Interaction of the diabetes insipidus locus alleles with the renal 120 kDa protein-encoding gene in rat development

Age-dependant dynamics of the kidney inner medullary 120-kDa protein content in vasopressin-deficient Brattleboro rats with di/di mutant genotype was studied in comparison with WAG rats with genotype and normal vasopressin expression. Age-dependant dynamics of vasopressin content in neurohypophysis of WAG rats was also examined. It was shown that 10-day-old WAG rats were unable to elevate the synthesis of the 120 kDa protein in respond to long-term dehydration, while this tendency was clearly observed in the 15-day-old rats and later in the development. In WAG rats the onset of this specific feature was time correlated with the development of the respond to hydration by the elevation of vasopressin synthesis and release from neurohypophysis into blood. In the di/di rats dehydration had no effect on the kidney 120-kDa protein synthesis in all ages examined. These results point to the interaction between the di alleles and the 120-kDa protein-encoding gene in the course of development.     

Interaction of the diabetes insipidusLocus Alleles with the Renal 120 kDa Protein-Encoding Gene in Rat Development

Age-dependent dynamics of the kidney inner medullary 120-kDa protein content in vasopressin-deficient Brattleboro rats with di/di mutant genotype was studied in comparison with WAG rats with +/+ genotype and normal vasopressin expression. Age-dependent dynamics of vasopressin content in neurohypophysis of WAG rats was also examined. It was shown that 10-day-old WAG rats were unable to elevate the synthesis of the 120 kDa protein in respond to long-term dehydration, while this tendency was clearly observed in the 15-day-old rats and later in the development. In WAG rats the onset of this specific feature was time correlated with the development of the respond to hydration by the elevation of vasopressin synthesis and release from neurohypophysis into blood. In the di/di rats dehydration had no effect on the kidney 120-kDa protein synthesis in all ages examined. These results point to the interaction between the di alleles and the 120-kDa protein-encoding gene in the course of development.     

Dynamics of renal medullary vinculin under changing of osmoregulating function

Reduction of vinculin occurs in the renal medulla under the long-lasting dehydration. The protein content measured in inner medulla of rats of the WAG line under hydration was 92.1 +/- 6.3 in relative units, but it was only 77.6 +/- 2.3 after a 3-day water deprivation. The vinculin content in the inner medullar layer from mutant rats of Brattleboro line incapable of synthesizing vasopressin is essentially higher: it is 188.9 +/- 8.5 in hydrated conditions and drops to 148.4 +/- 7.3 under a 3-day dehydration. The same high level of vinculin is in outer medulla from rats of Brattleboro line: 222.1 +/- 11.8 in hydrated animals and 174.9 +/- 11 after a 3-day dehydration. No differences were revealed in response of vinculin to alternative osmoregulating stimulation in cortex in both rat lines.     

Reaction of alpha-actinin from renal inner medulla on prolonged dehydration

A western-blot analysis using monoclonal antibodies revealed that a reduction of alpha-actinin occurred in the renal inner medulla under the long-lasting dehydration. The ratio between protein content measured in rats of WAG line being hydrated or after 3-days water deprivation consisted of 52.7+/-6.2 against 23.9+/-3.3 as evaluated in relative units. The alpha-actinin level changes similarly in mutant rats of Brattleboro line not capable of synthesizing vasopressin. It was 57.5+/-4.6 in hydrated animals, and statistically lower in rats being under 3-day water deprivation--26.4+/-5.7 in relative units of protein.     

Effect of the di mutation (non-sugar diabetes) on properties of kidney proteins in rats

Renal proteins were studied in Brattleboro rats of the didi genotype and in Wistar and Sprague-Dawley rats with normal alleles +2 of this loci. In animals of the +2 genotype maintained under conditions of water deprivation, the content of 120 kDa protein increased significantly in inner medulla of the kidney over 3 days. No changes in the amount of this protein were observed in rats of the didi genotype under the same conditions. We suggest that the congenital inability of didi mutants to synthesize vasopressin accounts for the distinctions observed in the reactions of rats with different genotypes.     

Expression of Tropomyosin-Encoding Gene in the Kidney Depends on Functioning of Vasopressin Gene

Brattleboro diabetes insipidus mutant rats and normal WAG rats were subjected to water loading or thirst during 3 days. It was found that tropomyosin-encoding gene expression has a tissue-specific pattern in the kidney. Northern blot and western blot analysis had shown that the main expression of Tpm3(3) takes place in the renal medulla, and its intensity differs in normal and mutant rats. The differences between mutants with an ineffective vasopressin synthesizing system and the rats having an intact vasopressin gene were more distinct under long-lasting dehydration. The ratio between renal medullary tropomyosin of Brattleboro and WAG lines of rats was 39.76 +/- 0.90 versus 18.29 +/- 0.86 under water loading, and 46.12 +/- 2.14 versus 13.83 +/- 0.66 in thirst.     

Activation of adenylate cyclase in renal medulla by bovine growth hormone. An artifact attributable to vasopressin.

The effect of bovine growth hormone on adenylate cyclase activity was studied in bovine and rat renal medulla. Highly purified growth hormone (lot B1003A) increased adenylate cyclase activity in plasma membranes from bovine renal medulla from 132+/-6 pmol cyclic AMP formed/mg protein per 10 min to 364+/-10 pmol cyclic AMP formed/mg protein per 10 min. Similar results were seen with homogenates of rat renal medulla. The minimum effective concentration of bovine growth hormone required to activate adenylate cyclase was 0.5 mug/ml and maximum activation was detected at 500 mug/ml. The amount of vasopressin determined by radioimmunoassay to contaminate the growth hormone caused an increase in adenylate cyclase activity comparable to that of the corresponding concentration of growth hormone that was tested. Dialysis of growth hormone and vasopressin resulted in parallel reductions in the effect of each hormone on adenylate cyclase activity. Similarly, both growth hormone and vasopressin produced increases in short circuit current in isolated toad bladders but these effects were not detectable after dialysis of the hormones. In contrast, the effect of growth hormone on the uptake of 35SO2-4 by cartilage from hypophysectomized rats was not decreased after dialysis. These results indicate that available preparations of growth hormone are contaminated by small but physiologically significant amounts of vasopressin and that the activation of adenylate cyclase activity in renal medulla in response to growth hormone can be explained by this contamination rather than by an effect of growth hormone per se.     

Effect of dDAVP, agonist of V2 vasopressin receptor on the hyaluronidase-1 and hyaluronidase-2 gene expression in the kidneys of Wistar and Brattleboro rats

Effect of vasopressin on the expression of Hyal-1 and Hyal-2 genes in different functional zones of Wistar and homozygous vasopressin-defficient Brattlboro rat kidneys was analysed using RT-PCR mehod. It was found that, in Wistar rats the content of Hyal-1 mRNA was higher in the medulla than in other kidney zones at the normal water and food regimen. The level of Hyal-1 mRNA in the cortex and the medulla of Brattlboro rat kidney exceeded that of papilla. There were no significant differences in the Hyal-2 mRNA content detected between functional zones of Wistar and Brattlboro rat kidneys. The treatment by dDAVP, the agonist of V2 vasopressin receptor (Desmopressin, 10 microg/100 g b.w.i.p. twice a day for two days) induced an increase in urine osmolality and significant increase in the Hyal-1 and Hyal-2 mRNA content in the medulla without changes in the cortex and papilla. The effect was more pronounced in Brattlboro rat kidney. These results demonstrate that, in control conditions, genes encoding Hyal-1 and Hyal-2 were expressed independently in all functional kidney zones in the both in normal Wistar and in vasopressin-defficient Brattlboro rats. Desmopressin (dDAVP) exerts a stimulating effect on Hyal-1 and Hyal-2 gene expression in the medulla.     

The effects of hypoosmotic infusion on the composition of renal tissue of the Australian brush-tailed possum Trichosurus vulpecula

Although the occurrence of organic osmolytes in the inner medulla of the marsupial kidney has been recently reported [Comp. Biochem. Physiol. (2002) 132B 635-644], changes in these substances, in response to water loading in vivo, has not been studied. Adult Trichosurus vulpecula, the Australian brush-tailed possum, were subjected to water deprivation for 48 h. Following anaesthesia and unilateral nephrectomy, the animals were perfused with hypo-osmotic saline (80 mmol l(-1); 1.5 ml min(-1)) for 60 min. This resulted in a rapid increase in urine volume and a corresponding fall in urine osmolality. At the end of the infusion the animals were killed and the second kidney removed. Analysis of the renal tissue revealed that water content of cortical, outer and inner medullary regions of the kidney increased slightly following infusion, while sodium, and chloride contents of all three regions fell. Potassium contents, on the other hand, were barely changed. Of the organic osmolytes determined, very significant decreases in the inner medulla, following infusion, were found for sorbitol (from 397+/-79 to 266+/-49 mmol kg(-1) protein), inositol (247+/-23 to 190+/-25 mmol kg(-1) protein), and betaine (464+/-70 to 356+/-21 mmol kg(-1) protein), while only inositol was significantly decreased in the outer medulla (197+/-22 to 150+/-16 mmol kg(-1) protein). Glycerophosphorylcholine levels were low throughout the kidney and were not significantly affected by the infusion. It was concluded that inositol and sorbitol play a significant role as compatible organic osmolytes in the possum kidney, while betaine functions as the principal counteracting osmolyte. Amino acid levels in the cortex and outer medulla showed no overall change in amount following infusion, although there were highly significant changes in individual amino acids. In the inner medulla there was a highly significant reduction in total amino acids with infusion, largely due to a fall in amounts of taurine (104+/-4 to 75+/-17 mmol kg(-1) protein), and glycine (97+/-15 to 71+/-18 mmol kg(-1) protein). A fall in free amino acid levels in the inner medulla appears to significantly contribute to the process of intracellular osmotic adjustment during an induced diuresis.     

Determination of the functional molecular size of vasopressin isoreceptors

The molecular size of vasopressin receptors in the intact membrane-bound state was determined by radiation inactivation (target size analysis). For the V1 receptor in rat liver a molecular size of (76 +/- 8) kDa was determined. For the V2 receptor in rat kidney and bovine kidney molecular sizes of (95 +/- 4) and (108 +/- 11) kDa were found. Statistical analysis gave evidence for size differences between rat liver and rat kidney receptors or differences between rat liver and bovine kidney receptors, but not between kidney receptors from different species. The results suggest that V1 and V2 receptors can be distinguished by functional properties as well as by their size.     

Hypertonicity is involved in redirecting the aquaporin-2 water channel into the basolateral,instead of the apical,plasma membrane of renal epithelial cells

In renal collecting ducts, vasopressin increases the expression of and redistributes aquaporin-2 (AQP2) water channels from intracellular vesicles to the apical membrane, leading to urine concentration. However, basolateral membrane expression of AQP2, in addition to AQP3 and AQP4, is often detected in inner medullary principal cells in vivo. Here, potential mechanisms that regulate apical versus basolateral targeting of AQP2 were examined. The lack of AQP2-4 association into heterotetramers and the complete apical expression of AQP2 when highly expressed in Madin-Darby canine kidney cells indicated that neither heterotetramerization of AQP2 with AQP3 and/or AQP4, nor high expression levels of AQP2 explained the basolateral AQP2 localization. However, long term hypertonicity, a feature of the inner medullary interstitium, resulted in an insertion of AQP2 into the basolateral membrane of Madin-Darby canine kidney cells after acute forskolin stimulation. Similarly, a marked insertion of AQP2 into the basolateral membrane of principal cells was observed in the distal inner medulla from normal rats and Brattleboro rats after acute vasopressin treatment of tissue slices that had been chronically treated with vasopressin to increase interstitial osmolality in the medulla, but not in tissues from vasopressin-deficient Brattleboro rats. These data reveal for the first time that chronic hypertonicity can program cells in vitro and in vivo to change the insertion of a protein into the basolateral membrane instead of the apical membrane.     

Putative osmolytes in the kidney of the Australian brush-tailed possum,Trichosurus vulpecula

The Australian brush-tailed possum, Trichosurus vulpecula, is capable of producing a moderately concentrated urine, at least up to 1300 mOsm l(-1). Kidneys of adult animals fed in captivity on a normal diet with ready access to water were analysed. The inner medullary regions were found to have moderately high concentrations of sodium (outer medulla, 367+/-37; inner medulla 975+/-93 mmol kg(-1) dry wt.), chloride (outer medulla 240+/-21; inner medulla 701+/-23 mmol kg(-1) dry wt.) and urea (outer medulla, 252+/-62; inner medulla, 714+/-69 mmol kg(-1) protein). When the animals were fed on a 'wet diet', amounts of these substances in the outer medulla and cortex were reduced, although with the exception of urea these changes were not significant. There were highly significant changes in amounts of Na(+), Cl(-) and urea in the inner medulla (Na(+), 566+/-7; Cl(-), 422+/-9 mmol kg(-1) dry wt.; urea 393+/-84 mmol kg(-1) protein). Likewise, the inner medulla of animals fed a 'dry diet' with limited access to water showed highly significant increases in the same substances (Na(+), 1213+/-167; Cl(-), 974+/-137 mmol kg(-1) dry wt.; urea, 1672+/-98 mmol kg(-1) protein). Inositol was found in the outer medulla (224+/-90 mmol kg(-1) protein) and inner medulla (282 mmol kg(-1) protein) as was sorbitol (outer medulla, 62+/-20; inner medulla, 274+/-72 mmol kg(-1) protein). Both these polyols were reduced in amount in renal tissue from 'wet diet' animals, and increased in 'dry diet' animals, but the changes were not statistically significant. The methylamines, betaine and glycerophosphorylcholine (GPC), showed a similar pattern, but both were significantly elevated in the inner medulla of 'dry diet' animals (betaine 154+/-57 to 315+/-29 mmol kg(-1) protein; GPC 35+/-7 to 47+/-10 mmol kg(-1) protein). It was concluded that in this marsupial the concentrating mechanism probably functions in a similar way to that in higher mammals, and that the mechanism of osmoprotection of the medulla of the kidney involves the same osmolytes. However, the high ratio of betaine to GPC in the inner medulla resembles the situation in the avian kidney.     

Hyperosmotic NaCl and urea synergistically regulate the expression of the UT-A2 urea transporter in vitro and in vivo

The UT-A2 urea transporter is involved in the recycling of urea through the kidney, a process required to maintain high osmotic gradients. Dehydration increases UT-A2 expression in vivo. The tissue distribution of UT-A2 suggested that hyperosmolarity, and not vasopressin, might mediate this effect. We have analyzed the regulation of UT-A2 expression by ambiant osmolarity both in vitro (mIMCD3 cell line) and in vivo (rat kidney medulla). The UT-A2 mRNA was found to be synergistically up-regulated by a combination of NaCl and urea. Curiously, the UT-A2 protein was undetectable in this hypertonic culture condition, or after transfection of the UT-A2 cDNA, whereas it could be detected in HEK-293 transfected cells. Treating rats with furosemide, a diuretic which decreases the kidney interstitium osmolarity without affecting vasopressin levels, led to decreased levels of the UT-A2 protein. Our results show that the UT-A2 urea transporter is regulated by hyperosmolarity both in vitro and in vivo.     

Salicylate nephropathy in the Gunn rat: potential role of prostaglandins

We examined the potential role of prostaglandins in the development of analgesic nephropathy in the Gunn strain of rat. The homozygous Gunn rats have unconjugated hyperbilirubinemia due to the absence of glucuronyl transferase, leading to marked bilirubin deposition in renal medulla and papilla. These rats are also highly susceptible to develop papillary necrosis with analgesic administration. We used homozygous (jj) and phenotypically normal heterozygous (jJ) animals. Four groups of rats (n = 7) were studied: jj and jJ rats treated either with aspirin 300 mg/kg every other day or sham-treated. After one week, slices of cortex, outer and inner medulla from one kidney were incubated in buffer and prostaglandin synthesis was determined by radioimmunoassay. The other kidney was examined histologically. A marked corticomedullary gradient of prostaglandin synthesis was observed in all groups. PGE2 synthesis was significantly higher in outer medulla, but not cortex or inner medulla, of jj (38 +/- 6 ng/mg prot) than jJ rats (15 +/- 3) (p less than 0.01). Aspirin treatment reduced PGE2 synthesis in all regions, but outer medullary PGE2 remained higher in jj (18 +/- 3) than jJ rats (9 +/- 2) (p less than 0.05). PGF2 alpha was also significantly higher in the outer medulla of jj rats with and without aspirin administration (p less than 0.05). The changes in renal prostaglandin synthesis were accompanied by evidence of renal damage in aspirin-treated jj but not jJ rats as evidenced by: increased incidence and severity of hematuria (p less than 0.01); increased serum creatinine (p less than 0.05); and increase in outer medullary histopathologic lesions (p less than 0.005 compared to either sham-treated jj or aspirin-treated jJ). These results suggest that enhanced prostaglandin synthesis contributes to maintenance of renal function and morphological integrity, and that inhibition of prostaglandin synthesis may lead to pathological renal medullary lesions and deterioration of renal function.     

Anomalous mobilities of Na,K-ATPase alpha subunit isoforms in SDS-PAGE: identification by N-terminal sequencing.

Three isoforms of the alpha subunit of Na,K-ATPase, alpha 1, alpha 2, and alpha 3 have been characterized at the DNA, mRNA and protein levels. In admixtures, isoforms migrate as doublets (i.e. alpha 1 and another band originally designated alpha +, comprising alpha 2 + alpha 3) when analyzed by SDS-PAGE. As deduced from cDNA sequences their masses range from 111.7 to 112.6 kDa. With conventional protein standards, however, SDS-PAGE yields nominal masses of 85-105 kDa. In this system, the presence of a doublet that reacted with a polyclonal anti-Na,K-ATPase antibody in the kidney was interpreted as indicating two molecular or conformational species of the kidney alpha sub-unit (Siegel, G.J. and Desmond, T.J. (1989) J. Biol. Chem. 264, 4751-4754). We report that Na,K-ATPase purified from dog, guinea pig and rat kidney medulla or from rat brain, can yield two distinct bands when analyzed by SDS-PAGE or STS-PAGE, migrating between 85 and 105 kDa. An additional band migrating at 117 and 120 kDa appears often in enzyme purified from rat and guinea pig kidney medulla. The apparent molecular weights and relative intensities of these bands vary with temperature and duration of incubation during sample preparation. N-terminal sequencing and monospecific antibody probes revealed that the two distinct bands obtained from the kidney enzyme consist only of the alpha 1 isoform. The band appearing at 117-120 kDa also contains only the alpha 1 N-terminal sequence. In contrast, as reported earlier (Sweadner, K.J. (1979) J. Biol. Chem. 254, 6060-6067), the doublet seen in brain preparations consists of alpha 1 and alpha 2 or (alpha 2 + alpha 3). We conclude that monospecific antibody probes or N-terminal sequencing must be used to identify Na,K-ATPase isoforms by SDS- or STS-PAGE. In addition, gel conditions that may affect the mobilities of the isoforms are discussed.     

Effect of vasopressin administration and deficiency upon 3H-AVP binding sites in the CNS and periphery during development.

Arginine8-vasopressin (AVP, 40 micrograms/100 g b.wt., SC) was administered to male Long-Evans (LE) pups from day 1 to 7 of life and the pups were sacrificed on day 8 or 60. 3H-AVP binding was performed on membranes prepared from the liver, kidney, and septum. No significant changes were observed in the kidney or septum of animals 8 or 60 days old. However, the chronic AVP treatment did result in a significant increase in the density of 3H-AVP binding sites in the liver when compared to control day 8 pups (control 44 +/- 2 vs. AVP 56 +/- 3 fmol/mg protein), with no change in affinity. This effect was maintained into adulthood, as the day 60 AVP-treated LE rats also showed a significant increase in liver 3H-AVP binding sites compared to control (control 186 +/- 9 vs. AVP 239 +/- 14 fmol/mg protein), with no change in affinity. A comparison of 3H-AVP binding sites in 8-day-old LE, heterozygous Brattleboro (HET-BB), and homozygous Brattleboro rats (HOM-BB) was performed to assess the effect of complete (HOM-BB) and partial (HET-BB) VP deficiency on binding sites in the CNS and periphery. The liver again was the only tissue in which a change in 3H-AVP binding characteristics was noted. The HOM-BB rat (Bmax 144 +/- 6 fmol/mg protein) displayed a significant increase in AVP binding sites from the LE rat (Bmax 100 +/- 7 fmol/mg protein), while the 3H-AVP binding sites in the HET-BB rat liver (Bmax 69.8 +/- 9 fmol/mg protein) were significantly lower than LE rats. Thus hepatic AVP receptors appear most sensitive to the presence or absence of vasopressin during the early postnatal period.     

Renal medullary nitric oxide deficit of Dahl S rats enhances hypertensive actions of angiotensin II

Studies were designed to examine the hypothesis that the renal medulla of Dahl salt-sensitive (Dahl S) rats has a reduced capacity to generate nitric oxide (NO), which diminishes the ability to buffer against the chronic hypertensive effects of small elevations of circulating ANG II. NO synthase (NOS) activity in the outer medulla of Dahl S rats (arginine-citrulline conversion assay) was significantly reduced. This decrease in NOS activity was associated with the downregulation of protein expression of NOS I, NOS II, and NOS III isoforms in this region as determined by Western blot analysis. In anesthetized Dahl S rats, we observed that a low subpressor intravenous infusion of ANG II (5 ng. kg(-1). min(-1)) did not increase the concentration of NO in the renal medulla as measured by a microdialysis with oxyhemoglobin trapping technique. In contrast, ANG II produced a 38% increase in the concentration of NO (87 +/- 8 to 117 +/- 8 nmol/l) in the outer medulla of Brown-Norway (BN) rats. The same intravenous dose of ANG II reduced renal medullary blood flow as determined by laser-Doppler flowmetry in Dahl S, but not in BN rats. A 7-day intravenous ANG II infusion at a dose of 3 ng. kg(-1). min(-1) did not change mean arterial pressure (MAP) in the BN rats but increased MAP in Dahl S rats from 120 +/- 2 to 138 +/- 2 mmHg (P < 0.05). ANG II failed to increase MAP after NO substrate was provided by infusion of L-arginine (300 microg. kg(-1). min(-1)) into the renal medulla of Dahl S rats. Intravenous infusion of L-arginine at the same dose had no effect on the ANG II-induced hypertension. These results indicate that an impaired NO counterregulatory system in the outer medulla of Dahl S rats makes them more susceptible to the hypertensive actions of small elevations of ANG II.     

Calmodulin binding and protein phosphorylation in adrenal medulla coated vesicles

Coated vesicles from bovine adrenal medulla contained clathrin and major detergent-insoluble polypeptides of 120-100, 51 and 49 kDa. Intact coated vesicles and vesicles lacking clathrin light chains were bound by immobilized calmodulin in the presence of Ca2+. Clathrin in the form of 700 A cages was not bound. The calmodulin binding components in intact coated vesicles are therefore contributed by the enclosed vesicle or by the 120-100, 50 or 49 kDa polypeptides. The 51 kDa component incorporated 32Pi from labelled ATP by an endogenous kinase activity; no other coat or vesicle membrane protein was phosphorylated in vitro, either by intrinsic or exogenous kinases.     

Urea transporter expression in aging kidney and brain during dehydration

Aging is commonly associated with defective urine-concentrating ability. The present study examined how the kidney and the brain of senescent (30-mo-old) female WAG/Rij rats respond to dehydration induced by 2 days of water deprivation in terms of urea transporter (UT) regulation. In euhydrated situation, senescent rats exhibited similar vasopressin plasma level but lower urine osmolality and papillary urea concentration and markedly reduced kidney UT-A1, UT-A3, and UT-B1 abundances compared with adult (10-mo-old) rats. Senescent rats responded to dehydration similarly to adult rats by a sixfold increase in vasopressin plasma level. Their papillary urea concentration was doubled, without, however, attaining that of dehydrated adult rats. Such an enhanced papillary urea sequestration occurred with a great fall of both UT-A1 and UT-A3 abundances in the tip of inner medulla and an increased UT-A1 abundance in the base of inner medulla. UT-A2 and UT-B1 were unchanged. These data suggest that the inability of control and thirsted senescent rats to concentrate urine as much as their younger counterparts derives from lower papillary urea concentration. In aging brain, UT-B1 abundance was increased twofold together with a fourfold increase in aquaporin-4 abundance. Dehydration did not alter the abundance of these transporters.     

Immunolocalization of betaine aldehyde dehydrogenase in porcine kidney.

Polyclonal anti-BADH serum was raised in rabbits against native BADH purified from porcine kidney. The antiserum cross-reacted strongly with BADH purified from kidney, Amaranthus palmierii, and Pseudomona aeuroginosa (1:1000), and weakly with Amaranthus hypochondriacus L (1:100). Antibodies bound to purified native kidney BADH in immunoblots showed a major band of an apparent molecular mass of 340 kDa and a subunit with an apparent molecular mass of 52 kDa. Data on activity assays showed higher activity in cortex sections (81.3 nmol/min/mg protein) than in medulla sections (21.3 nmol/min/mg protein). Immunolocalization of BADH in kidney tissue sections showed that BADH is found in cortex and medulla. In inner medulla, the enzyme was mainly localized in cells surrounding the tubules. Western blot analysis on extracts from the cortex and medulla sections showed higher concentration of BADH protein in cortex than in medulla. These results were in accordance with immunolocalization and activity analysis.