yrk (1967) 124. Stewart A. D. & Stewart J.: Amer. J. Physil. 277 (1969) Valtin H.: Amer. J. Med. 42 (1967) 814. Received n August 3rd, 1971.

A Plasma Humoral Factor of Extrarenal Origin Causing Release of Reninlike Activity in Hypotensive Dogs

A Plasma Humoral Factor of Extrarenal Origin Causing Release of Reninlike Activity in Hypotensive Dogs A Plasma Humral Factr f Extrarenal Origin Causing Release f Reninlike Activity in Hyptensive Dgs By E. De Vit, C. Wilsn, R. E. Shipley, R. P. Miller, and B. L. Mrtx ABSTRACT Plasma reninlike activity significantly