Sex differences in renal nitric oxide synthase, NAD(P)H oxidase, and blood pressure in obese Zucker rats.

BACKGROUND: By increasing renal oxidative stress, obesity may alter the protective effect of female sex on blood pressure (BP). OBJECTIVES: The aim of this study was to determine whether female rats had altered expression and activity of renal nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] oxidase and nitric oxide synthase (NOS), enzymes important in superoxide and nitric oxide generation, respectively, and whether this relationship was altered in obesity. METHODS: Male and female, lean and obese Zucker rats were fed progressively higher levels of NaCl over 54 days while BP was measured by radiotelemetry. Kidneys were harvested after euthanization. RESULTS: A total of 32 (n=8/body type/sex) Zucker rats were examined. On a high-salt diet (4% NaC1), male and obese rats had significantly higher mean arterial blood pressure relative to female and lean rats (mm Hg: lean male=108, lean female=99, obese male=129, and obese female=123) and reduced renal cortical NOS activity (determined by 2-way analysis of variance; P<0.05 for sex and body type). Immunoblotting revealed that cortical endothelial NOS protein abundance was reduced in obese but not in male rats. Surprisingly, lean female rats had the highest outer medullary protein levels of several NADPH oxidase subunits, including gp91phox, p47phox, and p67phox (% of lean male: 207, 196, and 151, respectively; P<0.01 for all). However, renal NADPH activity was not increased in lean females, but was significantly increased in obese rats of both sexes (P<0.05). CONCLUSIONS: High-NaCl diet increased BP modestly in obese females, but not at all in lean females, suggesting some loss of protection with obesity in female rats. Reduced cortical NOS activity (both in male and obese rats) and/or increased NADPH oxidase activity (obese rats) may have contributed to increased salt sensitivity of BP.

Reduced expression of insulin receptors in the kidneys of insulin-resistant rats.

Insulin resistance is accompanied by hyperinsulinemia and activation of the renin-angiotensin system, both of which are associated with hypertension. Because the kidney plays a major role in the regulation of blood pressure, we studied the regulation of insulin receptor expression in the kidney during states of insulin resistance. Using two rat models of insulin resistance, Western blot analysis demonstrated a significant reduction in the expression of insulin receptor subunits in the kidney compared to lean control rats. Treatment of insulin resistance in Zucker rats with the insulin-sensitizing drug rosiglitazone partially restored renal insulin receptor levels. Conversely, treatment with the angiotensin II type 1 receptor (AT1) antagonist candesartan increased renal insulin receptor expression compared to untreated rats. Streptozotocin-induced hyperglycemia, which results from hypoinsulinemia, reduced expression of renal insulin receptors. Hyperinsulinemia induced by insulin infusion, however, did not produce a similar effect. In conclusion, insulin receptors are downregulated in the kidneys of insulin resistant rats, possibly mediated by hyperglycemia and angiotensin II.

Insulin's impact on renal sodium transport and blood pressure in health, obesity, and diabetes.

Insulin has been shown to have antinatriuretic actions in humans and animal models. Moreover, endogenous hyperinsulinemia and insulin infusion have been correlated to increased blood pressure in some models. In this review, we present the current state of understanding with regard to the regulation of the major renal sodium transporters by insulin in the kidney. Several groups, using primarily cell culture, have demonstrated that insulin can directly increase activity of the epithelial sodium channel, the sodium-phosphate cotransporter, the sodium-hydrogen exchanger type III, and Na-K-ATPase. We and others have demonstrated alterations in the expression at the protein level of many of these same proteins with insulin infusion or in hyperinsulinemic models. We also discuss how this regulation is perturbed in type I and type II diabetes mellitus. Finally, we discuss a potential role for regulation of insulin receptor signaling in the kidney in contributing to sodium balance and blood pressure.

Trafficking of ENaC subunits in response to acute insulin in mouse kidney.

Studies done in cell culture have demonstrated that insulin activates the epithelial sodium channel (ENaC) via a variety of mechanisms. However, to date, upregulation of ENaC in native renal tissue by in vivo administration of insulin has not been demonstrated. To address this, we injected 6-mo-old male C57BL/CBA mice (n = 14/group) intraperitoneally with vehicle or 0.5 U/kg body wt insulin and examined short-term (1-2 h) sodium excretion and kidney ENaC subunits (alpha, beta, and gamma) and serum and glucocorticoid-induced kinase (SGK-1) regulation. Insulin resulted in a significant reduction in urine sodium (by approximately 80%) that was restored by intraperitoneal administration of the ENaC antagonist, benzamil (1.4 mg/kg body wt). Differential centrifugation followed by Western blotting of whole kidney revealed significantly increased band densities (by 26-103%) for insulin- relative to vehicle-treated mice for alpha- and gamma-ENaC in the homogenate (H), and plasma membrane-enriched fraction (MF), with no difference in the vesicle-enriched fraction (VF). Similarly, beta-ENaC was significantly increased in MF (by 45%) but no change in the H. It was, however, significantly decreased in the VF (by 28%) with insulin. In agreement, immunoperoxidase labeling demonstrated relatively stronger apical, relative to cytosolic, localization of alpha-, beta-, and gamma-ENaC with insulin, whereas, with vehicle, labeling was fairly evenly dispersed throughout collecting duct principal cells. Furthermore, Western blotting showed insulin increased SGK-1 (by 75%) and phosphorylated-SGK band densities (by 30%) but only in the MF. These studies demonstrate novel in vivo regulation of renal ENaC activity and subunit proteins and SGK-1 by insulin in the acute time frame in the mouse.

Rosiglitazone regulates ENaC and Na-K-2Cl cotransporter (NKCC2) abundance in the obese Zucker rat.

BACKGROUND/AIMS: Progressive diabetes is associated renal remodeling, which we previously showed correlated to reduced protein abundance of several major renal sodium transporters and channel subunits in the obese Zucker rat. Here we test whether rosiglitazone (RGZ), a peroxisome proliferator-activated subtype gamma receptor agonist, would be protective and attenuate these changes. METHODS: Male, obese and lean Zucker rats (9 weeks old) were randomly divided (n = 6/group) to receive control diet with or without RGZ at 3 mg/kg.bw/day for 12 weeks. RESULTS: RGZ normalized blood glucose and plasma fructosamine levels in obese rats. Obese control rats had relatively increased fractional excretion of sodium (FE(Na), sodium excretion relative to creatinine). Nonetheless, both obese and RGZ-treated rats had relatively higher 24-hour net sodium balances. Immunoblotting revealed obese rats had significantly reduced renal cortical protein abundances of the bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2) and the sodium hydrogen exchanger (NHE3). RGZ normalized NKCC2 abundance and increased the abundance of the alpha-subunit of the epithelial sodium channel (ENaC). In contrast, in the outer medulla, obese rats had increased abundance of NKCC2, gamma-ENaC (85-kDa), and endothelial NOS. Furthermore, RGZ caused a decrease in the abundance of cortical beta- and gamma-ENaC (85-kDa). Finally, the whole kidney abundances of alpha-1 Na-K-ATPase, alpha- beta-, and gamma-ENaC (70-kDa band) positively correlated with net sodium balance, whereas NKCC2 was negatively correlated to FE(Na). CONCLUSION: Chronic RGZ treatment of obese Zucker rats may preserve renal sodium reabsorptive capacity by its indirect actions to attenuate hyperglycemia as well as direct effects on transporter abundance and activity.

Sodium transporters in the distal nephron and disease implications.

The post-macula densa segments of the renal tubule--that is, the distal convoluted tubule, connecting tubule, and collecting duct--play a central role in determining final urine sodium excretion. The major regulated sodium transporters and channels in these cell types include the thiazide-sensitive (Na-Cl) cotransporter (NCC), the epithelial sodium channel (ENaC), and Na-K-ATPase. Furthermore, although not involved in sodium reabsorption, the anion exchanger, pendrin, and the basolateral bumetanide-sensitive Na-K-2Cl cotransporter (NKCC1 or BSC2) have roles in blood-volume maintenance. Mutations in several of these major sodium transporters, channel subunits, and their regulatory proteins have been linked to human diseases such as Liddle's syndrome, Gitelman's syndrome, and Gordon's syndrome, emphasizing the need for appropriate regulation of sodium at these sites for maintenance of sodium balance and normotension.

Aldosterone infusion with high-NaCl diet increases blood pressure in obese but not lean Zucker rats.

Insulin-resistant, obese Zucker rats have blunted pressure natriuresis and are mildly hypertensive. This may involve inappropriate regulation of the renin-angiotensin-aldosterone system. To evaluate mechanisms underlying this defect, we employed the model of aldosterone escape. Male lean (L) and obese (O) Zucker rats were infused with aldosterone (2.8 mug/g body wt(3/4)) via osmotic minipump while being fed a 0.02% NaCl diet (LS). After 4 days, six rats of each type were switched to a high-NaCl (HS) diet (4%) for 4 additional days. Mean arterial blood pressure measured by radiotelemetry was significantly increased by the HS diet only in obese rats (final mean mmHg): 104 (LLS), 99 (LHS), 103 (OLS), and 115 (OHS). Obese rats had relatively increased renal cortical abundance of the bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2) and whole kidney alpha- and beta-ENaC (epithelial sodium channel) relative to lean rats. However, band density for the thiazide-sensitive (Na-Cl) cotransporter (NCC) was similarly reduced by HS in lean and obese rats ( approximately 50%). Obese rats had relatively reduced creatinine clearances and plasma renin activities, effects exacerbated by HS. Furthermore, HS resulted in a 129% increase in urinary nitrates plus nitrites excretion in lean rats and led to, in contrast, a 46% reduction in obese rats. Plasma sodium and potassium concentrations were increased by HS in obese but not lean rats. Thus we demonstrate an impaired response to aldosterone infusion in obese relative to lean Zucker rats. This impairment may involve increased sodium reabsorption via NKCC2 or ENaC, decreased glomerular filtration rate, and/or nitric oxide bioavailability.

Increased renal alpha-ENaC and NCC abundance and elevated blood pressure are independent of hyperaldosteronism in vasopressin escape.

Previously, we demonstrated that rats undergoing vasopressin escape had increased mean arterial blood pressure (MAP), plasma and urine aldosterone, and increased renal protein abundance of the alpha-subunit of the epithelial sodium channel (ENaC), the thiazide-sensitive Na-Cl cotransporter (NCC), and the 70-kDa band of gamma-ENaC (Song J, Hu X, Khan O, Tian Y, Verbalis JG, and Ecelbarger CA. Am J Physiol Renal Physiol 287: F1076-F1083, 2004; Ecelbarger CA, Knepper MA, and Verbalis JG. J Am Soc Nephrol 12: 207-217, 2001). Here, we determine whether changes in these renal proteins and MAP require elevated aldosterone levels. We performed adrenalectomies (ADX) or sham surgeries on male Sprague-Dawley rats. Corticosterone and aldosterone were replaced to clamp these hormone levels. MAP was monitored by radiotelemetry. Rats were infused with 1-deamino-[8-D-arginine]-vasopressin (dDAVP) via osmotic minipumps (5 ng/h). At day 3 of dDAVP infusion, seven rats in each group were offered a liquid diet [water load (WL)] or continued on a solid diet (SD). Plasma aldosterone and corticosterone and urine aldosterone were increased by WL in sham rats. ADX-WL rats escaped, as assessed by early natriuresis followed by diuresis; however, urine volume and natriuresis were somewhat blunted. WL did not reduce the abundance or activity of 11-beta-hydroxsteroid dehydrogenase type 2. Furthermore, the previously observed increase in renal aldosterone-sensitive proteins and escape-associated increased MAP persisted in clamped rats. The densitometry of immunoblots for NCC, alpha- and gamma-70 kDa ENaC, respectively, were (% sham-SD): sham-WL, 159, 278, 233; ADX-SD, 69, 212, 171; ADX-WL, 116, 302, 161. However, clamping corticosteroids blunted the rise at least for NCC and gamma-ENaC (70 kDa). Overall, the increase in aldosterone observed in vasopressin escape is not necessary for the increased expression of NCC, alpha- or gamma-ENaC or increased MAP associated with "escape."

Regulation of blood pressure, the epithelial sodium channel (ENaC), and other key renal sodium transporters by chronic insulin infusion in rats.

Hyperinsulinemia is associated with hypertension. Dysregulation of renal distal tubule sodium reabsorption may play a role. We evaluated the regulation of the epithelial sodium channel (ENaC) and the thiazide-sensitive Na-Cl cotransporter (NCC) during chronic hyperinsulinemia in rats and correlated these changes to blood pressure as determined by radiotelemetry. Male Sprague-Dawley rats ( approximately 270 g) underwent one of the following three treatments for 4 wk (n = 6/group): 1) control; 2) insulin-infused plus 20% dextrose in drinking water; or 3) glucose water-drinking (20% dextrose in water). Mean arterial pressures were increased by insulin and glucose (mmHg at 3 wk): 98 +/- 1 (control), 107 +/- 2 (insulin), and 109 +/- 3 (glucose), P < 0.01. Insulin (but not glucose) increased natriuretic response to benzamil (ENaC inhibitor) and hydrochlorothiazide (NCC inhibitor) on average by 125 and 60%, respectively, relative to control rats, suggesting increased activity of these reabsorptive pathways. Neither insulin nor glucose affected the renal protein abundances of NCC or the ENaC subunits (alpha, beta, and gamma) in kidney cortex, outer medulla, or inner medulla in a major way, as determined by immunoblotting. However, insulin and to some extent glucose increased apical localization of these subunits in cortical collecting duct principal cells, as determined by immunoperoxidase labeling. In addition, insulin decreased cortical "with no lysine" kinase (WNK4) abundance (by 16% relative to control), which may have increased NCC activity. Overall, insulin infusion increased blood pressure, and NCC and ENaC activity in rats. Increased apical targeting of ENaC and decreased WNK4 expression may be involved.

Sex and body-type interactions in the regulation of renal sodium transporter levels, urinary excretion, and activity in lean and obese Zucker rats.

BACKGROUND: Female humans and rodents are relatively protected against the development of hypertension and renal disease. Whether this protection is modified during insulin resistance and obesity, however, is not known. OBJECTIVE: Because renal sodium reabsorption has a central role in determining blood pressure, we hypothesized that lean female rats would bave reduced renal expression, activity, and urinary excretion of 8 major sodium transporters/channels. METHODS: Lean and obese, male and female Zucker rats (n = 4-8 per group) were fed progressively higher levels of dietary NaCl over a period of 54 days. Urinary excretion of renal sodium transport proteins was determined for 3 different dietary levels (0.04%, 0.4%, and 4%) of NaCl. With the high-NaCl diet, natriuretic responses to benzamil, furosemide, and thiazide were used as in vivo markers for activity of the epithelial sodium channel (ENaC), the bumetanide-sensitive Na-K-2C1 cotransporter (NKCC2), and the thiazide-sensitive NaCl cotransporter (NCC), respectively. RESULTS: Female rats (of both body types) had lower plasma renin activity and insulin levels than their male counterparts. Likewise, immunoblotting revealed female rats had increased whole kidney abundance of NCC and of the alpha, beta, and gamma subunits of ENaC, as well as decreased abundance of the type 3 sodium hydrogen exchanger (NHE3), type 2 sodium phosphate cotransporter (NaPi-2), and alpha-1 sodium-potassium-adenosine triphosphatase (Na-K-ATPase), compared with males. Obese rats had reduced levels of NKCC2, NHE3, and gamma-ENaC, but higher levels of NaPi-2 and NCC. Urine excretion of sodium transporters in lean female rats was nearly undetectable, whereas obese rats of both sexes excreted markedly more NKCC2 and NCC, which agreed with greater natriuretic responses to thiazide and furosemide. CONCLUSIONS: Obese female rats are similar to lean female rats with regard to the sex-distinct pattern of renal sodium transporters. However, obese female rats are more like obese male rats with regard to increased natriuretic response tofurosemide and thiazide, and to urine excretion of several transporters including NCC. Our results suggest that, with obesity, there is some loss of the protective female advantage.

Regulation of the renal thiazide-sensitive Na-Cl cotransporter, blood pressure, and natriuresis in obese Zucker rats treated with rosiglitazone.

Previously, we showed an increase in protein abundance of the renal thiazide-sensitive Na-Cl cotransporter (NCC) in young, prediabetic, obese Zucker rats relative to lean age mates (Bickel CA, Verbalis JF, Knepper MA, and Ecelbarger CA. Am J Physiol Renal Physiol 281: F639-F648, 2001). To test whether this increase correlated with increased thiazide sensitivity (NCC activity) and blood pressure, and could be modified by insulin-sensitizing agents, we treated lean and obese Zucker rats (9 wk old) with either a control diet or this diet supplemented with 3 mg/kg body wt rosiglitazone (RGZ), a peroxisomal proliferator-activated receptor subtype gamma agonist and potent insulin-sensitizing agent, for 12 wk (n = 9/group). The rise in blood pressure, measured continuously by radiotelemetry, was significantly blunted in the RGZ-treated obese rats. Similarly, blood glucose and urinary albumin were markedly decreased in these rats. RGZ-treated rats whether lean or obese excreted a NaCl load faster but excreted less sodium in response to hydrochlorothiazide, applied as a novel in vivo measure of NCC activity. Obese rats had increased renal protein abundance and urinary excretion of NCC; however, this was not significantly reduced by RGZ (densitometry in cortex homogenate - %lean control): 100 +/- 9, 93 +/- 4, 124 +/- 9, and 141 +/- 14 for lean control, lean RGZ, obese control, and obese RGZ, respectively. Subcellular localization, as evaluated by confocal microscopy and immunoblotting following differential centrifugation, of NCC was not different between rat groups. Overall, RGZ reduced blood pressure and thiazide sensitivity; however, the mechanism(s) did not seem to involve a decrease in NCC protein abundance or cellular location. Decreased NCC activity may have contributed to the maintenance of normotension in RGZ-treated obese rats.

Effects of dietary fat, NaCl, and fructose on renal sodium and water transporter abundances and systemic blood pressure.

Dietary fructose, NaCl, and/or saturated fat have been correlated with mean arterial pressure (MAP) rises in sensitive strains of rats. Dysregulation of sodium and/or water reabsorption by the kidney may contribute. Using radiotelemetry and parallel semiquantitative immunoblotting, we examined the effects of various diets on MAP and the regulation of abundance of the major renal sodium and water transport proteins in male Sprague-Dawley rats. In study 1, rats ( approximately 275 g) were fed one of four diets for 4 wk (n = 6/group): 1) control, 2) 65% fructose, 3) control + added NaCl (2.59%), or 4) fructose + NaCl. In study 2, 5% butter (fat) was added to the above four diets. Both fat and NaCl, but not fructose, caused modest rises in MAP (5-10 mmHg) and increased the day-to-night ratio in diastolic blood pressure. NaCl or fructose increased kidney size. Creatinine clearance was increased by salt or fat, and fractional excretion of sodium was decreased by fat. In study 1, high NaCl markedly reduced plasma renin and aldosterone and its regulated proteins in whole kidney, i.e., the thiazide-sensitive Na-Cl cotransporter and the alpha- and gamma (70-kDa band)-subunits of the epithelial sodium channel. These effects were blunted by fat. Fructose increased the abundance of the sodium phosphate cotransporter, whereas it decreased the bumetanide-sensitive Na-K-2Cl cotransporter and aquaporin-2. Overall, doubling of dietary fat appeared to impair dietary sodium adaptation, i.e., blunt the downregulation of aldosterone-mediated effects, thus allowing blood pressure to rise at an accelerated rate.

Increased blood pressure, aldosterone activity, and regional differences in renal ENaC protein during vasopressin escape.

The syndrome of inappropriate antidiuretic hormone (SIADH) is associated with water retention and hyponatremia. The kidney adapts via a transient natriuresis and persistent diuresis, i.e., vasopressin escape. Previously, we showed an increase in the whole kidney abundance of aldosterone-sensitive proteins, the alpha- and gamma (70-kDa-band)-subunits of the epithelial Na(+) channel (ENaC), and the thiazide-sensitive Na-Cl cotransporter (NCC) in our rat model of SIADH. Here we examine mean arterial pressure via radiotelemetry, aldosterone activity, and cortical vs. medullary ENaC subunit and 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD-2) protein abundances in escape. Eighteen male Sprague-Dawley rats (300 g) were sham operated (n = 6) or infused with desmopressin (dDAVP; n = 12, a V(2) receptor-selective analog of AVP). After 4 days, one-half of the rats receiving dDAVP were switched to a liquid diet, i.e., water loaded (WL) for 5-7 additional days. The WL rats had a sustained increase in urine volume and blood pressure (122 vs. 104 mmHg, P < 0.03, at 7 days). Urine and plasma aldosterone levels were increased in the WL group to 844 and 1,658% of the dDAVP group, respectively. NCC and alpha- and gamma-ENaC (70-kDa band) were increased significantly in the WL group (relative to dDAVP), only in the cortex. Beta- and gamma-ENaC (85-kDa band) were increased significantly by dDAVP in cortex and medulla relative to control. 11beta-HSD-2 was increased by dDAVP in the cortex and not significantly affected by water loading. These changes may serve to attenuate Na(+) losses and ameliorate hyponatremia in vasopressin escape.

Targeted proteomics using immunoblotting technique for studying dysregulation of ion transporters in renal disorders.

Renal salt and water transport physiology has benefited tremendously from the rapid advance of proteomics. Proteomics developed as a fast-throughput means of screening for global changes in proteins in a selected tissue, organ or cell type, as a logical offshoot of similar comprehensive, messenger RNA array-type technology. Targeted proteomics utilizes similar techniques but examines a predetermined set of proteins. One approach that has been rigorously employed over the last 10 years to evaluate differences in renal protein abundances due to a treatment or genotype has been parallel semiquantitative immunoblotting using antibody arrays. This approach, and newer ones on the horizon, provide a rapid global overview of regulation of the individual proteins whose integrated action determines overall renal sodium or water reabsorption.

Rosiglitazone activates renal sodium- and water-reabsorptive pathways and lowers blood pressure in normal rats.

Synthetic agonists of the peroxisomal proliferator-activated receptor subtype gamma (PPAR-gamma) are highly beneficial in the treatment of type II diabetes. However, they are also associated with fluid retention and edema, potentially serious side effects of unknown origin. These studies were designed to test the hypothesis that rosiglitazone (RGZ, PPAR-gamma agonist) may activate sodium- and water-reabsorptive processes in the kidney, possibly in response to a drop in mean arterial blood pressure (MAP), as well as directly through PPAR-gamma. Targeted proteomics of the major renal sodium and water transporters and channel proteins was used to identify potentially regulated sites of renal sodium and water reabsorption. RGZ (47 or 94 mg/kg diet) was fed to male, Sprague-Dawley rats (approximately 270g) for 3 days. MAP, measured by radiotelemetry, was decreased significantly in rats fed either level of RGZ, relative to control rats. Delta MAP from baseline was -3.2 +/- 1.2 mm Hg in rats fed high-dose RGZ versus + 3.4 +/- 0.8 for rats fed control diet. RGZ did not affect feed or water intake, but rats treated with high-dose RGZ had decreased urine volume (by 22%), sodium excretion (44%), kidney weight (9%), and creatinine clearance (35%). RGZ increased whole kidney protein abundance of the alpha-1 subunit of Na-K-ATPase, the bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2), the sodium hydrogen exchanger (NHE3), the aquaporins 2 and 3, and endothelial nitric-oxide synthase. We conclude that both increases in renal tubule transporter abundance and a decrease in glomerular filtration rate likely contribute to the RGZ-induced sodium retention.

Increased renal ENaC subunit and sodium transporter abundances in streptozotocin-induced type 1 diabetes.

Uncontrolled diabetes mellitus (DM) is associated with copious water and sodium losses. We hypothesized that the kidney compensates for these losses by increasing the abundances of key sodium and water transporters and channels. Using targeted proteomic analysis via immunoblotting of kidney homogenates, we examined comprehensive regulation of transport proteins. In three studies, streptozotocin (STZ; 65 mg/kg) or vehicle was administered intraperitoneally to male Sprague-Dawley rats. In study 2, to control for potential renal toxicity of STZ, one group of STZ-treated rats was intensively treated with insulin to control diabetes. In another group, the reversibility of DM and related changes was assessed by treating animals with insulin for the final 4 days. In study 3, we correlated blood glucose to transporter changes by treating animals with different doses of insulin. In study 1, STZ treatment resulted in significantly increased band densities for the type 3 sodium/hydrogen exchanger (NHE3), the thiazide-sensitive Na-Cl cotransporter (NCC), and epithelial sodium channel (ENaC) subunits alpha, beta, and gamma (85- and 70-kDa bands) to 204, 125, 176, 132, 147, and 241% of vehicle mean, respectively. In study 2, aquaporin-2 (AQP2) and AQP3 were increased with DM, but not AQP1 or AQP4. Neither these changes, nor blood glucose itself, could be returned to normal by short-term intensive insulin treatment. Whole kidney abundance of AQP3, the bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2), and gamma-ENaC (85-kDa band) correlated most strongly with blood glucose in study 3. These comprehensive changes would be expected to decrease volume contraction accompanying large-solute and water losses associated with DM.

Regulation of renal salt and water transporters during vasopressin escape.

Hyponatremia, defined as a serum sodium < 135 mmol/l, is one of the most commonly encountered and serious electrolyte disorders of clinical medicine. The predominant cause of hyponatremia is an inappropriate elevation of circulating vasopressin levels relative to serum osmolality or the 'syndrome of inappropriate antidiuretic hormone secretion' (SIADH). Fortunately, the degree of the hyponatremia is limited by a process that counters the water-retaining action of vasopressin, namely 'vasopressin escape'. Vasopressin escape is characterized by a sudden increase in urine volume with a decrease in urine osmolality independent of circulating vasopressin levels. Until recently, little was known about the molecular mechanisms underlying escape. In the 1980s, we developed an animal model for vasopressin escape in which male Sprague-Dawley rats were infused with dDAVP, a V2-receptor-selective agonist of vasopressin, while being fed a liquid diet. Rats drank a lot of water in order to get the calories they desired. Using this model, we demonstrated that the onset of vasopressin escape (increased urine volume coupled to decreased urine osmolality) coincided temporally with a marked decrease in renal aquaporin-2 (water channel) protein and mRNA expression in renal collecting ducts. This protein reduction was reversible and correlated to decreased water permeability of the collecting duct. Studies examining the mechanisms underlying AQP2 decrease revealed a decrease in V2-receptor mRNA expression and binding, as well as a decrease in cyclic AMP production in response to acute-dDAVP challenge in collecting duct suspensions from these escape animals. Additional studies showed an increase in sodium transporters of the distal tubule. These changes, hypothetically, might help to attenuate the hyponatremia. Future studies are needed to fully elucidate systemic, intra-organ, and cellular signaling responsible for the physiological phenomenon of vasopressin escape.

Dysregulation of renal salt and water transport proteins in diabetic Zucker rats.

BACKGROUND: Diabetic nephropathy is commonly associated with renal salt and water retention and hypertension. The molecular mechanisms involved and how the kidney responds to this volume expansion, in terms of renal transporter regulation, are not understood. METHODS: Targeted proteomics employing semiquantitative immunoblotting were used to investigate regulation of abundance of the primary salt and water transport proteins of the kidney, in 6-month-old lean and obese Zucker rats, a model for Type II diabetes. RESULTS: Obese rats were significantly heavier, had larger kidneys, increased plasma creatinine and glucose levels and elevated blood pressures. Furthermore, they had a marked decrease in abundance of many pre-macula densa renal sodium transporters. Mean band densities (% lean) were: in cortex, sodium phosphate cotransporter (NaPi-2), 68%, and sodium hydrogen exchanger (NHE3), 66%; and in outer medulla, NHE3, 39%, and the bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2), 37%. Collecting duct proteins also were markedly reduced. In inner medulla, aquaporins-2, -3, and -4 were reduced to, 46, 48, and 46%, respectively, and the apical urea transporter, UTA1 to 52%. In contrast, post-macula densa sodium transporters were less affected. The thiazide-sensitive Na-Cl cotransporter (NCC) was 106% and the alpha- beta- and gamma-subunits of the epithelial sodium channel (ENaC), 54, 121, and 84% of lean, respectively. CONCLUSIONS: In obese rats, selective decreases for pre-macula densa sodium transporters may reflect decreased glomerular filtration rate and glomerulotubular balance. This potentially could reduce blood pressure by decreasing proximal tubule sodium reabsorption.