Abundance Of Sodium Transporters Research Articles

A7813 Variations in abundance of urinary exosomal renal sodium transporters in response to mineralocorticoid administration and oral salt loading and in relation to baseline plasma aldosterone and potassium

Objectives: To quantify changes in abundance of sodium transporters [Na-Cl cotransporter (NCC), its phosphorylated form (pNCC), Na-K-Cl cotransporter (NKCC2) and epithelial sodium channel g-subunit (g-ENaC)] and prostasin in urinary exosomes in response to mineralocorticoid administration and oral salt loading, and explore associations with plasma aldosterone (aldo) and K+, in patients undergoing fludrocortisone suppression testing (FST).Methods: Baseline (D0) and day 4 (D4) morning urine samples were collected from 13 patients, including 10 with hypertension and raised aldo/renin ratio undergoing FST to confirm or exclude primary aldosteronism (PA; confirmed in 9) and 3 cured of PA by unilateral adrenalectomy. Urinary exosomes were isolated by ultracentrifugation and analysed by immunoblotting.Results: Increases in abundance of NCC (1.48 fold, p = 0.03) and pNCC (1.52 fold, p = 0.04) and trends towards increases in prostasin (p = 0.07), NKCC2 (p = 0.09) and cleaved g-ENaC (p = 0.11) were observed among the 13 subjects after 4 days FST. Abundance of cleaved g-ENaC rose significantly (p = 0.02) in the 10 non-operated subjects. Among the 13 subjects, D0 aldo and K+ did not correlate significantly with abundance of the target proteins, although D0 pNCC showed a trend towards positive correlation with aldo (p = 0.09). In the 10 non-operated subjects, D0 pNCC showed a negative correlation with plasma K+ that almost reached significance (p = 0.06).Conclusion: FST is associated with significant increases in NCC and pNCC and trends towards increases in ENaC, prostasin and NKCC2. The negative correlation between baseline plasma K+ and abundance of pNCC observed in adrenal-intact subjects suggests a possible role for K+ in the regulation of tubular salt handling and blood pressure.

Increased Urinary Extracellular Vesicle Sodium Transporters in Cushing Syndrome With Hypertension.

Increased renal sodium reabsorption contributes to hypertension in Cushing syndrome (CS). Renal sodium transporters can be analyzed noninvasively in urinary extracellular vesicles (uEVs). To analyze renal sodium transporters in uEVs of patients with CS and hypertension. Observational study. University hospital. The uEVs were isolated by ultracentrifugation and analyzed by immunoblotting in 10 patients with CS and 7 age-matched healthy participants. In 7 patients with CS, uEVs were analyzed before and after treatment. Abundance of protein in uEVs. The 10 patients with CS were divided in those with suppressed and nonsuppressed renin-angiotensin-aldosterone system (RAAS; n = 5 per group). Patients with CS with suppressed RAAS had similar blood pressure but significantly lower serum potassium than patients with CS with nonsuppressed RAAS. Compared with healthy participants, only patients with suppressed RAAS had higher phosphorylated Na+-K+-Cl- cotransporter type 2 (pNKCC2) and higher total and phosphorylated Na+-Cl- cotransporter (pNCC) in uEVs. Serum potassium but not urinary free cortisol correlated with pNKCC2, pNCC, and Na+-Cl- cotransporter (NCC) in uEVs. Treatment of CS reversed the increases in pNKCC2, NCC, and pNCC. CS increases renal sodium transporter abundance in uEVs in patients with hypertension and suppressed RAAS. Potassium has recently been identified as an important driver of NCC activity, and low serum potassium may also contribute to increased renal sodium reabsorption and hypertension in CS. These results may also be relevant for hypertension induced by exogenous glucocorticoids.

Collecting Duct Nitric Oxide Synthase 1ß Activation Maintains Sodium Homeostasis During High Sodium Intake Through Suppression of Aldosterone and Renal Angiotensin II Pathways.

BackgroundDuring high sodium intake, the renin‐angiotensin‐aldosterone system is downregulated and nitric oxide signaling is upregulated in order to remain in sodium balance. Recently, we showed that collecting duct nitric oxide synthase 1β is critical for fluid‐electrolyte balance and subsequently blood pressure regulation during high sodium feeding. The current study tested the hypothesis that high sodium activation of the collecting duct nitric oxide synthase 1β pathway is critical for maintaining sodium homeostasis and for the downregulation of the renin‐angiotensin‐aldosterone system–epithelial sodium channel axis.Methods and ResultsMale control and collecting duct nitric oxide synthase 1β knockout (CDNOS1KO) mice were placed on low, normal, and high sodium diets for 1 week. In response to the high sodium diet, plasma sodium was significantly increased in control mice and to a significantly greater level in CDNOS1KO mice. CDNOS1KO mice did not suppress plasma aldosterone in response to the high sodium diet, which may be partially explained by increased adrenal AT1R expression. Plasma renin concentration was appropriately suppressed in both genotypes. Furthermore, CDNOS1KO mice had significantly higher intrarenal angiotensin II with high sodium diet, although intrarenal angiotensinogen levels and angiotensin‐converting enzyme activity were similar between knockout mice and controls. In agreement with inappropriate renin‐angiotensin‐aldosterone system activation in the CDNOS1KO mice on a high sodium diet, epithelial sodium channel activity and sodium transporter abundance were significantly higher compared with controls.ConclusionsThese data demonstrate that high sodium activation of collecting duct nitric oxide synthase 1β signaling induces suppression of systemic and intrarenal renin‐angiotensin‐aldosterone system, thereby modulating epithelial sodium channel and other sodium transporter abundance and activity to maintain sodium homeostasis.

Interleukin-17A Regulates Renal Sodium Transporters and Renal Injury in Angiotensin II-Induced Hypertension.

Angiotensin II-induced hypertension is associated with an increase in T-cell production of interleukin-17A (IL-17A). Recently, we reported that IL-17A(-/-) mice exhibit blunted hypertension, preserved natriuresis in response to a saline challenge, and decreased renal sodium hydrogen exchanger 3 expression after 2 weeks of angiotensin II infusion compared with wild-type mice. In the current study, we performed renal transporter profiling in mice deficient in IL-17A or the related isoform, IL-17F, after 4 weeks of Ang II infusion, the time when the blood pressure reduction in IL-17A(-/-) mice is most prominent. Deficiency of IL-17A abolished the activation of distal tubule transporters, specifically the sodium-chloride cotransporter and the epithelial sodium channel and protected mice from glomerular and tubular injury. In human proximal tubule (HK-2) cells, IL-17A increased sodium hydrogen exchanger 3 expression through a serum and glucocorticoid-regulated kinase 1-dependent pathway. In mouse distal convoluted tubule cells, IL-17A increased sodium-chloride cotransporter activity in a serum and glucocorticoid-regulated kinase 1/Nedd4-2-dependent pathway. In both cell types, acute treatment with IL-17A induced phosphorylation of serum and glucocorticoid-regulated kinase 1 at serine 78, and treatment with a serum and glucocorticoid-regulated kinase 1 inhibitor blocked the effects of IL-17A on sodium hydrogen exchanger 3 and sodium-chloride cotransporter. Interestingly, both HK-2 and mouse distal convoluted tubule 15 cells produce endogenous IL-17A. IL17F had little or no effect on blood pressure or renal sodium transporter abundance. These studies provide a mechanistic link by which IL-17A modulates renal sodium transport and suggest that IL-17A inhibition may improve renal function in hypertension and other autoimmune disorders.

Age‐dependent hypertension and renal sodium handling in the female intrauterine growth restricted rat

Hypertension in male offspring exposed to prenatal administration of glucocorticoids involves an increase in sodium transporter abundance. In a rodent model induced by placental insufficiency, female intrauterine growth‐restricted (IUGR) offspring are normotensive after puberty; however, there is an age‐dependent increase in blood pressure that occurs by one year of age, but the mechanism remains unknown. Thus, we hypothesized that an age‐dependent change in renal function and (Na+‐K+) ATPase activity may contribute to the development of hypertension in the female growth‐restricted rat. (Na+‐K+) ATPase activity in the basolateral membrane of the proximal tubule was similar in female growth‐restricted offspring relative to female control at one year of age (Control: 171±44 and IUGR: 76±18 nmol Pi/mg protein/min). Glomerular filtration rate also did not differ upon comparison of female growth‐restricted to female control (Control: 1.89±0.17 and IUGR: 1.57±0.17 ml/min/g kidney weight). In addition, hypertension at one year of age in female growth‐restricted offspring was not associated with changes in 24‐hour sodium or potassium excretion. Thus, these data indicate that alterations in renal sodium transport may not be an underlying mechanism of age‐induced hypertension in the female growth‐restricted rat. HL074927; HL51971; CAPES (Brazil)

Sex differences in adaptive downregulation of pre-macula densa sodium transporters with ANG II infusion in mice

An increase in blood pressure (BP) due to angiotensin II (ANG II) infusion or other means is associated with adaptive pressure natriuresis due to reduced sodium reabsorption primarily in proximal tubule (PT) and thick ascending limb (TAL). We tested the hypothesis that male and female mice would show differential response to ANG II infusion with regard to the regulation of the protein abundance of sodium transporters in the PT and TAL and that these responses would be modulated by aging. Young (approximately 3 mo) and old (approximately 21 mo) male and female mice were infused with ANG II at 800 ng x kg body wt(-1) x min(-1) by osmotic minipump for 7 days or received a sham operation. ANG II increased mean arterial pressure (MAP), measured by radiotelemetry, significantly more in male mice of both ages (increased approximately 30-40 mmHg), compared with females (increased approximately 15-25 mmHg). On day 1, MAP was also significantly increased in old mice, relative to young (P = 0.01). ANG II infusion was associated with a significant decline in plasma testosterone (to <30% of control male) in male mice and rise in young female mice (to 478% of control female). No sex differences were found in the upregulation of the sodium hydrogen exchanger abundance on Western blots observed with ANG II infusion or the downregulation of the sodium phosphate cotransporter; however, aging did impact on some of these changes. Male mice (especially young) also had significantly reduced levels of the TAL bumetanide-sensitive Na-K-2Cl cotransporter (to 60% of male control), while young females showed an increase (to 126% of female control) with ANG II infusion. These sex differences do not support impaired pressure natriuresis in male mice, but might reflect a greater need and attempt to mount an appropriately BP-metered natriuretic response by additional downregulation of TAL sodium reabsorption.

Reducing blood pressure in SHR with enalapril provokes redistribution of NHE3, NaPi2, and NCC and decreases NaPi2 and ACE abundance

To determine the effects of long-term angiotensin-converting enzyme inhibition (ACEI) and blood pressure (BP) lowering on renal sodium transporter abundance and distribution in spontaneously hypertensive rats (SHR), 9-wk SHR were treated with enalapril (30 mg.kg(-1).day(-1)) for 4 wk. BP decreased from 156 +/- 4 to 96 +/- 8 mmHg. Na(+)/H(+) exchanger isoform 3 (NHE3) and Na(+)-P(i) cotransporter type 2 (NaPi2) localized to the body of the microvilli (MV) in normotensive rat strains. In untreated SHR, NHE3 partially retracted from the body to base of the MV and NaPi2 retracted to subapical vesicles. After enalapril treatment of SHR, NHE3 fully retracted to the base of the MV and, by density gradient fractionation, NHE3, NaPi2, dipeptidyl peptidase IV, myosin VI, Na-Cl cotransporter, and cortical Na-K-Cl cotransporter redistributed from low-density (apical enriched) to high-density (endosome enriched) membranes. Enalapril decreased total abundance of myosin VI (to 0.51 +/- 0.18 of untreated), ACE (0.67 +/- 0.22), and cortical NaPi2 (0.83 +/- 0.10). Normalizing SHR BP with HRH (7.5 mg/day hydralazine, 0.15 mg/day reserpine, and 3 mg/day hydrochlorothiazide) did not change Na(+) transporter density distribution or abundance. We conclude that lowering BP to normal levels in SHR does not normalize Na(+) transporter distribution, rather, chronic ACEI treatment provokes retraction of Na(+) transporters and associated proteins from transport-relevant domains of apical membranes and/or reduces their abundance.

Detection of NHE3 and NCC in urine as biomarkers of renal function in extremely premature neonates

Hydronephrosis associated with neonatal ureter obstruction (NUO) is a common cause of renal failure in children. Medical management of these individuals is complicated by the lack of precise tools to assess early changes in kidney function associated with NUO. Urinary biomarkers may serve as indicators of early kidney changes. We isolated renal sodium transporter protein from the urine of neonates. Urine samples (1 ml to 36 ml) were obtained from 33 neonates, born at gestational ages between 24–40 weeks. Samples were preserved with protease inhibitors, then subjected to ultracentrifugation to isolate sodium transporter proteins present in low-density membrane fractions. Immunoblots were probed with rabbit antibodies raised to rat type 3 Na-H exchanger (NHE3) of the proximal tubule, and the Na-Cl cotransporter (NCC) of the distal convoluted tubule. Each of these sodium transporters was detectable in the low-density exosomal fraction of urine. Antibody to NHE3 labeled a defined band at an approximate MW of 85 KD; NCC antibody labeled a low molecular weight protein. In addition, each antibody labeled specific high molecular weight proteins, consistent with sodium transporters existing as complexes in low-density exosomal vesicles in urine. Further investigation is indicated to determine the change in urinary sodium transporter abundance with maturation of the kidney and in response to NUO.

Diminished Expression of Sodium Transporters in the Ureteral Obstructed Kidney in Rats

Background/Aims: Whether the postobstructive natriuresis and diuresis is related with an altered regulation of sodium transporters in the kidney was examined. Methods: Male Sprague-Dawley rats underwent either bilateral (BUO) or unilateral obstruction (UUO) of the proximal ureters for 24 h. The expression of Na,K-ATPase, type-3 sodium-hydrogen exchanger (NHE3), type-1 bumetanide-sensitive sodium cotransporter (BSC1), and thiazide-sensitive sodium cotransporter (TSC) proteins was determined in the obstructed kidney by Western blot analysis and immunohistochemistry. Catalytic activity of Na,K-ATPase was also determined. Results: The expression of α1 and β1 subunit proteins and the catalytic activity of Na,K-ATPase were significantly decreased in the obstructed kidney in BUO. The expressions of NHE3, BSC1 and TSC proteins were also significantly decreased. Immunohistochemistry confirmed the downregulation of these sodium transporters in the obstructed kidney. In UUO, the expression of sodium transporters was similarly decreased in the obstructed kidney. Conclusion: The postobstructive natriuresis and diuresis may in part be accounted for by a reduced abundance of sodium transporters in the kidney.

Effect of primary polydipsia on aquaporin and sodium transporter abundance.

Chronic primary polydipsia (POLY) in humans is associated with impaired urinary concentrating ability. However, the molecular mechanisms responsible for this finding have not been elucidated. The purpose of this study was to examine the effect of chronic primary POLY on water metabolism and renal aquaporin (AQP) water channels and sodium and urea transporter abundance in rats. Primary POLY was induced in male Sprague-Dawley rats by daily administration of 15 g powdered rat chow mixed in 100 ml water for 10 days. Control rats (CTL) received 15 g powdered rat chow per day and ad libitum drinking water. Rats were studied following this period before further intervention and with a 36-h period of water deprivation to examine maximal urinary concentrating ability. At baseline, POLY rats demonstrated significantly greater water intake (100 +/- 1 vs. 22 +/- 2 ml/day, P < 0.0001) and urinary output (80 +/- 1 vs. 11 +/- 1 ml/day, P < 0.0001) and decreased urinary osmolality (159 +/- 13 vs. 1,365 +/- 188 mosmol/kgH2O, P < 0.001) compared with CTL rats. These findings were accompanied by decreased inner medulla AQP-2 protein abundance in POLY rats compared with CTL rats before water deprivation (76 +/- 2 vs. 100 +/- 7% CTL mean, P < 0.007). With water deprivation, maximal urinary osmolality was impaired in POLY vs. CTL rats (2,404 +/- 148 vs. 3,286 +/- 175 mosmol/kgH2O, P < 0.0005). This defect occurred despite higher plasma vasopressin concentrations and similar medullary osmolalities in POLY rats. In response to 36-h water deprivation, inner medulla AQP-2 protein abundance was decreased in POLY rats compared with CTL rats (65 +/- 5 vs. 100 +/- 5% CTL mean, P < 0.0006). No significant differences were noted in renal protein abundance of either AQP-3 or AQP-4 or sodium and urea transporters. We conclude that the impaired urinary concentrating ability associated with primary POLY in rats is due to impaired osmotic equilibration in the collecting duct that is mediated primarily by decreased AQP-2 protein abundance.

Renal tubule sodium transporter abundance profiling in rat kidney: response to aldosterone and variations in NaCl intake.

Based on extensive physiological study of sodium transport mechanisms along the renal tubule, complementary DNAs for all of the major transporters and channels responsible for renal tubular sodium reabsorption have been cloned over the past decade. There is now a comprehensive set of cDNA and antibody probes that can be used to investigate physiological mechanisms on a molecular level. Using rabbit polyclonal antibodies to all of the major renal Na transport proteins, we have developed profiling methods allowing comprehensive, integrated analysis of sodium transporter protein abundance changes along the renal tubule in response to physiological and pathophysiological perturbations. Here, we review some of our recent findings with this approach, focusing on renal responses to aldosterone and to variations in NaCl intake.

Alveolar epithelial fluid transport in acute lung injury: new insights.

Pulmonary oedema is a life-threatening condition that frequently leads to acute respiratory failure. From a physiological perspective, pulmonary oedema develops either because of an increase in lung vascular hydrostatic pressure or an increase in lung vascular permeability. Resolution of alveolar oedema depends on the active removal of salt and water from the distal air spaces of the lung across the distal lung epithelial barrier. Much has been learned about the molecular and cellular basis for oedema fluid reabsorption, including the role of apical ion transporters for sodium (epithelial sodium channel) and chloride (cystic fibrosis transmembrane conductance regulator), as well as the central importance of the sodium pump. The rate of fluid clearance can be upregulated by both catecholamine-dependent and -independent mechanisms. Injury to the alveolar epithelium can disrupt the integrity of the alveolar barrier or downregulate ion transport pathways, thus, reducing net alveolar fluid reabsorption and enhancing the extent of alveolar oedema. Endogenous catecholamines upregulate alveolar fluid clearance in several experimental models of acute lung injury, but this upregulation may be short term and insufficient to counterbalance alveolar flooding. There is new evidence, however, that pharmacological treatment with beta2-adrenergic agonists and/or epithelial growth factors may influence a more sustained stimulation of alveolar fluid reabsorption and in turn facilitate recovery from experimental pulmonary oedema. Similar results have been achieved experimentally by gene transfer to enhance the abundance of sodium transporters in the alveolar epithelium. Clinical studies show that impaired alveolar fluid transport mechanisms contribute to the development, severity and outcome of pulmonary oedema in humans. Very recent data suggest that mechanisms that augment transepithelial sodium transport and enhance the clearance of alveolar oedema may lead to more effective prevention or treatment for some types of pulmonary oedema.

Sodium transporter abundance profiling in kidney: effect of spironolactone.

Renal tubule profiling studies were carried out to investigate the long-term effects of administration of spironolactone, a mineralocorticoid receptor antagonist, on abundances of the major Na transporter and Na channel proteins along the rat renal tubule. Oral administration of spironolactone for 7 days to NaCl-restricted rats did not significantly alter abundances of Na transporters expressed proximal to the macula densa, while substantially decreasing the abundances of the thiazide-sensitive Na-Cl cotransporter (NCC), the alpha-subunit of the amiloride-sensitive epithelial Na channel (ENaC), and the 70-kDa form of the gamma-subunit of ENaC. A dependency of NCC expression on aldosterone was confirmed by showing increased NCC expression in response to aldosterone infusion in adrenalectomized rats. Immunoperoxidase labeling of ENaC in renal cortex confirmed that dietary NaCl restriction causes a redistribution of ENaC to the apical domain of connecting tubule cells and showed that high-dose spironolactone administration does not block this apical redistribution. In contrast, spironolactone completely blocked the increase in alpha-ENaC abundance in response to dietary NaCl restriction. We conclude that the protein abundances of NCC, alpha-ENaC, and the 70-kDa form of gamma-ENaC are regulated via the classical mineralocorticoid receptor, but the subcellular redistribution of ENaC in response to dietary NaCl restriction is not prevented by blockade of the mineralocorticoid receptor.

Increased renal Na-K-ATPase, NCC, and beta-ENaC abundance in obese Zucker rats.

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.

Transepithelial sodium and water transport in the lung. Major player and novel therapeutic target in pulmonary edema.

Active transepithelial transport of sodium from the airspaces to the lung interstitium is a primary mechanism driving alveolar fluid clearance. This mechanism depends on sodium uptake by amiloride-sensitive sodium channels on the apical membrane of alveolar type II cells followed by extrusion of sodium on the basolateral surface by the Na-K-ATPase. Injury to the alveolar epithelium can disrupt the integrity of the alveolar barrier or downregulate ion transport pathways thus reducing net alveolar fluid reabsorption, and enhancing the extent of alveolar edema. Endogenous catecholamines upregulate alveolar fluid clearance in several experimental models of acute lung injury, but this upregulation is short-term and often not sufficient to counterbalance alveolar flooding. There is new evidence, however, that pharmacological treatment with beta-adrenergic agonists and/or epithelial growth factors may induce a more sustained stimulation of alveolar fluid reabsorption and in turn facilitate recovery from experimental pulmonary edema. Similar results have been achieved experimentally by gene transfer enhancing the abundance of sodium transporters in the alveolar epithelium. Clinical studies show that impaired alveolar fluid transport mechanisms contribute to the development, severity and outcome of pulmonary edema in humans. Very recent data suggest that mechanisms that augment transepithelial sodium transport and enhance the clearance of alveolar edema may lead to more effective prevention or treatment for pulmonary edema and acute lung injury.

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