Regulation Of Renal Na Research Articles

Inwardly rectifying K+ channels 4.1 and 5.1 (Kir4.1/Kir5.1) in the renal distal nephron.

The inwardly rectifying potassium channel (Kir) 4.1 (encoded by KCNJ10) interacts with Kir5.1 (encoded by KCNJ16) to form a major basolateral K+ channel in the renal distal convoluted tubule (DCT), connecting tubule (CNT), and the cortical collecting duct (CCD). Kir4.1/Kir5.1 heterotetramer plays an important role in regulating Na+ and K+ transport in the DCT, CNT, and CCD. A recent development in the field has firmly established the role of Kir4.1/Kir5.1 heterotetramer of the DCT in the regulation of thiazide-sensitive Na-Cl cotransporter (NCC). Changes in Kir4.1/Kir5.1 activity of the DCT are an essential step for the regulation of NCC expression/activity induced by dietary K+ and Na+ intakes and play a role in modulating NCC by type 2 angiotensin II receptor (AT2R), bradykinin type II receptor (BK2R), and β-adrenergic receptor. Since NCC activity determines the Na+ delivery rate to the aldosterone-sensitive distal nephron (ASDN), a distal nephron segment from late DCT to CCD, Kir4.1/Kir5.1 activity plays a critical role not only in the regulation of renal Na+ absorption but also in modulating renal K+ excretion and maintaining K+ homeostasis. Thus, Kir4.1/Kir5.1 activity serves as an important component of renal K+ sensing mechanism. The main focus of this review is to provide an overview regarding the role of Kir4.1 and Kir5.1 of the DCT and CCD in the regulation of renal K+ excretion and Na+ absorption.

Renal Proximal Tubule‐Specific Ablation of Atp1a1Reveals a Novel Tonic Inhibitory Mechanism of Sodium Reabsorption

Systemic levels of endogenous cardiotonic steroids (CTS) increase markedly during salt loading, volume expansion, and renal insufficiency, suggesting a physiological role in the regulation of renal Na+ handling. Rather than the classic CTS‐mediated inhibition of Na+/K+‐ATPase (NKA)‐mediated ion transport in the renal proximal tubule (RPT), in vitro pharmacological approaches have suggested that low concentrations of CTS (in the range of those reported in the blood) may initiate NKA/Src‐mediated signaling to reduce apical Na+/H+ Exchanger‐3 (NHE3) and transepithelial Na+ flux in the RPT.To obtain genetic evidence of this putative NKA/Src mechanism in the RPT and asses its physiological impact, we used a knockdown and rescue approach in pig renal epithelial cells (LLC‐PK1) and generated a PT‐specific NKA α1 knockout mouse (RPTα1−/−) by crossing SGLT2 (sodium glucose co‐transporter 2)‐Cre mice with Floxed Atp1a1 mice. A SGLT2‐Cre/Rosa 26 system was then used to re‐introduce expression of wild‐type NKA α1 (RPT α1WT) or a Src‐null mutant NKA α1Y260A (RPTα1Y260A) with intact ion‐pumping.In cells with 90% NKA α1 knockdown compared to the parent LLC‐PK1 cell line, we observed a 50% decrease in phosphorylated NHE3 (inactive form) without change in total NHE3, and a 50% increase in total Sodium‐Bicarbonate cotransporter‐1A (NBCe1A) expression. Comparable NHE3 activation with NBCe1A increase was observed when NKA α1 knockdown cells were rescued with a Src‐binding NKA α1 null‐mutant or non‐src binding NKA α2, but not with Src‐binding gain‐of‐function α2 mutant or the WT NKA α1, suggesting a role for NKA/Src receptor function in the tonic inhibition of NHE3.RPT‐specific KO and rescue confirmed by immunohistochemistry in kidney cross‐section from RPTα1−/−, RPTα1WT, and RPTα1Y260A mice did not alter kidney size, morphology or overall structure as assessed by periodic acid shift (PAS) and Masson’s trichrome staining. Western blot analyses of RPTα1−/− cortex indicated a decrease in phosphorylated NHE3 with no change in total NHE3 and an increase in NBCe1A expression comparable to those observed in vitro. Functionally, a 65% decrease in daily urine output and absolute Na+ excretion was observed in RPTα1−/− mice (n=6–10). Consistent with a NKA‐dependent tonic inhibition of NHE3, increased PT Na+ reabsorption was indicated by a 65% decrease in urinary lithium clearance in RPTα1−/−, with no change in glomerular filtration rate measured by FITC‐sinistrin clearance (n=10–12). The absolute Na+ excretion was rescued in the RPTα1WT but not in the RPTα1Y260A mouse, which supported the role of NKA α1/Src signaling in the PT Na+ reabsorption (n=5).These studies reveal a novel mechanism of tonic inhibition of NHE3 and NBCe1A by Atp1a1. In vitro results provide genetic evidence that NKA/Src receptor function is critical to this mechanism, which was corroborated in vivo. Animal studies further indicate a significant physiologically impact of this hitherto unrecognized regulation of Na+ reabsorption in the PT, which may be regulated by endogenous CTS in health and disease.Support or Funding InformationAHA fellowship #19PRE34450095

Kidney dopamine D1-like receptors and angiotensin 1–7 interaction inhibits renal Na+ transporters

The role of dopamine D1-like receptors (DR) in the regulation of renal Na+ transporters, natriuresis, and blood pressure is well established. However, the involvement of the angiotensin 1-7 (ANG 1-7)-Mas receptor in the regulation of Na+ balance and blood pressure is not clear. The present study aimed to investigate the hypothesis that ANG 1-7 can regulate Na+ homeostasis by modulating the renal dopamine system. Sprague-Dawley rats were infused with saline alone (vehicle) or saline with ANG 1-7, ANG 1-7 antagonist A-779, DR agonist SKF38393, and antagonist SCH23390. Infusion of ANG 1-7 caused significant natriuresis and diuresis compared with saline alone. Both natriuresis and diuresis were blocked by A-779 and SCH23390. SKF38393 caused a significant, SCH23390-sensitive natriuresis and diuresis, and A-779 had no effect on the SKF38393 response. Concomitant infusion of ANG 1-7 and SKF38393 did not show a cumulative effect compared with either agonist alone. Treatment of renal proximal tubules with ANG 1-7 or SKF38393 caused a significant decrease in Na+-K+-ATPase and Na+/H+ exchanger isoform 3 activity. While SCH23390 blocked both ANG 1-7- and SKF38393-induced inhibition, the DR response was not sensitive to A-779. Additionally, ANG 1-7 activated PKG, enhanced tyrosine hydroxylase activity via Ser40 phosphorylation, and increased renal dopamine production. These data suggest that ANG 1-7, via PKG, enhances tyrosine hydroxylase activity, which increases renal dopamine production and activation of DR and subsequent natriuresis. This study provides evidence for a unidirectional functional interaction between two G protein-coupled receptors to regulate renal Na+ transporters and induce natriuresis.

The Influence of β1‐Adrenergic Receptor Genotype of Renal Sodium Handling and Blood Pressure Response to Angiotensin Receptor Blockers in Hypertension Patients

IntroductionThe β1‐adrenergic receptors (ADRB1) are found primarily in the heart and the juxtaglomerular cells of the kidneys. Within the kidneys, ADRB1 promote the release of renin and the activation of the renin‐angiotensin‐aldosterone system (RAAS). The RAAS plays a primary role in the regulation of renal Na+ handling and blood pressure (BP). Specifically, within the RAAS, aldosterone preferentially increases renal tubular luminal Na+ transport via increasing apical Na+ permeability. As a result, systolic and diastolic blood pressures are impacted by RAAS activity. Additionally, baseline renin levels predict Na+ sensitivity. Multiple polymorphisms of the ADRB1 have been identified as including a glycine (Gly) for arginine (Arg) substitution at amino acid 389. The Gly389 polymorphism is associated with a lower risk of hypertension and increased plasma renin levels compared to the Arg389 polymorphism. The influence of ADRB1 genotype on renin release and blood pressure suggests the functional role it may play in renal Na+ handling.PurposeThe purpose of this study was to identify the influence of ADRB1 genotype on renal Na+ handling and response to angiotensin receptor blocker (ARB) therapy in hypertension patients.Methods158 patients with controlled hypertension on a prescribed ARB completed one study visit consisting of a buccal swab collection and a medical chart review for objective BP history. Buccal swabs were analyzed for ADRB1 389 genotype (Gly389Gly = 14, Gly389Arg = 69, and Arg389Arg = 75). The relationship between genotype and Δ systolic blood pressure (SBP), Δ diastolic blood pressure (DBP), Δ mean arterial blood pressure (MAP), and the average SBP, DBP, and MAP for the last year was assessed.ResultsThere were no differences between genotype groups for age, height, weight, or BMI. There were no differences between genotype groups for ΔSBP, ΔDBP, ΔMAP (ΔSBP = −29.29 ± 2.91, −34.36 ± 3.59, −32.30 ± 3.21, ΔDBP = −16.43 ± 2.48, −11.89 ± 3.84, −13.71 ± 2.72, ΔMAP = −20.71 ± 2.22, −16.91 ± 3.71, −16.83 ± 2.81 for Gly389Gly, Gly389Arg, and Arg389Arg respectively). Additionally, there was no differences in one‐year average for DBP and MAP (DBP = 79.17 ± 2.65, 77.97 ± 0.99, 76.98 ± 0.93, MAP = 98.22 ± 2.85, 94.12 ± 0.93, 94.32 ± 0.83 for Gly389Gly, Gly389Arg, and Arg389Arg respectively). There was a statistically significant difference in one‐year average for SBP between genotype groups (136.32 ± 3.62, 126.67 ± 1.35, 128.98 ± 1.15 for Gly389Gly, Gly389Arg, and Arg389Arg respectively)ConclusionsThe present study suggests ADRB1 389 genotype influences BP response to ARB therapy in hypertension patients, particularly the one‐year average of SBP.Support or Funding InformationGeneticure Inc.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Role of Na/K‐ATPase Non‐Enzymatic Function in Proximal Tubule Sodium Handling

Systemic levels of endogenous cardiotonic steroids (CTS) increase markedly during salt loading, volume expansion and renal insufficiency, suggesting a physiological role in the regulation of renal Na+ handling. CTS are classically known as specific inhibitors of Na/K‐ATPase (NKA) α1 ion‐pumping function, which is critical for Na+ and water reabsorption in the renal proximal tubule (PT). At much lower concentrations, closer to the range of those reported for endogenous CTS in the blood, they also initiate NKA/Src‐mediated signaling in renal cell cultures. To investigate the respective roles of PT NKA α1/Src signaling and ion‐pumping function in physiological salt handling, we developed a PT‐specific NKA α1 knockout (RPTα1−/−) mouse with PT‐specific rescue by wild‐type NKA α1 (RPT α1WT) or a mutant NKA α1 with intact ion‐pumping function and blunted NKA/Src signaling (RPTα1Y260A).The PT‐specific NKA α1 knockout mouse was generated by crossing SGLT2 (sodium glucose co‐transporter 2)‐Cre mice with Floxed ATP1a1 mice. A SGLT2‐Cre/Rosa 26 system was then used to re‐introduce α1WT or α1Y260A expression at the Rosa26 locus. Mice were born with the expected Mendelian ratio and appeared phenotypically normal, with no detectable change in water and food intake, or body weight. Renal phenotyping was further assessed using metabolic cages in 2–4 month‐old male and female mice.PT‐specific KO and rescue was confirmed by immunohistochemistry in PT cells isolated from RPTα1−/−, RPTα1WT, and RPTα1Y260A mice. Kidney size, morphology and structure appeared overall normal following periodic acid shift (PAS) and trichrome staining. Functionally, in contrast with the predicted outcome based solely on the classical enzymatic function of NKA, a significant decrease in daily urine output (0.55±0.2 vs 1.76±0.3 mL/24h in RPTα1+/+, n=8–10, p<0.01) and absolute Na+ excretion (0.13±0.07 vs 0.35±0.0.5 mmol/24h in RPTα1+/+, n=6, p<0.01) were observed in the absence of PT NKA. The urinary clearance of lithium was significantly decreased in RPTα1−/− (1.42±0.3 vs 4.13±0.5 mL/min in RPTα1+/+, n=3, p<0.05), suggesting that the observed increase in Na+ reabsorption originated in the PT. The urinary output was rescued in the RPTα1WT (2.12±0.3 vs 2.16±0.4 mL/24h in RPTα1WTcontrol, n=7–10, p>0.05) but not in the RPTα1Y260A (0.42±0.1 vs 1.56±0.2 mL/24h in RPTα1Y260Acontrol, n=6–8, p<0.01), which supported the role of NKA α1/Src signaling in the PT Na+ reabsorption.These results support a previously unascertained physiological role of NKA/Src signaling in PT Na+ and water reabsorption, and warrant further investigation to assess the mechanism and possible impact on blood pressure regulation.Support or Funding InformationMarshall Institute of Interdisciplinary Research FundsThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Regulation of renal Na-(K)-Cl cotransporters by vasopressin.

Vasopressin (AVP) induces antidiuresis, thus playing an essential role in body water and electrolyte homeostasis. Its antidiuretic effects are mediated chiefly by V2 vasopressin receptors (V2R) expressed along the distal nephron and collecting duct epithelia. NaCl reabsorption in the distal nephron, which includes the thick ascending limb (TAL) and distal convoluted tubule (DCT), largely depends on the activity of two structurally related Na-(K)-Cl cotransporters, NKCC2 in TAL and NCC in DCT. AVP-induced activation of these transporters contributes to urine concentration and renal electrolyte reabsorption. Previous work has specified molecular pathways mediating the effects of V2R activation in TAL and DCT, and protein networks involved in intracellular trafficking and phosphoregulation of the two transporters have been identified. This review summarizes recent progress in understanding AVP signalling mechanisms that are responsible for the activation of NKCC2 and NCC. Implications in the pathophysiology of diseases such as nephrogenic diabetes insipidus, diabetes mellitus and salt-sensitive hypertension are discussed in this context.

Role of Serum and Glucocorticoid-Inducible Kinase (SGK)-1 in Senescence: A Novel Molecular Target Against Age-Related Diseases.

Senescence is a phenomenon characterized by a progressive decline of body homeostasis. Premature senescence acts when the cellular system is not able to adequately respond to noxious stimuli by synthesis of stressor molecules. Among those, serum-and-glucocorticoidinducible kinase-1 (SGK-1) dramatically increases under typical physiopathological conditions, such as glucocorticoid or mineralcorticoids exposure, inflammation, hyperglycemia, and ischemia. SGK-1 has been implicated in mechanism regulating oxidative stress, apoptosis, and DNA damage, which are all leading to a state of accelerating aging. Moreover, SGK-1-sensitive ion channels participate in the regulation of renal Na(+)/K(+) regulation, blood pressure, gastric acid secretion, cardiac action potential, and neuroexcitability. Recently, we demonstrated in endothelial cells as an increase in SGK-1 activity and expression reduces oxidative stress, improves cell survival and restores insulin-mediated nitric oxide production after hyperglycemia. Moreover, we showed as SGK-1 delays the onset of senescence by increasing telomerase activity, significantly decreasing reactive oxygen species (ROS) production, and by directly interacting with hTERT. Therefore, SGK-1 may represent a specific target to further develop novel therapeutic options against chronic diseases such as diabetes typical of aging. SGK-1 has been also associated with cancer, neurodegenerative diseases, and cardiovascular disease, among other age-related diseases. However, to date, the data available on SGK-1 and aging, are sparse, controversial, and only from C. elegans experimental models. In this review we sought to discuss the possible implication of SGK-1 in mechanisms regulating senescence and age-related diseases. Moreover, we aimed to discuss and identify the possible role of SGK-1 as possible molecular target to counteract and prevent aging.

PP.42.01

Objective: Serum/Glucocorticoid Regulated Kinase 1 (SGK1) plays a significant role in the regulation of renal Na+ reabsorption and K+ secretion as well as blood pressure (BP). The aim of this study was to investigate whether common variants in SGK1 gene are involved in BP responses to dietary sodium intervention or potassium supplementation. Design and method: 344 subjects from 126 families were recruited from rural areas of Northern China. After a 3-day baseline observation, they were sequentially maintained on a 7-day low-sodium diet (3 g of NaCl or 51.3mmol of sodium per day), a 7-day high-sodium diet (18 g of NaCl or 307.8mmol of sodium per day) and a 7-day high-sodium plus potassium supplementation intervention (4.5 g of KCl or 60mmol/d potassium per day). Six single nucleotide polymorphisms (SNPs) were selected from the SGK1 gene. Results: SGK1 SNP rs3813344 was significantly associated with baseline systolic blood pressure (SBP), diastolic blood pressure (DBP) and mean arterial blood pressure (MAP) (P = 0.049, 0.015 and 0.018, respectively), and rs9389154 was associated with SBP at baseline (P = 0.040) after adjustment for multiple testing. In addition, rs9376026 was associated with DBP and MAP responses to low-sodium intervention significantly (P = 0.018 and 0.022, respectively). However, no associations between 6 SNPs in the SGK1 gene and BP responses to high-sodium or high-sodium plus potassium-supplementation intervention reached statistical significance. Conclusions: Our study indicates that SGK1 genetic variants may have an important role in BP response to dietary sodium intervention or potassium supplementation and BP regulation.

Lack of an effect of collecting duct-specific deletion of adenylyl cyclase 3 on renal Na+and water excretion or arterial pressure

cAMP is a key mediator of connecting tubule and collecting duct (CD) Na(+) and water reabsorption. Studies performed in vitro have suggested that CD adenylyl cyclase (AC)3 partly mediates the actions of vasopressin; however, the physiological role of CD AC3 has not been determined. To assess this, mice were developed with CD-specific disruption of AC3 [CD AC3 knockout (KO)]. Inner medullary CDs from these mice exhibited 100% target gene recombination and had reduced ANG II- but not vasopressin-induced cAMP accumulation. However, there were no differences in urine volume, urinary urea excretion, or urine osmolality between KO and control mice during normal water intake or varying degrees of water restriction in the presence or absence of chronic vasopressin administration. There were no differences between CD AC3 KO and control mice in arterial pressure or urinary Na(+) or K(+) excretion during a normal or high-salt diet, whereas plasma renin and vasopressin concentrations were similar between the two genotypes. Patch-clamp analysis of split-open cortical CDs revealed no difference in epithelial Na(+) channel activity in the presence or absence of vasopressin. Compensatory changes in AC6 were not responsible for the lack of a renal phenotype in CD AC3 KO mice since combined CD AC3/AC6 KO mice had similar arterial pressure and renal Na(+) and water handling compared with CD AC6 KO mice. In summary, these data do not support a significant role for CD AC3 in the regulation of renal Na(+) and water excretion in general or vasopressin regulation of CD function in particular.

Regulation of ion channels by the serum‐ and glucocorticoid‐inducible kinase SGK1

The ubiquitously expressed serum- and glucocorticoid-inducible kinase-1 (SGK1) is genomically regulated by cell stress (including cell shrinkage) and several hormones (including gluco- and mineralocorticoids). SGK1 is activated by insulin and growth factors through PI3K and 3-phosphoinositide-dependent kinase PDK1. SGK1 activates a wide variety of ion channels (e.g., ENaC, SCN5A, TRPV4-6, ROMK, Kv1.3, Kv1.5, Kv4.3, KCNE1/KCNQ1, KCNQ4, ASIC1, GluR6, ClCKa/barttin, ClC2, CFTR, and Orai/STIM), which participate in the regulation of transport, hormone release, neuroexcitability, inflammation, cell proliferation, and apoptosis. SGK1-sensitive ion channels participate in the regulation of renal Na(+) retention and K(+) elimination, blood pressure, gastric acid secretion, cardiac action potential, hemostasis, and neuroexcitability. A common (∼3-5% prevalence in Caucasians and ∼10% in Africans) SGK1 gene variant is associated with increased blood pressure and body weight as well as increased prevalence of type II diabetes and stroke. SGK1 further contributes to the pathophysiology of allergy, peptic ulcer, fibrosing disease, ischemia, tumor growth, and neurodegeneration. The effect of SGK1 on channel activity is modest, and the channels do not require SGK1 for basic function. SGK1-dependent ion channel regulation may thus become pathophysiologically relevant primarily after excessive (pathological) expression. Therefore, SGK1 may be considered an attractive therapeutic target despite its broad range of functions.

SGK regulation of renal sodium transport.

The serum and glucocorticoid regulated kinase (SGK) family of protein kinases shares similar biochemical and hormonal signaling properties; however, the SGK kinases also exhibit distinct differences in regulating renal sodium (Na(+)) transport. This review will highlight recent advances in our understanding of the specificity of SGK kinase signaling and regulation of renal Na(+) transport. Differential expression of SGK kinases at the cellular and subcellular levels contributes to signaling specificity. New evidence indicates that SGK1 associates with the apical cell membrane of cortical collecting duct cells to regulate open probability of the epithelial Na(+) channel (ENaC). Scaffold proteins can also recruit SGK1 to multiprotein complexes for regulation of ENaC expression in the apical membrane. Recent SGK1 knockout models have implicated the NaCl co-transporter (NCC) as another target of SGK1 regulation. Less is known about the function of SGK2 or SGK3, but both kinases can regulate Na(+)/H(+) exchanger 3 (NHE3) activity. The SGK kinases assume distinct roles in regulating Na transport in both proximal and distal elements of the kidney tubule. Future examination of the molecular mechanisms by which the SGK kinases regulate specific substrates will inform our understanding of how these kinases contribute to the physiology of renal Na(+) transport.

Sorting protein‐related receptor SorLA is involved in regulation of renal Na+‐K+‐2Cl‐cotransporter through interaction with an isoform of calcineurin phosphatase

Activity of the Na+‐K+‐2Cl‐cotransporter (NKCC2) of the thick ascending limb (TAL) is dependent on its N‐terminal phosphorylation. Sorting protein‐related receptor SorLA was shown to facilitate this process. To clarify the interference between SorLA and NKCC2 we have evaluated the profile of relevant kinases and phosphatases in TAL of SorLA‐deficient (Sorla−/−) compared to wild‐type (WT) mice.SorLA‐deficiency was associated with nearly absent NKCC2 phosphorylation (−97%). Levels of the SPAK‐OSR1 kinases were not changed while the abundance of the calcineurin phosphatase PP2BAβ was significantly increased in SorLA‐deficiencient animals (+201%). Confocal analysis revealed apical accumulation of the phosphatase in TAL of Sorla−/− mice. The interaction of NKCC2 with PP2BAβ was verified by co‐immunoprecipitation. Application of a calcineurin inhibitor cyclosporine in cultured TAL cells induced an increase of NKCC2 phosphorylation.Our results establish PP2BAβ as a relevant phosphatase responsible for the dephosphorylation of NKCC2 and provide evidence that SorLA may mediate degradation of PP2BAβ thus facilitating the phosphorylation of the transporter.

Nedd4-2 Modulates Renal Na+-Cl− Cotransporter via the Aldosterone-SGK1-Nedd4-2 Pathway

Regulation of renal Na(+) transport is essential for controlling blood pressure, as well as Na(+) and K(+) homeostasis. Aldosterone stimulates Na(+) reabsorption by the Na(+)-Cl(-) cotransporter (NCC) in the distal convoluted tubule (DCT) and by the epithelial Na(+) channel (ENaC) in the late DCT, connecting tubule, and collecting duct. Aldosterone increases ENaC expression by inhibiting the channel's ubiquitylation and degradation; aldosterone promotes serum-glucocorticoid-regulated kinase SGK1-mediated phosphorylation of the ubiquitin-protein ligase Nedd4-2 on serine 328, which prevents the Nedd4-2/ENaC interaction. It is important to note that aldosterone increases NCC protein expression by an unknown post-translational mechanism. Here, we present evidence that Nedd4-2 coimmunoprecipitated with NCC and stimulated NCC ubiquitylation at the surface of transfected HEK293 cells. In Xenopus laevis oocytes, coexpression of NCC with wild-type Nedd4-2, but not its catalytically inactive mutant, strongly decreased NCC activity and surface expression. SGK1 prevented this inhibition in a kinase-dependent manner. Furthermore, deficiency of Nedd4-2 in the renal tubules of mice and in cultured mDCT(15) cells upregulated NCC. In contrast to ENaC, Nedd4-2-mediated inhibition of NCC did not require the PY-like motif of NCC. Moreover, the mutation of Nedd4-2 at either serine 328 or 222 did not affect SGK1 action, and mutation at both sites enhanced Nedd4-2 activity and abolished SGK1-dependent inhibition. Taken together, these results suggest that aldosterone modulates NCC protein expression via a pathway involving SGK1 and Nedd4-2 and provides an explanation for the well-known aldosterone-induced increase in NCC protein expression.

Expression and Activity of SGLT2 in Diabetes Induced by Streptozotocin: Relationship with the Lipid Environment

Background/Aims: Diabetes mellitus may impact on the regulation of renal Na⁺-glucose cotransporter type 2 (SGLT2), however, previous studies have yielded conflicting results on the effects of streptozotocin (STZ)-induced diabetes on SGLT-mediated glucose transport. Methods: Diabetes was induced in male Wistar rats. The studies were performed at 3 (D3), 7 (D7) and 14 (D14) days after a single i.p. injection of STZ. SGLT2 activity was measured using α-¹⁴C-methyl glucose uptake in brush-border vesicles (BBV) from renal cortex, and SGLT2 expression was assessed by immunoblotting. Phospholipids were quantified by a modification of Fiske-Subarow‘s method after being separated by thin-layer chromatography. Results: Glucose uptake was reduced in all groups of diabetic rats. SGLT2 expression decreased in D3 and D7. There was a decrease in sphingomyelin (SM) content and an increase in phosphatidylcholine (PC) content in BBV from D14 versus control, without differences in phosphatidylinositol (PI), phosphatidylserine (PS) and phosphatidylethanolamine (PE). Conclusion: The downregulation of SGLT2 activity during STZ-induced diabetes may be a protective mechanism to control the excess of circulating glucose and could be a consequence of a decrease in SGLT2 expression in D3 and D7, whereas altered activity of SGLT2 in D14 could be a consequence of changes in membrane lipid composition. However, we cannot discard the possibility that the decrease in SGLT2 activity could be due to a covalent modification of the active site of the protein.

Akt Mediates the Effect of Insulin on Epithelial Sodium Channels by Inhibiting Nedd4-2

The epithelial sodium channel (ENaC) plays an important role in transepithelial Na(+) absorption; hence its function is essential for maintaining Na(+) and fluid homeostasis and regulating blood pressure. Insulin is one of the hormones that regulates activity of ENaC. In this study, we investigated the contribution of two related protein kinases, Akt (also known as protein kinase B) and the serum- and glucocorticoid-dependent kinase (Sgk), on insulin-induced ENaC activity in Fisher rat thyroid cells expressing ENaC. Overexpression of Akt1 or Sgk1 significantly increased ENaC activity, whereas expression of a dominant-negative construct of Akt1, Akt1(K179M), decreased basal activity of ENaC. Inhibition of the endogenous expression of Akt1 and Sgk1 by short interfering RNA not only inhibited ENaC but also disrupted the stimulatory effect on ENaC of insulin and of the downstream effectors of insulin, phosphatidylinositol 3-kinase and PDK1. Conversely, overexpression of Akt1 or Sgk1 increased expression of ENaC at the cell membrane and overcame the inhibitory effect of Nedd4-2 on ENaC. Furthermore, mutation of consensus phosphorylation sites on Nedd4-2 for Akt1 and Sgk1, Ser(342) and Ser(428), completely abolished the inhibitory effect of Sgk1 and Akt1 on Nedd4-2 action. Together these data suggest that both Akt and Sgk are components of an insulin signaling pathway that increases Na(+) absorption by up-regulating membrane expression of ENaC via a regulatory system that involves inhibition of Nedd4-2.

Reactive oxygen species and the regulation of renal Na+-K+-ATPase in opossum kidney cells

Culture of opossum kidney (OK) cells over 40 passages resulted in marked overexpression of alpha(1)-subunit and beta(1)-subunit Na(+)-K(+)-ATPase, which paralleled with increases in Na(+)-K(+)-ATPase activity, and enhanced availability in hydrogen peroxide (H(2)O(2)) (J Membr Biol 212: 163-175, 2006). The present study evaluated the mechanisms involved in increased H(2)O(2) production in OK cells cultured over 40 passages (up to passage 80) and whether H(2)O(2) plays a role in the regulation of Na(+)-K(+)-ATPase expression and activity. The accumulation of H(2)O(2) in the extracellular medium over 24 h (236.5 +/- 2.7 vs. 319.3 +/- 2.9 nM) and the rate of H(2)O(2) production (5.21 +/- 0.02 vs. 7.37 +/- 0.02 nM/min) in cells with 40 passages was lower (P < 0.05) than in cells with 80 passages. The increase in H(2)O(2) production in cells with 80 passages was accompanied by overexpression of NOX1, but not of NOX2, and overexpression of SOD1, SOD2, and SOD3. Apocynin, a NADPH inhibitor, markedly attenuated the increase in H(2)O(2) production in cells with 80 passages and significantly decreased Na(+)-K(+)-ATPase activity (26% reduction) and alpha(1)-subunit Na(+)-K(+)-ATPase overexpression. Increases in Na(+)-K(+)-ATPase alpha(1)-subunit mRNA abundance were also observed in OK cells with increased number of cell passages, but apocynin did not affect transcript abundance. In conclusion, the increased availability of H(2)O(2) in the intracellular milieu plays an important role in the long-term regulation of Na(+)-K(+)-ATPase expression and activity. These cells may constitute an interesting model to study events related to oxidative stress, in which adaptation to increases in H(2)O(2) had progressively adapted in a more natural manner.

Renal tubular transport and the genetic basis of hypertensive disease

Several monogenic hypertensive disorders are caused by genetic mutations leading to the deranged function and/or regulation of renal tubular NaCl transport, such as mutations of the renal epithelial Na+ channel (ENaC) in Liddle syndrome, of the kinase WNK1 (with no K) in Gordon syndrome, and of the mineralocorticoid receptor, or of 11beta-hydroxysteroid dehydrogenase. Moreover, excessive formation of aldosterone in glucocorticoid-remediable hypertension leads to severe hypertension. Conversely, impaired function of the Na+,K+,2Cl- cotransporter (NKCC2), the renal outer medullary K+ channel (ROMK1), and the renal epithelial Cl- channel ClCKb/Barttin causes Bartter syndrome and defective Na+,Cl+ cotransporter (NCCT) Gitelman syndrome, salt-wasting disorders with hypotension. These monogenic disorders are rare, but illustrate the significance of renal tubular transport in blood pressure regulation. There is little doubt, however, that deranged renal salt reabsorption significantly contributes to essential hypertension polymorphisms of several genes participating in the regulation of renal Na+ transport have been shown to be associated with blood pressure and prevalence of hypertension. Two common genes will be discussed in more detail. The first encodes the renal Cl- channel ClCKb. A gain-of-function mutation of ClCKb, increasing channel activity by 7- to 20-fold is found in approximately 20% of unselected Caucasians and 40% of an unselected African population. The second common gene variant (prevalence, 3%-5% in unselected Caucasians), to be discussed in more detail, affects the serum and glucocorticoid inducible kinase SGK1, a kinase upregulated by mineralocorticoids and enhancing the activity of ENaC, ROMK, and Na+/K+ATPase. Both gene variants are associated with slightly increased blood pressure. SGK1 further stimulates the glucose transporter SGLT1, and the SGK1 gene variant correlates, in addition, with increased body mass index.

Role of cGMP in natriuresis and pressure-natriuresis

Cyclic GMP (cGMP) is synthesized by renal proximal tubule (RPT) cells and is exported from these cells into the renal interstitial (RI) compartment by an organic anion transporter system. We have demonstrated that RI gGMP inhibits renal Na+ reabsorption at the renal tubule. We have further demonstrated that cGMP-induced natriuresis can be blocked by intrarenal pharmacological inhibition of protein kinase G (PKG). In order to demonstrate that RI cGMP is a potent physiological regulator of Na+ excretion, we selectively decreased RI cGMP using an RI infusion of cGMP-specific phosphodiesterase (PDE), which does not cross the cell membrane and is confined to the extracellular space because of its molecular size. cGMP PDE selectively decreased extracellular RI cGMP levels, without influencing cAMP levels, and caused significant antinatriuresis. On the other hand, blockade of cGMP degradation with selective cGMP PDE inhibitor, zaprinast, increased both RI cGMP and sodium excretion, independently of systemic or renal hemodynamic changes. Therefore, extracellular RI cGMP plays an important role in the regulation of renal Na+ excretion.

Regulation of renal Na+/HCO3- cotransporter stimulation by CO2: role of phosphorylation, exocytosis and protein synthesis.

The sodium bicarbonate cotransporter (NBC1) mediates bicarbonate reabsorption in the renal proximal tubule. NBC1 activity is stimulated by 10% CO2, however, the mechanism is poorly understood. Here, we examined the mechanism of NBC1 regulation by 10% CO2 using an immortalized human proximal tubule cell line (HK2). In cells exposed to 10% CO2, the cotransporter activity (measured as deltapH/min) increased within minutes and this increase was maintained for 6 to 24 h. Early NBC1 stimulation was accompanied by increased NBC1 phosphorylation. Basolateral membrane NBC1 protein increased by 30 min and reached a maximum at 6 h. Increased NBC activity at 6 h was accounted for by increased NBC exocytosis to the basolateral membrane and not by decreased endocytosis. Latruncullin B (an actin cytoskeleton inhibitor) did not prevent CO2-induced stimulation, while nocodazole (a microtubule-disrupting agent) abrogated the stimulatory effect of 10% CO2. A significant increase in NBC1 mRNA expression level was observed at 6 h and maintained for 24 h. Total NBC1 protein increased at 12 to 24 h with 10% CO2 incubation and this effect was blocked by cycloheximide. In summary, the present study demonstrates that early activation of NBC1 activity by 10% CO2 was mediated by NBC1 phosphorylation. The stimulation of cotransporter activity observed at 6 h was due to exocytosis, while the late effect starting from 12 h was accounted for by increased protein synthesis.

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.