Effects of potassium on expression of renal sodium transporters in salt-sensitive hypertensive rats induced by uninephrectomy

Dietary potassium is an important modulator of systemic blood pressure (BP). The purpose of this study was to determine whether dietary potassium is associated with an altered abundance of major renal sodium transporters that may contribute to the modulation of systemic BP. A unilateral nephrectomy (uNx) was performed in male Sprague-Dawley rats, and the rats were fed a normal-salt diet (0.3% NaCl) for 4 wk. Thereafter, the rats were fed a high-salt (HS) diet (3% NaCl) for the entire experimental period. The potassium-repleted (HS+KCl) group was given a mixed solution of 1% KCl as a substitute for drinking water. We examined the changes in the abundance of major renal sodium transporters and the expression of mRNA of With-No-Lysine (WNK) kinases sequentially at 1 and 3 wk. The systolic BP of the HS+KCl group was decreased compared with the HS group (140.3 ± 2.97 vs. 150.9 ± 4.04 mmHg at 1 wk; 180.3 ± 1.76 vs. 207.7 ± 6.21 mmHg at 3 wk). The protein abundances of type 3 Na(+)/H(+) exchanger (NHE3) and Na(+)-Cl(-) cotransporter (NCC) in the HS+KCl group were significantly decreased (53 and 45% of the HS group at 1 wk, respectively; 19 and 8% of HS group at 3 wk). WNK4 mRNA expression was significantly increased in the HS+KCl group (1.4-fold of control at 1 wk and 1.9-fold of control at 3 wk). The downregulation of NHE3 and NCC may contribute to the BP-attenuating effect of dietary potassium associated with increased urinary sodium excretion.     

Effect of high-NaCl or high-KCl diet on hepatic Na+- and K+-receptor sensitivity and NKCC1 expression in rats

We previously reported that the bumetanide-sensitive Na(+)-K(+)-2Cl- cotransporter (NKCC1) is involved in the hepatic Na+ and K+ sensor mechanism. In the present study, we examined the effects of a high-NaCl or high-KCl diet on hepatic Na+ and K+ receptor sensitivity and NKCC1 expression in the liver of Sprague-Dawley rats. RT-PCR and Western blots were used to measure NKCC1 mRNA and protein expression, respectively. Infusion of hypertonic NaCl or isotonic KCl + NaCl solutions into the portal vein increased hepatic afferent nerve activity (HANA) in a Na+ or K+ dose-dependent manner. After 4 wk on a high-NaCl or high-KCl diet, HANA responses were attenuated compared with animals fed a normal diet, and NKCC1 expression was reduced. These results show that a high-NaCl or high-KCl diet decreases NKCC1 expression in the liver, and it might cause a reduction in hepatic Na(+)- and K(+)-receptor sensitivity.     

Na+, K+, and Cl- transport in resting pancreatic acinar cells

To understand the role of Na+, K+, and Cl- transporters in fluid and electrolyte secretion by pancreatic acinar cells, we studied the relationship between them in resting and stimulated cells. Measurements of [Cl-]i in resting cells showed that in HCO3(-)-buffered medium [Cl- ]i and Cl- fluxes are dominated by the Cl-/HCO3- exchanger. In the absence of HCO3-, [Cl-]i is regulated by NaCl and NaK2Cl cotransport systems. Measurements of [Na+]i showed that the Na(+)-coupled Cl- transporters contributed to the regulation of [Na+]i, but the major Na+ influx pathway in resting pancreatic acinar cells is the Na+/H+ exchanger. 86Rb influx measurements revealed that > 95% of K+ influx is mediated by the Na+ pump and the NaK2Cl cotransporter. In resting cells, the two transporters appear to be coupled through [K+]i in that inhibition of either transporter had small effect on 86Rb uptake, but inhibition of both transporters largely prevented 86Rb uptake. Another form of coupling occurs between the Na+ influx transporters and the Na+ pump. Thus, inhibition of NaK2Cl cotransport increased Na+ influx by the Na+/H+ exchanger to fuel the Na+ pump. Similarly, inhibition of Na+/H+ exchange increased the activity of the NaK2Cl cotransporter. The combined measurements of [Na+]i and 86Rb influx indicate that the Na+/H+ exchanger contributes twice more than the NaK2Cl cotransporter and three times more than the NaCl cotransporter and a tetraethylammonium-sensitive channel to Na+ influx in resting cells. These findings were used to develop a model for the relationship between the transporters in resting pancreatic acinar cells.     

Na+- and K+-dependent oligomeric interconversion among alphabeta-protomers, diprotomers and higher oligomers in solubilized Na+/K+-ATPase

Protein fractions of a higher-oligomer (H), (alphabeta)(2)-diprotomer (D) and alphabeta-protomer (P) were separated from dog kidney Na(+)/K(+)-ATPase solubilized in the presence of NaCl and KCl. Na(+)/K(+)-dependent interconversion of the oligomers was analysed using HPLC at 0 degrees C. With increasing KCl concentrations, the content or amount of D increased from 27.6 to 54.3% of total protein, i.e. DeltaC(max) = 26.7%. DeltaC(max) for the sum of D and H was equivalent to the absolute value of DeltaC(max) for P, regardless of the anion present, indicating that K(+) induced the conversion of P into D and/or H, and Na(+) had the opposite effect. When enzymes that had been denatured to varying degrees by aging were solubilized, DeltaC(max) increased linearly with the remaining ATPase activity. The magnitude of the interconversion could be explained based on an equilibrium of D <==> 2P, assuming 50-fold difference in the K(d) between KCl and NaCl, and coexistence of unconvertible oligomers, which comprised as much as 39% of the eluted protein. Oligomeric interconversion, determined as a function of the KCl or NaCl concentration, showed K(0.5)s of 64.8 microM and 6.50 mM for KCl and NaCl, respectively, implying that oligomeric interconversion was coupled with Na(+)/K(+)-binding to their active transport sites.     

Nitric oxide decreases renal medullary Na+, K+-ATPase activity through cyclic GMP-protein kinase G dependent mechanism.

The aim of this study was to investigate the effect of nitric oxide on renal Na+,K(+)-ATPase and ouabain-sensitive H+,K(+)-ATPase activities. The study was performed in male Wistar rats. The investigated substances were infused under general anaesthesia into abdominal aorta proximally to the renal arteries. The activity of ATPases was assayed in isolated microsomal fraction. NO donor, S-nitroso-N-acetylpenicillamine (SNAP), infused at doses of 10(-7) and 10(-6)mol/kg/min decreased medullary Na+,K(+)-ATPase activity by 29.4% and 45.2%, respectively. Another NO donor, spermine NONOate, administered at the same doses reduced Na+,K(+)-ATPase activity in the renal medulla by 31.7% and 46.5%, respectively. Neither of NO releasers had any effect on Na+,K(+)-ATPase in the renal cortex and on either cortical or medullary ouabain-sensitive H+,K(+)-ATPase. Infusion of NO precursor, L-arginine (100 micromol/kg/min), decreased medullary Na+,K(+)-ATPase activity by 32.2%, whereas inhibitor of nitric oxide synthase, L-NAME (10 nmol/kg/min), increased this activity by 20.7%. The effect of synthetic NO donors was mimicked by 8-bromo-cGMP and blocked by inhibitors of soluble guanylate cyclase, ODQ or methylene blue, as well as by specific inhibitor of protein kinase G, KT5823. In addition, inhibitory effect of either SNAP or 8-bromo-cGMP on medullary Na+,K(+)-ATPase was abolished by 17-octadecynoic acid (17-ODYA), which inhibits cytochrome P450-dependent metabolism of arachidonic acid. These data suggest that NO decreases Na+,K(+)-ATPase activity in the renal medulla through the mechanism involving cGMP, protein kinase G, and cytochrome P450-dependent arachidonate metabolites. In contrast, NO has no effect on Na+,K(+)-ATPase in the renal cortex and on either cortical or medullary ouabain-sensitive H+,K(+)-ATPase.     

Conservation of Na+ vs. K+ by the rat cortical collecting duct

Regulation of transport by principal cells of the distal nephron contributes to maintenance of Na(+) and K(+) homeostasis. To assess which of these ions is given a higher priority by these cells, we investigated the upregulation of epithelial Na(+) channels (ENaC) in the rat cortical collecting duct (CCD) during Na depletion with and without simultaneous K depletion. ENaC activity, assessed as whole cell amiloride-sensitive current in split-open tubules, was 260 ± 40 pA/cell in K-repleted but virtually undetectable (3 ± 1 pA/cell) in K-depleted animals. This difference was confirmed biochemically by the reduced amounts of the cleaved forms of both the α-ENaC and γ-ENaC subunits measured in immunoblots. In contrast, in K-depleted rats, simultaneously reducing Na intake did not affect the activity of ROMK channels, assessed as tertiapin-Q-sensitive whole cell currents, in the CCDs. The lack of Na current in K-depleted animals was the result of reduced levels of aldosterone in plasma, rather than a reduced sensitivity to the hormone. However, rats on a low-Na, low-K diet for 1 wk did not excrete more Na than those on a low-Na, control-K diet for the same period of time. Immunoblot analysis indicated increased levels of the thiazide-sensitive NaCl cotransporter and the apical Na-H exchanger NHE3. This suggests that with reduced K intake, Na balance is maintained despite reduced aldosterone and Na(+) channel activity by upregulation of Na(+) transport in upstream segments. Under these conditions, Na(+) transport by the aldosterone-sensitive distal nephron is reduced, despite the low-Na intake to minimize K(+) secretion and urinary K losses.     

Leptin decreases renal medullary Na(+), K(+)-ATPase activity through phosphatidylinositol 3-kinase dependent mechanism.

We examined the effect of leptin on renal function and renal Na(+),K(+)-ATPase and ouabain-sensitive H(+),K(+)-ATPase activities in the rat. Leptin was infused under general anaesthesia into the abdominal aorta proximally to the renal arteries. Leptin infused at doses of 1 and 10 microg/kg/min increased urine output by 40% and 140%, respectively. Urinary Na(+) excretion increased in rats receiving leptin at doses of 0.1, 1, and 10 microg/kg/min by 57.6%, 124.2% and 163.6%, respectively. Leptin had no effect on creatinine clearance, potassium excretion and phosphate excretion. Na(+),K(+)-ATPase activity in the renal medulla of rats treated with 1 and 10 microg/kg/min leptin was lower than in control animals by 25.5% and 33.2%, respectively. In contrast, cortical Na(+),K(+)-ATPase as well as either cortical or medullary ouabain-sensitive H(+),K(+)-ATPase activities did not differ between leptin-treated and control animals. The effect of leptin on Na(+),K(+)-ATPase activity was abolished by actin depolymerizing agents, cytochalazin D and latrunculin B, and by phosphatidylinositol 3-kinase (PI3K) inhibitors, wortmannin and LY294002. These results indicate that: 1). natriuretic effect of leptin is mediated, at least in part, by decrease in renal medullary Na(+),K(+)-ATPase activity, 2). inhibition of medullary Na(+),K(+)-ATPase by leptin is mediated by PI3K and requires integrity of actin cytoskeleton.     

AtHKT1 facilitates Na+ homeostasis and K+ nutrition in planta

Genetic and physiological data establish that Arabidopsis AtHKT1 facilitates Na(+) homeostasis in planta and by this function modulates K(+) nutrient status. Mutations that disrupt AtHKT1 function suppress NaCl sensitivity of sos1-1 and sos2-2, as well as of sos3-1 seedlings grown in vitro and plants grown in controlled environmental conditions. hkt1 suppression of sos3-1 NaCl sensitivity is linked to higher Na(+) content in the shoot and lower content of the ion in the root, reducing the Na(+) imbalance between these organs that is caused by sos3-1. AtHKT1 transgene expression, driven by its innate promoter, increases NaCl but not LiCl or KCl sensitivity of wild-type (Col-0 gl1) or of sos3-1 seedlings. NaCl sensitivity induced by AtHKT1 transgene expression is linked to a lower K(+) to Na(+) ratio in the root. However, hkt1 mutations increase NaCl sensitivity of both seedlings in vitro and plants grown in controlled environmental conditions, which is correlated with a lower K(+) to Na(+) ratio in the shoot. These results establish that AtHKT1 is a focal determinant of Na(+) homeostasis in planta, as either positive or negative modulation of its function disturbs ion status that is manifested as salt sensitivity. K(+)-deficient growth of sos1-1, sos2-2, and sos3-1 seedlings is suppressed completely by hkt1-1. AtHKT1 transgene expression exacerbates K(+) deficiency of sos3-1 or wild-type seedlings. Together, these results indicate that AtHKT1 controls Na(+) homeostasis in planta and through this function regulates K(+) nutrient status.     

Hepatoportal bumetanide-sensitive K(+)-sensor mechanism controls urinary K(+) excretion

To determine whether a K(+)-sensor mechanism exists in the hepatoportal region, periarterial hepatic afferent nerve activity responses to intraportal injection of KCl were examined in anesthetized rats. Hepatic afferent nerve activity increased in response to intraportal injection in a K(+) concentration-dependent manner, and the increase was attenuated by inhibition of the Na(+)-K(+)-2Cl(-) cotransporter by bumetanide in a dose-dependent manner. These results suggest that a bumetanide-sensitive K(+)-sensor mechanism exists in the hepatoportal region. Stimulation of this mechanism by intraportal KCl infusion elicited an immediate and powerful kaliuresis with no significant change in the plasma K(+) concentration; this was significantly greater than the kaliuresis induced by intravenous KCl infusion and was attenuated by severing the periarterial hepatic nervous plexus. These results indicate that a hepatoportal bumetanide-sensitive K(+)-sensor mechanism senses the portal venous K(+) concentration and that stimulation of this sensor mechanism causes kaliuresis, which is mainly mediated by the periarterial hepatic nervous plexus.     

Regulation of renal Na(+),K(+)-ATPase and ouabain-sensitive H(+),K(+)-ATPase by the cyclic AMP-protein kinase A signal transduction pathway

We investigated the effect of the cyclic AMP-protein kinase A (PKA) signalling pathway on renal Na(+),K(+)-ATPase and ouabain-sensitive H(+),K(+)-ATPase. Male Wistar rats were anaesthetized and catheter was inserted through the femoral artery into the abdominal aorta proximally to the renal arteries for infusion of the investigated substances. Na(+),K(+)-ATPase activity was measured in the presence of Sch 28080 to block ouabain-sensitive H(+),K(+)-ATPase and improve specificity of the assay. Dibutyryl-cyclic AMP (db-cAMP) administered at a dose of 10(-7) mol/kg per min and 10(-6) mol/kg per min increased Na(+),K(+)-ATPase activity in the renal cortex by 34% and 42%, respectively, and decreased it in the renal medulla by 30% and 44%, respectively. db-cAMP infused at 10(-6) mol/kg per min increased the activity of cortical ouabain-sensitive H(+),K(+)-ATPase by 33%, and medullary ouabain-sensitive H(+),K(+)-ATPase by 30%. All the effects of db-cAMP were abolished by a specific inhibitor of protein kinase A, KT 5720. The stimulatory effect on ouabain-sensitive H(+),K(+)-ATPase and on cortical Na(+),K(+)-ATPase was also abolished by brefeldin A which inhibits the insertion of proteins into the plasma membranes, whereas the inhibitory effect on medullary Na(+),K(+)-ATPase was partially attenuated by 17-octadecynoic acid, an inhibitor of cytochrome p450-dependent arachidonate metabolism. We conclude that the cAMP-PKA pathway stimulates Na(+),K(+)-ATPase in the renal cortex as well as ouabain-sensitive H(+),K(+)-ATPase in the cortex and medulla by a mechanism requiring insertion of proteins into the plasma membrane. In contrast, medullary Na(+),K(+)-ATPase is inhibited by cAMP through a mechanism involving cytochrome p450-dependent arachidonate metabolites.     

Regulation of epithelial Na+ channels by adrenal steroids: mineralocorticoid and glucocorticoid effects

Epithelial Na+ channels (ENaC) can be regulated by both mineralocorticoid and glucocorticoid hormones. In the mammalian kidney, effects of mineralocorticoids have been extensively studied, but those of glucocorticoids are complicated by metabolism of the hormones and cross-occupancy of mineralocorticoid receptors. Here, we report effects of dexamethasone, a synthetic glucocorticoid, on ENaC in the rat kidney. Infusion of dexamethasone (24 μg/day) for 1 wk increased the abundance of αENaC 2.26 ± 0.04-fold. This was not accompanied by an induction of Na+ currents (I(Na)) measured in isolated split-open collecting ducts. In addition, hormone treatment did not increase the abundance of the cleaved forms of either αENaC or γENaC or the expression of βENaC or γENaC protein at the cell surface. The absence of hypokalemia also indicated the lack of ENaC activation in vivo. Dexamethasone increased the abundance of the Na+ transporters Na+/H+ exchanger 3 (NHE3; 1.36 ± 0.07-fold), Na(+)-K(+)-2Cl(-) cotransporter 2 (NKCC2; 1.49 ± 0.07-fold), and Na-Cl cotransporter (NCC; 1.72 ± 0.08-fold). Surface expression of NHE3 and NCC also increased with dexamethasone treatment. To examine whether glucocorticoids could either augment or inhibit the effects of mineralocorticoids, we infused dexamethasone (60 μg/day) together with aldosterone (12 μg/day). Dexamethasone further increased the abundance of αENaC in the presence of aldosterone, suggesting independent effects of the two hormones on this subunit. However, I(Na) was similar in animals treated with dexamethasone+aldosterone and with aldosterone alone. We conclude that dexamethasone can occupy glucocorticoid receptors in cortical collecting duct and induce the synthesis of αENaC. However, this induction is not sufficient to produce an increase in functional Na+ channels in the apical membrane, implying that the abundance of αENaC is not rate limiting for channel formation in the kidney.     

Time-dependent effect of leptin on renal Na+,K+-ATPase activity

Leptin, secreted by adipose tissue, is involved in the pathogenesis of arterial hypertension, however, the mechanisms through which leptin increases blood pressure are incompletely elucidated. We investigated the effect of leptin, administered for different time periods, on renal Na(+),K(+)-ATPase activity in the rat. Leptin was infused under anesthesia into the abdominal aorta proximally to the renal arteries for 0.5-3 h. Leptin administered at doses of 1 and 10 microg/min per kg for 30 min decreased the Na(+),K(+)-ATPase activity in the renal medulla. This effect disappeared when the hormone was infused for > or =1 h. Leptin infused for 3 h increased the Na(+),K(+)-ATPase activity in the renal cortex and medulla. The stimulatory effect was abolished by a specific inhibitor of Janus kinases (JAKs), tyrphostin AG490, as well as by an NAD(P)H oxidase inhibitor, apocynin. Leptin increased urinary excretion of hydrogen peroxide (H(2)O(2)) between 2 and 3 h of infusion. The effect of leptin on renal Na(+),K(+)-ATPase and urinary H(2)O(2) was augmented by a superoxide dismutase mimetic, tempol, and was abolished by catalase. In addition, infusion of H(2)O(2) for 30 min increased the Na(+),K(+)-ATPase activity. Inhibitors of extracellular signal regulated kinases (ERKs), PD98059 or U0126, prevented Na(+),K(+)-ATPase stimulation by leptin and H(2)O(2). These data indicate that leptin, by acting directly within the kidney, has a delayed stimulatory effect on Na(+),K(+)-ATPase, mediated by JAKs, H(2)O(2) and ERKs. This mechanism may contribute to the abnormal renal Na(+) handling in diseases associated with chronic hyperleptinemia such as diabetes and obesity.     

Nitric oxide -- superoxide cooperation in the regulation of renal Na(+),K(+)-ATPase

The aim of this study was to investigate whether endogenous superoxide anion is involved in the regulation of renal Na(+),K(+)-ATPase and ouabain-sensitive H(+),K(+)-ATPase activities. The study was performed in male Wistar rats. Compounds modulating superoxide anion concentration were infused under general anaesthesia into the abdominal aorta proximally to the renal arteries. The activity of ATPases was assayed in isolated microsomal fraction. We found that infusion of a superoxide anion-generating mixture, xanthine oxidase (1 mU/min per kg) + hypoxanthine (0.2 mumol/min per kg), increased the medullary Na(+),K(+)-ATPase activity by 49.5% but had no effect on cortical Na(+),K(+)-ATPase and either cortical or medullary ouabain-sensitive H(+),K(+)-ATPase. This effect was reproduced by elevating endogenous superoxide anion with a superoxide dismutase inhibitor, diethylthiocarbamate. In contrast, a superoxide dismutase mimetic, TEMPOL, decreased the medullary Na(+),K(+)-ATPase activity. The inhibitory effect of TEMPOL was abolished by inhibitors of nitric oxide synthase (L-NAME), soluble guanylate cyclase (ODQ) and protein kinase G (KT5823). The stimulatory effect of diethylthiocarbamate was not observed in animals pretreated with a synthetic cGMP analogue, 8-bromo-cGMP. An inhibitor of NAD(P)H oxidase, apocynin (1 mumol/min per kg), decreased the Na(+),K(+)-ATPase activity in the renal medulla and its effect was prevented by L-NAME, ODQ or KT5823. In contrast, a xanthine oxidase inhibitor, oxypurinol, administered at the same dose was without effect. These data suggest that NAD(P)H oxidase-derived superoxide anion increases Na(+),K(+)-ATPase activity in the renal medulla by reducing the availability of NO. Excessive intrarenal generation of superoxide anion may upregulate medullary Na(+),K(+)-ATPase leading to sodium retention and blood pressure elevation.     

Tyrosine 537 within the Na+,K+-ATPase alpha-subunit is essential for AP-2 binding and clathrin-dependent endocytosis

In renal epithelial cells endocytosis of Na(+),K(+)-ATPase molecules is initiated by phosphorylation of its alpha(1)-subunit, leading to activation of phosphoinositide 3-kinase and adaptor protein-2 (AP-2)/clathrin recruitment. The present study was performed to establish the identity of the AP-2 recognition domain(s) within the Na(+),K(+)-ATPase alpha(1)-subunit. We identified a conserved sequence (Y(537)LEL) within the alpha(1)-subunit that represents an AP-2 binding site. Binding of AP-2 to the Na(+),K(+)-ATPase alpha(1)-subunit in response to dopamine (DA) was increased in OK cells stably expressing the wild type rodent alpha-subunit (OK-WT), but not in cells expressing the Y537A mutant (OK-Y537A). DA treatment was associated with increased alpha(1)-subunit abundance in clathrin vesicles from OK-WT but not from OK-Y537A cells. In addition, this mutation also impaired the ability of DA to inhibit Na(+),K(+)-ATPase activity. Because phorbol esters increase Na(+),K(+)-ATPase activity in OK cells, and this effect was not affected by the Y537A mutation, the present results suggest that the identified motif is specifically required for DA-induced AP-2 binding and Na(+),K(+)-ATPase endocytosis.     

Potassium excretion during antinatriuresis: perspective from a distal nephron model

Renal excretion of Na(+) and K(+) must be regulated independently within the distal nephron, but is complicated by the fact that changing excretion of one solute requires adjustments in the transport of both. It is long known that hypovolemia increases Na(+) reabsorption while impairing K(+) excretion, even when distal Na(+) delivery is little changed. Renewed interest in this micropuncture observation came with identification of the molecular defects underlying familial hyperkalemic hypertension (FHH), which also increases distal Na(+) reabsorption and impairs K(+) excretion. In this work, a mathematical model of the distal nephron (Weinstein AM. Am J Physiol Renal Physiol 295: F1353-F1364, 2008), including the distal convoluted tubule (DCT), connecting segment (CNT), and collecting duct (CD), is used to examine renal K(+) excretion during antinatriuresis. Within the model, Na(+) avidity is represented as the modulation of DCT NaCl reabsorption, and the K(+) secretion signal is an aldosterone-like effect on principal cells of the CNT and CD. The first model prediction is that changes in DCT NaCl reabsorption are not mediated by NaCl cotransporter density alone, but require additional adjustments of both peritubular Na-K-ATPase and KCl cotransport. A second observation is that the CNT response to increased DCT Na(+) reabsorption should not only stabilize CD K(+) delivery but also compensate for the compromise of K(+) excretion downstream, as low Na(+) delivery increases CD K(+) reabsorption. Such anticipatory regulation is seen with the aldosterone response of hypovolemia, while the FHH phenotype manifests enhanced DCT NaCl transport but a blunted aldosterone effect. The model emphasizes the need for two distinct signals to the distal nephron, regulating Na(+) excretion and K(+) excretion, in contrast to a single switch apportioning NaCl reabsorption and Na(+)-for-K(+) exchange.     

Spectrophotometric assay of renal ouabain-resistant Na(+)-ATPase and its regulation by leptin and dietary-induced obesity

Apart from Na(+),K(+)-ATPase, a second sodium pump, Na(+)-stimulated, K(+)-independent ATPase (Na(+)-ATPase) is expressed in proximal convoluted tubule of the mammalian kidney. The aim of this study was to develop a method of Na(+)-ATPase assay based on the method previously used by us to measure Na(+),K(+)-ATPase activity. The ATPase activity was assayed as the amount of inorganic phosphate liberated from ATP by isolated microsomal fraction. Na(+)-ATPase activity was calculated as the difference between the activities measured in the presence and in the absence of 50 mM NaCl. Na(+)-ATPase activity was detected in the renal cortex (3.5 +/- 0.2 mumol phosphate/h per mg protein), but not in the renal medulla. Na(+)-ATPase was not inhibited by ouabain or an H(+),K(+)-ATPase inhibitor, Sch 28080, but was almost completely blocked by 2 mM furosemide. Leptin administered intraperitoneally (1 mg/kg) decreased the Na(+),K(+)-ATPase activity in the renal medulla at 0.5 and 1 h by 22.1% and 27.1%, respectively, but had no effect on Na(+)-ATPase in the renal cortex. Chronic hyperleptinemia induced by repeated subcutaneous leptin injections (0.25 mg/kg twice daily for 7 days) increased cortical Na(+),K(+)-ATPase, medullary Na(+),K(+)-ATPase and cortical Na(+)-ATPase by 32.4%, 84.2% and 62.9%, respectively. In rats with dietary-induced obesity, the Na(+),K(+)- ATPase activity was higher in the renal cortex and medulla by 19.7% and 34.3%, respectively, but Na(+)-ATPase was not different from control. These data indicate that both renal Na(+)-dependent ATPases are separately regulated and that up-regulation of Na(+)-ATPase may contribute to Na(+) retention and arterial hypertension induced by chronic hyperleptinemia.     

Renal medullary endothelin-1 is decreased in Dahl salt-sensitive rats

Although it is well established that the renal endothelin (ET-1) system plays an important role in regulating sodium excretion and blood pressure through activation of renal medullary ET(B) receptors, the role of this system in Dahl salt-sensitive (DS) hypertension is unclear. The purpose of this study was to determine whether the DS rat has abnormalities in the renal medullary endothelin system when maintained on a high sodium intake. The data indicate that Dahl salt-resistant rats (DR) on a high-salt diet had a six-fold higher urinary endothelin excretion than in the DR rats with low Na(+) intake (17.8 ± 4 pg/day vs. 112 ± 44 pg/day). In sharp contrast, urinary endothelin levels increased only twofold in DS rats in response to a high Na(+) intake (13 ± 2 pg/day vs. 29.8 ± 5.5 pg/day). Medullary endothelin concentration in DS rats on a high-Na(+) diet was also significantly lower than DR rats on a high-Na(+) diet (31 ± 2.8 pg/mg vs. 70.9 ± 5 pg/mg). Furthermore, DS rats had a significant reduction in medullary ET(B) receptor expression compared with DR rats while on a high-Na(+) diet. Finally, chronic infusion of ET-1 directly into the renal medulla blunted Dahl salt-sensitive hypertension. These data indicate that a decrease in medullary production of ET-1 in the DS rat could play an important role in the development of salt-sensitive hypertension observed in the DS rat.     

Salt-sensing mechanisms in blood pressure regulation and hypertension

High salt consumption contributes to the development of hypertension and is considered an independent risk factor for vascular remodeling, cardiac hypertrophy, and stroke incidence. In this review, we discuss the molecular origins of primary sensors involved in the phenomenon of salt sensitivity. Based on the analysis of literature data, we conclude that the kidneys and central nervous system (CNS) are two major sites for salt sensing via several distinct mechanisms: 1) [Cl(-)] sensing in renal tubular fluids, primarily by Na(+)-K(+)-Cl(-) cotransporter (NKCC) isoforms NKCC2B and NKCC2A, whose expression is mainly limited to macula densa cells; 2) [Na(+)] sensing in cerebrospinal fluid (CSF) by a novel isoform of Na(+) channels, Na(x), expressed in subfornical organs; 3) sensing of CSF osmolality by mechanosensitive, nonselective cation channels (transient receptor potential vanilloid type 1 channels), expressed in neuronal cells of supraoptic and paraventricular nuclei; and 4) osmolarity sensing by volume-regulated anion channels in glial cells of supraoptic and paraventricular nuclei. Such multiplicity of salt-sensing mechanisms likely explains the differential effects of Na(+) and Cl(-) loading on the long-term maintenance of elevated blood pressure that is documented in experimental models of salt-sensitive hypertension.     

Structural peculiarities of Na+,K+-ATPase isozymes from the calf brain

Functionally active preparations of Na+,K(+)-ATPase isozymes from calf brain that contain catalytic subunits of three types (alpha 1, alpha 2, and alpha 3) were obtained using two approaches: a selective removal of contaminating proteins by the Jorgensen method and a selective solubilization of the enzyme with subsequent reconstitution of the membrane structure by the Esmann method. The ouabain inhibition constants were determined for the isozymes. The real isozyme composition of the Na+ pump from the grey matter containing glial cells and the brain stem containing neurons was determined. The plasma membranes of glial cells were shown to contain mainly Na+,K(+)-ATPase of the alpha 1 beta 1 type and minor amounts of isozymes of the alpha 2 beta 2 (beta 1) and the alpha 3 beta 1 (beta 2) type. The axolemma contains alpha 2 beta 1- and alpha 3 beta 1 isozymes. A carbohydrate analysis indicated that alpha 1 beta 1 enzyme preparations from the brain grey matter substantially differ from the renal enzymes of the same composition in the glycosylation of the beta 1 isoform. An enhanced sensitivity of the alpha 3 catalytic subunit of Na+,K(+)-ATPase from neurons to endogenous proteolysis was found. A point of specific proteolysis in the amino acid sequence PNDNR492 decreases Y493 was localized (residue numbering is that of the human alpha 3 subunit). This sequence corresponds to one of the regions of the greatest variability in alpha 1, alpha 2, alpha 3, and alpha 4-subunits, but at the same time, it is characteristic of the alpha 3 isoforms of various species. The presence of the beta 3 isoform of tubulin (cytoskeletal protein) was found for the first time in the high-molecular-mass Na+,K(+)-ATPase alpha 3 beta 1 isozyme complex isolated from the axolemma of brain stem neurons, and its binding to the alpha 3 catalytic subunit was shown.