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High fructose intake has been associated with increased incidences of renal disease and hypertension, among other pathologies. Most fructose is cleared by the portal system and metabolized in the liver; however, systemic levels of fructose can rise with increased consumption. We tested whether there were sex differences in the renal responses to a high-fructose diet in mice. Two-month-old male and female C57BL6/129/SV mice (n = 6 mice per sex per treatment) were randomized to receive control or high-fructose (65% by weight) diets as pelleted chow ad libitum for 3 mo. Fructose feeding did not significantly affect body weight but led to a 19% and 10% increase in kidney weight in male and female mice, respectively. In male mice, fructose increased the expression (∼50%) of renal cortical proteins involved in metabolism, including glucose transporter 5 (facilitative fructose transporter), ketohexokinase, and the insulin receptor (β-subunit). Female mice had lower basal levels of glucose transporter 5, which were unresponsive to fructose. However, female mice had increased urine volume and plasma K+ and decreased plasma Na+ with fructose, whereas male mice were less affected. Likewise, female mice showed a two- to threefold reduction in the expression Na+-K+-2Cl- cotransporter 2 in the thick ascending limb and aquaporin-2 in the collecting duct with fructose relative to female control mice, whereas male mice had no change. Overall, our results support greater proximal metabolism of fructose in male animals and greater distal tubule/collecting duct (electrolyte homeostasis) alterations in female animals. These sex differences may be important determinants of the specific nature of pathologies that develop in association with high fructose consumption. [ABSTRACT FROM AUTHOR]

Riazi S, Tiwari S, Sharma N, Rash A, Ecelbarger CM. Abundance of the Na-K-2C1 cotransporter NKCC2 is increased by high-fat feeding in Fischer 344 X Brown Norway (Fl) rats. Am J Physiol Renal Physiol 296: F762-F770, 2009. First published February 4, 2009; doi: 10.11 52/ajprenal.90484.2008.-Insulin resistance is associated with hypertension by mechanisms likely involving the kidney. To determine how the major apical sodium transporter of the thick ascending limb, the bumetanide-sensitive Na-K-2C1 cotransporter (NKCC2) is regulated by high-fat feeding, we treated young male, Fischer 344 X Brown Norway (F344BN) rats for 8 wk with diets containing either normal (NF, 4%) or high (HF, 36%) fat, by weight, primarily as lard. HF-fed rats had impaired glucose tolerance, increased urine excretion of 8-isoprostane (a marker of oxidative stress), increased protein levels for NKCC2 (50-125%) and the renal outer medullary potassium channel (106%), as well as increased natriuretic response to furosemide (20-40%). To test the role of oxidative stress in this response, in study 2, rats were fed the NF or HF diet plus plain drinking water, or water containing N[supG]-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor (100 mg/I), or tempol, a superoxide dismutase mimetic (I mmolll). The combination of tempol with HF nullified the increase in medullary NKCC2, while L-NAME with HF led to the highest expression of medullary NKCC2 (to 498% of NF mean). However, neither of these drugs dramatically affected the elevated natriuretic response to furosemide with HF. Finally, L-NAME led to a marked increase in blood pressure (measured by radiotelemetry), which was significantly enhanced with HF. Mean arterial blood pressure at 7 wk was as follows (mmHg): NF, 100 ± 2; NF plus L-NAME, 122 ± 3; and HF plus L-NAME, 131 ± 2. Overall, HF feeding increased the abundance of NKCC2. Inappropriately high sodium reabsorption in the thick ascending limb via NKCC2 may contribute to hypertension with insulin resistance. [ABSTRACT FROM AUTHOR]

BACKGROUND: The incidence of cardiovascular and renal disease is lower in premenopausal women than in aged-matched men. However, in the setting of diabetes mellitus (DM), this 'female advantage' no longer exists: the incidence and progression of DM and its associated end-organ complications are equal in men and women, regardless of age. We have recently reported that estrogen supplementation attenuates the progression of diabetic nephropathy, suggesting that lack of estrogen may nullify female sex as a protective factor against DM. OBJECTIVE: This study examined circulating levels of estradiol in DM and expression of estrogen receptor subtypes (ERa and ERP) in the nondiabetic (ND) and diabetic (D) kidney. METHODS: The study was performed in ND and streptozotocin-induced D Sprague-Dawley rats after 2 weeks (male and female) and 12 weeks (female) of DM. The animals (N = 8/group) were kept either intact, ovariectomized (OVX), or OVX with 17beta-estradiol (E(2)) supplementation (OVX + E(2), 5 mug/kg/d). Plasma estradiol levels were measured by enzyme-linked immunosorbent assay, and expression of renal ERalpha and ERbeta was measured by immunohistochemistry and Western blot analysis. RESULTS: DM was associated with reduced circulating estradiol levels (ND: mean [SEM] 37.1 [7.2]; D: 24.5 [9.3] pg/mL; P < 0.05). The diabetic kidney exhibited increased expression of ERalpha protein (ND: 0.82 [0.06]; D: 1.15 [0.09] arbitrary units; P < 0.05), but no differences in ERP were observed. This resulted in an overall increase in the ratio of ERalpha/ERbeta protein expression in the diabetic kidney. No differences in the expression of ERa were observed in either females or males with similar glycemic levels after 2 weeks of DM. CONCLUSIONS: Reduced circulating levels of estradiol and imbalance in the expression of estrogen receptor subtypes in the diabetic kidney may explain why female sex is no longer a protective factor in the setting of DM. Thus, estradiol supplementation may be an effective regimen in attenuating the onset and progression of diabetic renal complications. [ABSTRACT FROM AUTHOR]

Background: Cisplatin (CP) induced polyuria in rats is associated with a reduction in medullary hypertonicity, normally generated by the thick ascending limb (TAL) salt transporters, and the collecting duct urea transporters (UT). To investigate the molecular basis of this abnormality, we determined the protein abundance of major salt and UT isoforms in rat kidney during CP-induced polyuria.Methods: Male Sprague-Dawley rats received either a single injection of CP (5 mg/kg, N = 6) or saline (N = 6) intraperitoneally five days before sacrifice. Urine, blood, and kidneys were collected and analyzed.Results: CP-treated rats developed polyuric acute renal failure as assessed by increased blood urea nitrogen (BUN), urine volume and decreased urine osmolality. Western analysis of kidney homogenates revealed a marked reduction in band density of the bumetanide-sensitive Na-K-2Cl cotransporter in cortex (60% of control values, P < 0.05), but not in outer medulla (OM) (106% of control values). There were no differences in band densities for the renal outer medullary potassium channel (ROMK), the type III Na-H exchanger (NHE3), the alpha-subunit of Na,K-ATPase in the OM; or for UT-A1, UT-A2 or UT-A4 in outer or inner medulla. However, the band pattern of UT-A2 and UT-A4 proteins in the OM of CP-treated rats was different from the control rats, suggesting a qualitative modification of these proteins.Conclusions: Changes in the abundance of outer or inner medullary salt or urea transporters are unlikely to play a role in the CP-induced reduction in medullary hypertonicity. However, qualitative changes in UT proteins may affect their functionality and thus may have a role. [ABSTRACT FROM AUTHOR]

Vasopressin plays a role in both salt and water balance in the kidney. Classic studies, utilizing isolated perfused tubules, have revealed that vasopressin increases sodium reabsorption in the kidney thick ascending limb and the collecting duct. Furthermore, the activity of several sodium transport proteins expressed in these segments, such as the bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2) and the epithelial sodium channel (ENaC), have been shown to be directly increased by vasopressin. Increased protein abundance might be one means through which sodium transporter and channel activity is enhanced. We have used immunoblotting and immunohistochemistry in order to investigate the regulation of abundance of the major sodium transporters and channels expressed along the renal tubule in response to vasopressin. Chronic (7-day) studies were performed in which vasopressin levels were elevated either endogenously by water restriction of Sprague-Dawley rats or exogenously through infusion of the vasopressin V2-receptor-selective agonist, dDAVP (1-deamino-8d-arginine-vasopressin), to Brattleboro rats. We found a significant increase in protein abundance for NKCC2 and the beta- and gamma-subunits of ENaC with either water restriction or dDAVP infusion. The alpha-subunit of Na-K-ATPase was increased by water restriction, but not by dDAVP infusion, and alpha-ENaC and the thiazide-sensitive cotransporter (NCC) were increased by dDAVP infusion but not by water restriction. Acute (60-min) in vivo exposure to dDAVP led to an increase in both beta- and gamma-ENaC abundance in kidney cortex homogenates, displaying the rapid nature of some of these changes. Overall these increases in sodium transporter and channel abundances likely contribute to both the antidiuretic and antinatriuretic actions of vasopressin., (Copyright 2001 Academic Press.)

Renal sodium retention, as a result of increased abundance of sodium transporters, may play a role in the development and/or maintenance of the increased blood pressure in obesity. To address this hypothesis, we evaluated the relative abundances of renal sodium transporters in lean and obese Zucker rats at 2 and 4 mo of age by semiquantitative immunoblotting. Mean systolic blood pressure was higher in obese rats relative to lean at 3 mo, P < 0.02. Furthermore, circulating insulin levels were 6- or 13-fold higher in obese rats compared with lean at 2 or 4 mo of age, respectively. The abundances of the alpha(1)-subunit of Na-K-ATPase, the thiazide-sensitive Na-Cl cotransporter (NCC or TSC), and the beta-subunit of the epithelial sodium channel (ENaC) were all significantly increased in the obese rats' kidneys. There were no differences for the sodium hydrogen exchanger (NHE3), the bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2 or BSC1), the type II sodium-phosphate cotransporter (NaPi-2), or the alpha-subunit of ENaC. These selective increases could possibly increase sodium retention by the kidney and therefore could play a role in obesity-related hypertension.

UT-A1 is an extremely hydrophobic 929-amino acid integral membrane protein, expressed in the renal inner medullary collecting duct, with a central role in the urinary concentrating mechanism. Previous immunoblotting studies in rats have revealed that UT-A1 is present in kidney in 97- and 117-kDa monomeric forms and that the relative abundance of the two forms is altered by vasopressin treatment and other treatments that altered urinary inner medullary urea concentration. The present studies were carried out using protein chemistry techniques to determine the origin of the two forms. Peptide-directed polyclonal antibodies targeted to five sites along the polypeptide sequence from the NH2 to the COOH terminus labeled both forms, thus failing to demonstrate a significant deletion in the primary amino acid chain. The 97- and 117-kDa monomeric forms were both reduced to 88 kDa by deglycosylation with N-glycosidase F, indicating that a single polypeptide chain is glycosylated to two different extents. Studies using nonionic detergents for membrane solubilization or using homobifunctional cross-linkers demonstrated that UT-A1 exists as a 206-kDa protein complex in native kidney membranes. The mobility of this complex was also increased by deglycosylation. Both the 97- and 117-kDa proteins, as well as the 206-kDa complex, were immunoprecipitated with UT-A1 antibodies. We conclude that UT-A1 is a glycoprotein and that the two monomeric forms (97 and 117 kDa) in inner medullary collecting duct are the consequence of different states of glycosylation.

The V2 vasopressin receptor (V2R) plays a key role in the maintenance of a normal body water balance. To generate an in vivo model that allows the physiological and molecular analysis of the role of V2Rs in kidney function, we have created mouse lines that lack functional V2Rs by using targeted mutagenesis in mouse embryonic stem cells. Specifically, we introduced a nonsense mutation known to cause X-linked nephrogenic diabetes insipidus (XNDI) in humans (Glu242stop) into the mouse genome. V2R-deficient hemizygous male pups showed a decrease in basal urine osmolalities and were unable to concentrate their urine. These pups also exhibited an enlargement of renal pelvic space, failed to thrive, and died within the first week after birth due to hypernatremic dehydration. Interestingly, female mice heterozygous for the V2R mutation showed normal growth but displayed an XNDI-like phenotype, characterized by reduced urine concentrating ability of the kidney, polyuria, and polydipsia. Western blot analysis and immunoelectron microscopic studies showed that the loss of functional V2Rs had no significant effect on the basal expression levels of aquaporin-2 and the bumetanide-sensitive Na-K-2Cl cotransporter (BSC-1). The V2R mutant mice described here should serve as highly useful tools for the development of novel therapeutic strategies for the treatment of XNDI.

Sodium transport is increased by vasopressin in the cortical collecting ducts of rats and rabbits. Here we investigate, by quantitative immunoblotting, the effects of vasopressin on abundances of the epithelial sodium channel (ENaC) subunits (alpha, beta, and gamma) in rat kidney. Seven-day infusion of 1-deamino-[8-D-arginine]-vasopressin (dDAVP) to Brattleboro rats markedly increased whole kidney abundances of beta- and gamma-ENaC (to 238% and 288% of vehicle, respectively), whereas alpha-ENaC was more modestly, yet significantly, increased (to 142% of vehicle). Similarly, 7-day water restriction in Sprague-Dawley rats resulted in significantly increased abundances of beta- and gamma- but no significant change in alpha-ENaC. Acute administration of dDAVP (2 nmol) to Brattleboro rats resulted in modest, but significant, increases in abundance for all ENaC subunits, within 1 h. In conclusion, all three subunits of ENaC are upregulated by vasopressin with temporal and regional differences. These changes are too slow to play a major role in the short-term action of vasopressin to stimulate sodium reabsorption in the collecting duct. Long-term increases in ENaC abundance should add to the short-term regulatory mechanisms (undefined in this study) to enhance sodium transport in the renal collecting duct.

The heterotrimeric G protein G(s) is required for hormone-stimulated intracellular cAMP generation because it couples hormone receptors to the enzyme adenylyl cyclase. Hormones that activate G(s) in the kidney include parathyroid hormone, glucagon, calcitonin, and vasopressin. Recently, it has been demonstrated that the G(s)alpha gene is imprinted in a tissue-specific manner, leading to preferential expression of G(s)alpha from the maternal allele in some tissues. In the kidney, G(s)alpha is imprinted in the proximal tubule but not in more distal nephron segments, such as the thick ascending limb or collecting duct. This most likely explains why in both humans and mice heterozygous mutations in the maternal allele lead to parathyroid hormone resistance in the proximal tubule whereas mutations in the paternal allele do not. In contrast, heterozygous mutations have little effect on vasopressin action in the collecting ducts. In mice with heterozygous null G(s)alpha mutations (both those with mutations on the maternal or paternal allele), expression of the Na-K-2Cl cotransporter was decreased in the thick ascending limb, suggesting that its expression is regulated by cAMP. The G(s)alpha genes also generate alternative, oppositely imprinted transcripts encoding XLalphas, a G(s)alpha isoform with a long NH(2)-terminal extension, and NESP55, a chromogranin-like neurosecretory protein. The role, if any, of these proteins in renal physiology is unknown.

The renal urea transporter gene (UT-A) produces different transcripts in the inner medullary collecting ducts (UT-A1) and thin descending limbs of Henle's loop (UT-A2), coding for distinct proteins. Peptide-directed rabbit polyclonal antibodies were used to identify the UT-A2 protein in renal medulla of mouse and rat. In the inner stripe of outer medulla, an antibody directed to the COOH terminus of UT-A recognized a membrane protein of 55 kDa. The abundance of this 55-kDa protein was strongly increased in response to chronic infusion of the vasopressin analog 1-deamino-[8-D-arginine]vasopressin (DDAVP) in Brattleboro rats, consistent with previous evidence that UT-A2 mRNA abundance is markedly increased. Immunofluorescence labeling with the COOH-terminal antibody in Brattleboro rats revealed labeling in the lower portion of descending limbs from short-looped nephrons (in the aquaporin-1-negative portion of this segment). This UT-A labeling was increased in response to DDAVP. Increased labeling was also seen in descending limbs of long-looped nephrons in the base of the inner medulla. These results indicate that UT-A2 is expressed as a 55-kDa protein in portions of the thin descending limbs of Henle's loop and that the abundance of this protein is strongly upregulated by vasopressin.

Recent results indicate that renal escape from vasopressin-induced antidiuresis is accompanied by a marked downregulation of whole kidney aquaporin-2 (AQP-2) protein and mRNA expression. However, in those studies, the escaped animals were also markedly hypo-osmolar compared to controls as a result of water loading during antidiuresis. The present studies evaluated whether systemic or local osmolality contributes to the downregulation of AQP-2 expression in this model. In the first study, two groups of 1-deamino-[8-D-arginine]-vasopressin (dDAVP)-infused rats were water-loaded; after establishment of escape, one group was then water-restricted for 4 d to reverse the escape, whereas the other group continued daily water loading. Whole kidney AQP-2 protein was measured by Western blotting, and inner medulla AQP-2 mRNA was determined by Northern blotting. Results were compared to dDAVP-infused rats fed solid chow. After 4 d of water restriction, urine volume decreased to the same level as in the rats on solid chow; however, plasma sodium concentrations and plasma osmolality remained low. Despite maintenance of significant hypo-osmolality, rats in which escape was subsequently reversed by water restriction reestablished high dDAVP-stimulated kidney levels of AQP-2 after 4 d of water restriction. In the second study, AQP-2 expression was evaluated in different regions of kidneys from water-loaded rats undergoing escape from antidiuresis. Despite markedly different interstitial osmolalities, significant downregulation of AQP-2 expression compared to dDAVP-infused control rats was seen in the inner medulla, outer medulla, and cortex. Thus, neither systemic nor interstitial osmolality appears to appreciably be correlated with downregulation of kidney AQP-2 expression during escape from antidiuresis. These results therefore suggest that additional vasopressin- and osmolality-independent factors, likely related to the effects of extracellular fluid volume expansion, also regulate kidney AQP-2 expression in rats.

Tubuloglomerular feedback (TGF) stabilizes nephron function by causing changes in single-nephron GFR (SNGFR) to compensate for changes in late proximal flow (VLP). TGF responds within seconds and reacts over a narrow range of VLP that surrounds normal VLP. To accommodate sustained increases in VLP, TGF must reset around the new flow. We studied TGF resetting by inhibiting proximal reabsorption with benzolamide (BNZ; administered repeatedly over a 24-hour period) in Wistar-Froemter rats. BNZ acutely activates TGF, thereby reducing SNGFR. Micropuncture was performed 6-10 hours after the fourth BNZ dose, when diuresis had subsided. BNZ caused glomerular hyperfiltration, which was prevented with inhibitors of macula densa nitric oxide synthase (NOS). Because of hyperfiltration, BNZ increased VLP and distal flow, but did not affect the basal TGF stimulus (early distal salt concentration). BNZ slightly blunted normalized maximum TGF response and the basal state of TGF activation. BNZ sensitized SNGFR to reduction by S-methyl-thiocitrulline (SMTC) and caused the maximum TGF response to be strengthened by SMTC. Sensitization to type I NOS (NOS-I) blockers correlated with increased macula densa NOS-I immunoreactivity. Tubular transport measurements confirmed that BNZ affected TGF within the juxtaglomerular apparatus. During reduced proximal reabsorption, TGF resets to accommodate increased flow and SNGFR through a mechanism involving macula densa NOS.

Cyclooxygenase inhibitors, such as indomethacin and diclofenac, have well-described effects to enhance renal water reabsorption and urinary concentrating ability. Concentrating ability is regulated in part at the level of the thick ascending limb of Henle's loop, where active NaCl absorption drives the countercurrent multiplication mechanism. We used semiquantitative immunoblotting to test the effects of indomethacin and diclofenac, given over a 48-h period, on the expression levels of the ion transporters responsible for active NaCl transport in the thick ascending limb. Both agents strongly increased the expression level of the apical Na-K-2Cl cotransporter in both outer medulla and cortex. Neither agent significantly altered outer medullary expression levels of other thick ascending limb proteins, namely, the type 3 Na/H exchanger (NHE-3), Tamm-Horsfall protein, or alpha1- or beta1-subunits of the Na-K-ATPase. Administration of the EP3-selective PGE(2) analog, misoprostol, to indomethacin-treated rats reversed the stimulatory effect of indomethacin on Na-K-2Cl cotransporter expression. We conclude that cyclooxygenase inhibitors enhance urinary concentrating ability in part through effects to increase Na-K-2Cl cotransporter expression in the thick ascending limb of Henle's loop. This action is most likely due to elimination of an EP3-receptor-mediated tonic inhibitory effect of PGE(2) on cAMP production.

Transport processes along the nephron are regulated in part by hormone stimulation of adenylyl cyclases mediated by the heterotrimeric G protein G(s). To assess the role of this pathway in the regulation of Na-K-2Cl cotransporter abundance in the renal thick ascending limb (TAL), we studied mice with heterozygous disruption of the Gnas gene, which codes for the alpha-subunit of G(s). Outer medullary G(s)alpha protein abundance (as assessed by semiquantitative immunoblotting) and glucagon-stimulated cAMP production were significantly reduced in the heterozygous G(s)alpha knockout mice (GSKO) relative to their wild-type (WT) littermates. Furthermore, Na-K-2Cl cotransporter protein abundance in the outer medulla was significantly reduced (band density, 48% of WT). In addition, GSKO mice had a significantly reduced (72% of WT) urinary osmolality in response to a single injection of 1-deamino-[8-D-arginine]vasopressin (DDAVP), a vasopressin analog. In contrast, outer medullary protein expression of the type 3 Na/H exchanger (NHE-3) or Tamm-Horsfall protein did not differ between the GSKO mice and their WT littermates. However, abundance of type VI adenylyl cyclase was markedly decreased in the outer medullas of GSKO mice, suggesting a novel feed-forward regulatory mechanism. We conclude that expression of the Na-K-2Cl cotransporter of the TAL is dependent on G(s)alpha-mediated hormone stimulation, most likely due to long-term changes in cellular cAMP levels.

In the renal inner medullary collecting duct (IMCD), vasopressin regulates two key transporters, namely aquaporin-2 (AQP2) and the vasopressin-regulated urea transporter (VRUT). Both are present in intracellular vesicles as well as the apical plasma membrane. Short-term regulation of AQP2 has been demonstrated to occur by vasopressin-induced trafficking of AQP2-containing vesicles to the apical plasma membrane. Here, we have carried out studies to determine whether short-term regulation of VRUT occurs by a similar process. Cell surface labeling with NHS-LC-biotin in rat IMCD suspensions revealed that vasopressin causes a dose-dependent increase in the amount of AQP2 labeled at the cell surface, whereas VRUT labeled at the cell surface did not increase in response to vasopressin. Immunoperoxidase labeling of inner medullary thin sections from Brattleboro rats treated with 1-desamino-8-D-arginine vasopressin (DDAVP) for 20 min revealed dramatic translocation of AQP2 to the apical region of the cell, with no change in the cellular distribution of VRUT. In addition, differential centrifugation of inner medullary homogenates from Brattleboro rats treated with DDAVP for 60 min revealed a marked depletion of AQP2 from the low-density membrane fraction (enriched in intracellular vesicles) but did not alter the quantity of VRUT in this fraction. Finally, AQP2-containing vesicles immunoisolated from a low-density membrane fraction from renal inner medulla did not contain immunoreactive VRUT. Thus vasopressin-mediated regulation of AQP2, but not of VRUT, depends on regulated vesicular trafficking to the plasma membrane.

To investigate whether the enhancement of thick ascending limb (TAL) NaCl transport in response to long-term increases in circulating vasopressin concentration is associated with increased expression levels of the apical Na-K-2Cl cotransporter in the rat TAL, we have carried out immunoblotting and immunofluorescence studies using affinity-purified, peptide-directed antibodies. Semiquantitative immunoblotting studies demonstrated a marked increase (193% of controls) in Na-K-2Cl cotransporter band density in response to restriction of water intake to 15 ml/day for 7 days. In contrast, the expression levels of two other apical proteins of the TAL (the type 3 Na/H exchanger and Tamm-Horsfall protein) were unchanged in the outer medulla. A 7-day subcutaneous infusion of the V2 receptor-selective vasopressin analog, 1-desamino-[8-D-arginine]vasopressin (DDAVP), to Brattleboro rats also markedly increased Na-K-2Cl cotransporter expression in the outer medulla (183% of controls). Immunofluorescence localization in outer medullary tissue sections confirmed the increase in Na-K-2Cl cotransporter expression in response to DDAVP. We conclude that vasopressin strongly upregulates the expression of the Na-K-2Cl cotransporter of the TAL and that it is likely to play an important role in the long-term regulation of the countercurrent multiplication system.

A bumetanide-sensitive Na-K-2Cl cotransporter, BSC-1, is believed to mediate the apical component of transcellular NaCl absorption in the thick ascending limb (TAL) of Henle's loop. To study its ultrastructural localization in kidney, we used an affinity-purified, peptide-derived polyclonal antibody against rat BSC-1. Immunoblots from rat kidney cortex and outer medulla revealed a solitary 161-kDa band in membrane fractions. Immunocytochemistry of 1-micrometer cryosections demonstrated strong BSC-1 labeling of the apical and subapical regions of medullary and cortical TAL cells. Notably, macula densa cells also exhibited distinct labeling. Distal convoluted tubules and other renal tubule segments were unlabeled. Immunoelectron microscopy demonstrated that BSC-1 labeling was associated with the apical plasma membrane and with subapical intracellular vesicles in medullary and cortical TAL and in macula densa cells. Smooth-surfaced TAL cells, in particular, had extensive BSC-1 labeling of intracellular vesicles. These results support the view that BSC-1 provides the apical pathway for NaCl transport across the TAL and that an extensive intracellular reservoir of BSC-1 is present in a subpopulation of TAL cells. Furthermore, the BSC-1 localization in the apical plasma membrane of macula densa cells is consistent with its proposed role in tubuloglomerular feedback.

Background: Several pathophysiological states associated with deranged water balance are associated with altered expression and/or intracellular distribution of aquaporin water channels. The possible role of dysregulation of thick ascending limb NaCl transporters, which are responsible for countercurrent multiplication in the kidney, has not been evaluated., Methods: Semiquantitative immunoblotting and immunocytochemistry were carried out in the kidneys of rat with adriamycin-induced nephrotic syndrome and in vehicle-injected control rats., Results: Preliminary studies confirmed the presence of a severe concentrating defect. Semiquantitative immunoblotting of outer medullary homogenates demonstrated a marked decrease in the abundance of three thick ascending limb Na+ transporters in nephrotic rats, namely the bumetanide-sensitive Na-K-2Cl cotransporter (BSC-1), the type 3 Na/H exchanger (NHE-3), and the alpha 1-subunit of the Na-K-ATPase. These results are predictive of a decrease in the NaCl transport capacity of the medullary thick ascending limb and therefore a decrease in countercurrent multiplication. Immunocytochemistry of outer medullary thin sections demonstrated broad (but highly variable) suppression of BSC-1 expression in the outer medullas of adriamycin-nephrotic rats. There was also a large decrease in outer medullary expression of two collecting duct water channels (aquaporin-2 and -3) and the major water channel of the thin descending limb of Henle's loop (aquaporin-1)., Conclusion: The concentrating defect in adriamycin-induced nephrotic syndrome in rats is a consequence of multiple defects in water and solute transporter expression, which would alter both the generation of medullary interstitial hypertonicity and osmotic equilibration in the collecting duct. Whether a similar widespread defect in transporter expression is present in idiopathic nephrotic syndrome is, at this point, untested.

Previously, we demonstrated that escape from vasopressin-induced antidiuresis ("vasopressin escape") in rats is associated with a large, selective decrease in whole kidney expression of aquaporin-2, the vasopressin-regulated water channel. Here, we show that isolated perfused inner medullary collecting ducts (IMCDs) from vasopressin-escape rats desamino-[D-arginine]vasopressin (DDAVP)/water-loaded have dramatically reduced vasopressin-dependent osmotic water permeabilities [46% of control rats (DDAVP alone)], which coincides with a fall in inner medullary aquaporin-2 protein abundance as measured by immunoblotting in the opposite kidney. Furthermore, we demonstrate in IMCD suspensions that cAMP accumulation in response to DDAVP is substantially reduced in the vasopressin-escape rats both in the presence and absence of the phosphodiesterase inhibitor IBMX. By immunoblotting, we show that the abundance of two proteins important in cAMP generation: the stimulatory heterotrimeric G protein subunit Gs and adenylyl cyclase type VI, do not change. We conclude that vasopressin escape is associated with relative vasopressin resistance of the collecting duct cells manifested by decreased intracellular cAMP levels. The decreased cAMP levels can contribute to the demonstrated decrease in collecting duct water permeability in two ways: 1) by causing a decrease in aquaporin-2 expression and 2) by limiting the acute action of vasopressin to increase collecting duct water permeability.

Nephrotic syndrome is associated with abnormal regulation of renal water excretion. To investigate the role of collecting duct water channels and solute transporters in this process, we have carried out semiquantitative immunoblotting of kidney tissues from rats with adriamycin-induced nephrotic syndrome. These experiments demonstrated that adriamycin-induced nephrotic syndrome is associated with marked decreases in expression of aquaporin-2, aquaporin-3, aquaporin-4, and the vasopressin-regulated urea transporter in renal inner medulla, indicative of a suppression of the capacity for water and urea absorption by the inner medullary collecting duct. In contrast, expression of the alpha(1)-subunit of the Na,K-ATPase in the inner medulla was unaltered. Light and electron microscopy of perfusion-fixed kidneys demonstrated that the collecting ducts are morphologically normal and unobstructed. Inner medullary expression of the descending limb water channel, aquaporin-1, was not significantly altered, pointing to a selective effect on the collecting duct. Aquaporin-2 and aquaporin-3 expression was also markedly diminished in the renal cortex, indicating that the effect is not limited to the inner medullary collecting duct. Differential centrifugation studies and immunocytochemistry in inner medullary thin sections demonstrated increased targeting of aquaporin-2 to the plasma membrane, consistent with the expected short-term action of vasopressin on aquaporin-2 trafficking. The extensive down-regulation of aquaporin and urea transporter expression may represent an appropriate renal response to the extracellular volume expansion observed in nephrotic syndrome, but may occur at the expense of decreased urinary concentrating and diluting capacity.

In animal models of the syndrome of inappropriate antidiuresis (SIADH), sustained administration of vasopressin and water results in free-water retention and progressive hyponatremia for several days, which is then followed by escape from the vasopressin-induced antidiuresis. With the onset of vasopressin escape, water excretion increases despite sustained administration of vasopressin, allowing water balance to be re-established and the serum sodium to be stabilized at a steady, albeit decreased, level. Studies from our laboratories have investigated whether this escape phenomenon can be attributed to altered regulation of aquaporin water channels. After four-day pre-treatment with 1-deamino-[8-D-arginine]-vasopressin (dDAVP) by osmotic minipump, rats were divided into control (continued dDAVP) and water-loaded (continued dDAVP plus a daily oral water load) groups. A significant increase in urine volume in the water-loaded rats was observed by the second day of water loading, indicating escape from antidiuresis. The onset of escape coincided temporally with a marked decrease in renal aquaporin-2 protein (measured by semi-quantitative immunoblotting), which began at day 2 and fell to 17% of control levels by day 3. In contrast, there was no decrease in the renal expression of aquaporins 1, 3, or 4. The marked suppression of whole kidney aquaporin-2 protein was accompanied by a concomitant suppression of whole kidney aquaporin-2 mRNA levels. Immunocytochemical localization and differential centrifugation studies demonstrated that trafficking of aquaporin-2 to the plasma membrane remained intact during vasopressin escape. Additional studies have indicated that the observed down-regulation of aquaporin-2 expression also occurs in the renal cortex as well as the inner and outer medullas, and can be reversed simply by water restriction despite maintenance of hyponatremia. Our results therefore suggest that escape from vasopressin-induced antidiuresis is attributable, at least in part, to a vasopressin-independent and osmolality-independent decrease in aquaporin-2 water channel expression in the renal collecting duct. Similar mechanisms likely contribute to the phenomenon of escape from antidiuresis seen clinically in patients with SIADH as well.

Aquaporins (AQPs) are a newly recognized family of transmembrane proteins that function as molecular water channels. At least four aquaporins are expressed in the kidney where they mediate rapid water transport across water-permeable epithelia and play critical roles in urinary concentrating and diluting processes. AQP1 is constitutively expressed at extremely high levels in the proximal tubule and descending limb of Henle's loop. AQP2, -3 and -4 are expressed predominantly in the collecting duct system. AQP2 is the predominant water channel in the apical plasma membrane and AQP3 and -4 are found in the basolateral plasma membrane. Short-term regulation of collecting duct water permeability by vasopressin is largely a consequence of regulated trafficking of AQP2-containing vesicles to and from the apical plasma membrane.

Four studies were conducted to examine the effect of dietary citrate and calcium and modest reductions in kidney function on aluminum utilization in rats. Ingestion of citrate increased retention of aluminum in bones of rats fed 1 mg Al/g diet and increased apparent absorption of zinc. The increased retention of aluminum was not linearly related to dietary citrate levels. These data suggest that citrate had a general effect on the solubility of trace elements in the gut that promoted absorption. When dietary calcium intake was increased from 67 to 250 mumol/g diet, aluminum concentrations in bone were reduced without a change in growth of rats. A reduction (approximately 30%) in kidney function, which was insufficient to alter growth, increased aluminum retention in bone by 34% in rats injected with aluminum and by 13% in rats fed aluminum. Rats fed aluminum seemed to retain in tissues only 0.01 to 0.05% as much aluminum as those injected with aluminum. Thus, tissue concentrations of aluminum, and presumably toxicity, can be altered by moderate changes in diet and kidney function even though overall retention of orally administered aluminum is extremely low.