Sodium-hydrogen Exchanger Type Research Articles

Functional NHE1 expression is critical to blood brain barrier integrity and sumatriptan blood to brain uptake.

Disruption of blood-brain barrier integrity and dramatic failure of brain ion homeostasis including fluctuations of pH occurs during cortical spreading depression (CSD) events associated with several neurological disorders, including migraine with aura, traumatic brain injury and stroke. NHE1 is the primary regulator of pH in the central nervous system. The goal of the current study was to investigate the role of sodium-hydrogen exchanger type 1 (NHE1) in blood brain barrier (BBB) integrity during CSD events and the contributions of this antiporter on xenobiotic uptake. Using immortalized cell lines, pharmacologic inhibition and genetic knockdown of NHE1 mitigated the paracellular uptake of radiolabeled sucrose implicating functional NHE1 in BBB maintenance. In contrast, loss of functional NHE1 in endothelial cells facilitated uptake of the anti-migraine therapeutic, sumatriptan. In female rats, cortical KCl but not aCSF selectively reduced total expression of NHE1 in cortex and PAG but increased expression in trigeminal ganglia; no changes were seen in trigeminal nucleus caudalis. Thus, in vitro observations may have a significance in vivo to increase brain sumatriptan levels. Pharmacological inhibition of NHE1 prior to cortical manipulations enhanced the efficacy of sumatriptan at early time-points but induced facial sensitivity alone. Overall, our results suggest that dysregulation of NHE1 contributes to breaches in BBB integrity, drug penetrance, and the behavioral sensitivity to the antimigraine agent, sumatriptan.

Strong and Sustained Antihypertensive Effect of Small Interfering RNA Targeting Liver Angiotensinogen

Small interfering RNAs (siRNAs) targeting hepatic angiotensinogen ( Agt) may provide long-lasting antihypertensive effects, but the optimal approach remains unclear. Here, we assessed the efficacy of a novel AGT siRNA in spontaneously hypertensive rats. Rats were treated with vehicle, siRNA (10 mg/kg fortnightly; subcutaneous), valsartan (31 mg/kg per day; oral), captopril (100 mg/kg per day; oral), valsartan+siRNA, or captopril+valsartan for 4 weeks (all groups, n=8). Mean arterial pressure (recorded via radiotelemetry) was lowered the most by valsartan+siRNA (-68±4 mm Hg), followed by captopril+valsartan (-54±4 mm Hg), captopril (-23±2 mm Hg), siRNA (-14±2 mm Hg), and valsartan (-10±2 mm Hg). siRNA and captopril monotherapies improved cardiac hypertrophy equally, but less than the dual therapies, which also lowered NT-proBNP (N-terminal pro-B-type natriuretic peptide). Glomerular filtration rate, urinary NGAL (neutrophil gelatinase-associated lipocalin), and albuminuria were unaffected by treatment. siRNA lowered circulating AGT by 97.9±1.0%, and by 99.8±0.1% in combination with valsartan. Although siRNA greatly reduced renal Ang (angiotensin) I, only valsartan+siRNA suppressed circulating and renal Ang II. This coincided with decreased renal sodium hydrogen exchanger type 3 and phosphorylated sodium chloride cotransporter abundances. Renin and plasma K+ increased with every treatment, but especially during valsartan+siRNA; no effects on aldosterone were observed. Collectively, these data indicate that Ang II elimination requires >99% suppression of circulating AGT. Maximal blockade of the renin-angiotensin system, achieved by valsartan+siRNA, yielded the greatest reduction in blood pressure and cardiac hypertrophy, whereas AGT lowering alone was as effective as conventional renin-angiotensin system inhibitors. Given its stable and sustained efficacy, lasting weeks, RNA interference may offer a unique approach to improving therapy adherence and treating hypertension.

GPR37L1 Regulates the Expression and Function of NHE3 in Renal Proximal Tubule Cells

The orphan G protein‐coupled receptor 37L1 (GPR37L1) has strong homology with the endothelin‐B receptor but it does not interact with endothelin. GPR37L1 is known to be expressed in several regions in the brain, but its expression profile and function in peripheral tissues are poorly understood. We determined the expression profile of GPR37L1 in mouse organs by RT‐PCR and immunoblot analyses and further used immunohistochemical analyses to locate the sites of GPR37L1 expression within the tissues. Quantitative RT‐PCR revealed that the expression of GPR37L1 was highest in the brain, heart, stomach, testis, and ovary and less in the kidney, liver, lung, spleen, pancreas, and skeletal muscle. Immunohistochemical analyses revealed the expression of GPR37L1 in specific regions within some organs, e.g., neurons in the granular layer of the cerebellum, epithelial cells lining the airway of the lung, epithelial cells lining the gastric mucosa, apical membrane of renal proximal tubule cells (RPTCs), early spermatids in seminiferous tubule of the testis, and granulosa cells in the secondary follicle of the ovary. Transient expression of GPR37L1 in human (h)RPTCs increased sodium transport whereas silencing of GPR37L1 decreased sodium transport. Inhibition of sodium hydrogen exchanger type 3 (NHE3) expression abrogated the GPR37L1‐mediated increase in sodium transport. Renal‐restricted silencing of Gpr37l1 in mice increased urine output and sodium excretion and decreased systolic and diastolic blood pressures. The renal‐selective silencing of GPR37L1 decreased the expression of NHE3 but not the expression of Na+/K+‐ATPase or sodium/glucose cotransporter 2. Over‐expression of GPR37L1 in hRPTCs increased the phosphorylation of ERK1/2 (1.73 ± 0.11‐fold, P<0.05) and AKT (1.89 ± 0.12‐fold, P<0.01) and decreased forskolin‐induced cAMP production (38% ± 4.9). Our findings show that in the kidney GPR37L1 increases luminal RPT sodium transport and blood pressure by increasing NHE3 expression, via the ERK1/2 and AKT pathway and increasing NHE3 function by suppressing cAMP production via Gai. Taken together, these results show that GPR37L1 expressed in RPTCs plays an important role in increasing renal sodium transport; GPR37L1 may be a novel target to design drugs to treat hypertension.Support or Funding InformationUS National Institute of Health R56DK116828, R37HL023081, P01HL074940, R01HL092196, R01DK039308, and R01DK090918, and National Kidney Foundation NKF‐MD‐Mini.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Urinary concentrating defect in mice lacking Epac1 or Epac2.

cAMP is a universal second messenger regulating a plethora of processes in the kidney. Two downstream effectors of cAMP are PKA and exchange protein directly activated by cAMP (Epac), which, unlike PKA, is often linked to elevation of [Ca2+]i. While both Epac isoforms (Epac1 and Epac2) are expressed along the nephron, their relevance in the kidney remains obscure. We combined ratiometric calcium imaging with quantitative immunoblotting, immunofluorescent confocal microscopy, and balance studies in mice lacking Epac1 or Epac2 to determine the role of Epac in renal water-solute handling. Epac1-/- and Epac2-/- mice developed polyuria despite elevated arginine vasopressin levels. We did not detect major deficiencies in arginine vasopressin [Ca2+]i signaling in split-opened collecting ducts or decreases in aquaporin water channel type 2 levels. Instead, sodium-hydrogen exchanger type 3 levels in the proximal tubule were dramatically reduced in Epac1-/- and Epac2-/- mice. Water deprivation revealed persisting polyuria, impaired urinary concentration ability, and augmented urinary excretion of Na+ and urea in both mutant mice. In summary, we report a nonredundant contribution of Epac isoforms to renal function. Deletion of Epac1 and Epac2 decreases sodium-hydrogen exchanger type 3 expression in the proximal tubule, leading to polyuria and osmotic diuresis.-Cherezova, A., Tomilin, V., Buncha, V., Zaika, O., Ortiz, P. A., Mei, F., Cheng, X., Mamenko, M., Pochynyuk, O. Urinary concentrating defect in mice lacking Epac1 or Epac2.

Avian leukosis virus subgroup J induces its receptor--chNHE1 up-regulation.

sBackgroundAvian leukosis virus subgroup J (ALV-J) is an oncogenic retrovirus which causes immunosuppression and neoplasia in meat-type and egg-type chickens. ALV-J infects host cells via specific interaction between the viral Env and the cell surface receptor —chicken sodium hydrogen exchanger type 1 (chNHE1). NHE1 involved in altering the cellular pH and playing a critical role in tumorigenesis. However, little is known about the other relationship between ALV-J and chNHE1.Methods and resultsIn ALV-J infected DF-1 cells, the mRNA level of chNHE1 was up-regulated with time-dependent manner tested by real time PCR, and accordingly, intracellular pH was increased tested by spectrofluorometer. In vivo, the mRNA level of chNHE1 was determined by real time PCR in ALV-J infected experimental chickens and field cases. The result showed that the mRNA level of chNHE1 was up-regulated after virus shedding, especially in continuous viremic shedders (CS group). However, no significant difference was found between non-shedding group (NS group) and control group. In field cases, mRNA level of chNHE1 was positively correlated with increasing ALV-J load in tumor bearing and immune tolerance chickens. Furthermore, immunohistochemistry results showed that the protein expression of chNHE1 was up-regulated in different organs of both experimental chickens and tumor bearing chickens compared with the control.ConclusionTaken together, we conclude that ALV-J induces chNHE1 up-regulation in viremia and neoplasia chickens.

Gastrorenal communication: sniffing and tasting.

A video slideshow introduction to the Symposium by organiser Pedro A. Jose can be found here.

HK-2 Human Renal Proximal Tubule Cells as a Model for G Protein–Coupled Receptor Kinase Type 4–Mediated Dopamine 1 Receptor Uncoupling

HK-2 human renal proximal tubule cells (RPTC) are commonly used in the in vitro study of "normal" RPTCs. We discovered recently that HK-2 cells are uncoupled from dopamine 1 receptor (D(1)R) adenylyl cyclase (AC) stimulation. We hypothesized that G protein-coupled receptor kinase type 4 (GRK4) single nucleotide polymorphisms may be responsible for the D(1)R/AC uncoupling in HK-2. This hypothesis was tested by genotyping GRK4 single nucleotide polymorphisms, measuring D(1)-like receptor agonist (fenoldopam)-stimulated cAMP accumulation, quantifying D(1)R inhibition of sodium transport, and testing the ability of GRK4 small interfering RNA to reverse the D(1)R/AC uncoupling. We compared HK-2 with 2 normally coupled human RPTC cell lines and 2 uncoupled RPTC cell lines. The HK-2 cell line was found to have 4 of 6 potential GRK4 single nucleotide polymorphisms known to uncouple the D(1)R from AC (namely, R65L, A142V, and A486V). AC response to fenoldopam stimulation was increased in the 2 normally coupled human RPTC cell lines (FEN: 2.02+/-0.05-fold and 2.33+/-0.19-fold over control; P<0.001; n=4) but not in the 2 uncoupled or HK-2 cell lines. GRK4 small interfering RNA rescued the fenoldopam-mediated AC stimulation in the uncoupled cells, including HK-2. The expected fenoldopam-mediated inhibition of sodium hydrogen exchanger type 3 was absent in HK-2 (n=6) and uncoupled RPTC cell lines (n=6) but was observed in the 2 normally coupled human RPTC cell lines (-25.41+/-4.7% and -27.36+/-2.70%; P<0.001; n=6), which express wild-type GRK4. Despite the fact that HK-2 cells retain many functional characteristics of RPTCs, they are not normal from the perspective of dopaminergic function.

Upregulation of Renal Sodium Transporters in D 5 Dopamine Receptor–Deficient Mice

D(5) dopamine receptor (D(5)R)-deficient (D(5)(-/-)) mice have hypertension that is aggravated by an increase in sodium intake. The present experiments were designed to test the hypothesis that a dysregulation of renal sodium transporters is related to the salt sensitivity in D(5)(-/-) mice. D(5)R was expressed in the renal proximal tubule, thick ascending limb, distal convoluted tubule, and cortical and outer medullary collecting ducts in D(5)(+/+) mice. On a control Na(+) diet, renal protein expressions of NKCC2 (sodium-potassium-2 chloride cotransporter), sodium chloride cotransporter, and alpha and gamma subunits of the epithelial sodium channel were greater in D(5)(-/-) than in D(5)(+/+) mice. Renal renin abundance and urine aldosterone levels were similar but renal angiotensin II type 1 receptor (AT(1)R) protein expression was increased in D(5)(-/-) mice. An elevated Na(+) diet increased further the elevated blood pressure of D(5)(-/-) mice but did not affect the normal blood pressure of D(5)(+/+) mice. The increased levels of NKCC2, sodium chloride cotransporter, and alpha and gamma subunits of the epithelial sodium channel persisted with the elevated Na(+) diet and unaffected by chronic AT(1)R blockade (losartan) in D(5)(-/-) mice. The expressions of proximal sodium transporters NHE3 (sodium hydrogen exchanger type 3) and NaPi2 (sodium phosphate cotransporter type 2) were increased by the elevated Na(+) diet in D(5)(-/-) mice; the increased expression of NHE3 but not NaPi2 was abolished by AT(1)R blockade. Our findings suggest that the increased protein expression of sodium transporters/channels in distal nephron segments may be the direct consequence of the disruption of D(5)R, independent of the renin-angiotensin aldosterone system.

Dipeptidyl peptidase IV inhibition downregulates Na+-H+exchanger NHE3 in rat renal proximal tubule

In the microvillar microdomain of the kidney brush border, sodium hydrogen exchanger type 3 (NHE3) exists in physical complexes with the serine protease dipeptidyl peptidase IV (DPPIV). The purpose of this study was to explore the functional relationship between NHE3 and DPPIV in the intact proximal tubule in vivo. To this end, male Wistar rats were treated with an injection of the reversible DPPIV inhibitor Lys [Z(NO2)]-pyrrolidide (I40; 60 mg.kg(-1).day(-1) ip) for 7 days. Rats injected with equal amounts of the noninhibitory compound Lys[Z(NO2)]-OH served as controls. Na(+) - H(+) exchange activity in isolated microvillar membrane vesicles was 45 +/- 5% decreased in rats treated with I40. Membrane fractionation studies using isopycnic centrifugation revealed that I40 provoked redistribution of NHE3 along with a small fraction of DPPIV from the apical enriched microvillar membranes to the intermicrovillar microdomain of the brush border. I40 significantly increased urine output (67 +/- 9%; P < 0.01), fractional sodium excretion (63 +/- 7%; P < 0.01), as well as lithium clearance (81 +/- 9%; P < 0.01), an index of end-proximal tubule delivery. Although not significant, a tendency toward decreased blood pressure and plasma pH/HCO(3)(-) was noted in I40-treated rats. These findings indicate that inhibition of DPPIV catalytic activity is associated with inhibition of NHE3-mediated NaHCO3 reabsorption in rat renal proximal tubule. Inhibition of apical Na(+) - H(+) exchange is due to reduced abundance of NHE3 protein in the microvillar microdomain of the kidney brush border. Moreover, this study demonstrates a physiologically significant interaction between NHE3 and DPPIV in the intact proximal tubule in vivo.

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

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

NHE3 phosphorylation at serines 552 and 605 does not directly affect NHE3 activity

Direct phosphorylation of sodium hydrogen exchanger type 3 (NHE3) is a well-established physiological phenomenon; however, the exact role of NHE3 phosphorylation in its regulation remains unclear. The objective of this study was to evaluate whether NHE3 phosphorylation at serines 552 and 605 is physiologically regulated in vivo and, if so, whether changes in phosphorylation at these sites are tightly coupled to changes in transport activity. To this end, we directly compared PKA-induced NHE3 inhibition with site-specific changes in NHE3 phosphorylation in vivo and in vitro. In vivo, PKA was activated using an intravenous infusion of parathyroid hormone in Sprague-Dawley rats. In vitro, PKA was activated directly in opossum kidney (OKP) cells using forskolin and IBMX. NHE3 activity was assayed in microvillar membrane vesicles in the rat model and by (22)Na uptake in the OKP cell model. In both cases, NHE3 phosphorylation at serines 552 and 605 was determined using previously characterized monoclonal phosphospecific antibodies directed to these sites. In vivo, we found dramatic changes in NHE3 phosphorylation at serines 552 and 605 with PKA activation but no corresponding alteration in NHE3 activity. This dissociation between NHE3 phosphorylation and activity was further verified in OKP cells in which phosphorylation clearly preceded transport inhibition. We conclude that although phosphorylation of NHE3 at serines 552 and 605 is regulated by PKA both in vivo and in vitro, phosphorylation of these sites does not directly alter Na(+)/H(+) exchange activity.

Initial Rate Analysis of Zoniporide Hydrolysis Degradants Using High-Performance Liquid Chromatography Coupled with Mass Spectrometric Detection

Zoniporide, N-(5-Cyclopropyl-1-quinolin-5-yl-1H-pyrazole-4-carbonyl)-guanidine methanesulfonic acid, is a sodium-hydrogen exchanger type 1 (NHE-1) inhibitor. This compound forms two major hydrolysis degradants (Degradants I and II) and therefore was formulated as an IV concentrate lyophile. The purpose of this study was to perform initial rate analysis on formation of Degradants I and II, to determine if a liquid Zoniporide formulation is feasible. Solutions of Zoniporide were placed on stability at ambient temperature (30°C), refrigerated temperature (5°C), and frozen temperature (−20°C). Initial formation rates were determined for Degradants I and II by using high-performance liquid chromatography/mass spectrometry (HPLC/MS). The initial formation rates were used to predict the time required for a degradant level of 0.1% relative to Zoniporide to be reached. The predicted times for Degradant I to reach 0.1% were 9 days, 330 days, and 30,962 days at temperatures of 30°C, 5°C, and −20°C, respectively, indicating that refrigerated or frozen storage would be required for a liquid Zoniporide formulation to be feasible and reach the target shelf life. The initial formation rates of Degradant II were approximately 1-order of magnitude lower. In Addition, samples of the Zoniporide solutions were assayed for concentrations of Degradant I and Zoniporide after 2.5 years of storage at 5°C and −20°C to determine the validity of the initial formation rate predictions. The mean experimentally determined Degradant I concentration at 5°C was within 13% of the predicted concentration, and the mean experimentally determined Degradant I concentration at −20°C was within 37% of the predicted concentration. This agreement between the experimentally determined and predicted Degradant I concentrations indicates that the initial rate method is sufficiently accurate and can be used to rapidly evaluate various formulation conditions with respect to stability.

Molecular pathogenetic mechanisms of nephrotic edema: progress in understanding

Molecular and pathogenetic mechanisms in sodium retention and water reabsorption of nephrotic edema are discussed. Are reported and analyzed molecular mechanisms about sodium retention in collecting duct cells regarding activation and surface expression of epithelial sodium channels (ENaC) and sodium–potassium-ATPase (Na,K-ATPase) by aldosterone, vasopressin, natriuretic peptide system (underfill theory): is necessary a better understanding about the dysregulation of ENaC and Na,K-ATPase surface expression and the resistance to natriuretic peptide system. Are also reported and analyzed molecular mechanisms of sodium retention in proximal tubule cells regarding intrinsic albumin toxicity upon type 3 sodium–hydrogen exchanger ionic pump and the activity of sodium–hydrogen exchanger regulatory factor protein (overfill theory): a better knowledge about the link between albumin, sodium–hydrogen exchanger type 3 (NHE3) ionic pump, sodium–hydrogen exchanger regulatory factor protein is necessary. Then molecular mechanisms of vasopressin free water retention through acquaporin water channels in collecting duct cells are discussed: further studies are necessary to understand vasopressin release pathway (osmotic/nonosmotic) and V2 receptor activation with cell surface expression of renal acquaporins water channel.

ChemInform Abstract: Discovery of Zoniporide: A Potent and Selective Sodium—Hydrogen Exchanger Type 1 (NHE‐1) Inhibitor with High Aqueous Solubility.

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Discovery of zoniporide: A potent and selective sodium–hydrogen exchanger type 1 (NHE-1) inhibitor with high aqueous solubility

Zoniporide (CP-597,396) is a potent and selective inhibitor of NHE-1, which exhibits high aqueous solubility and acceptable pharmacokinetics for intravenous administration. The discovery, synthesis, activities, and rat and dog pharmacokinetics of this compound are presented. The potency and selectivity of zoniporide may be due to the conformation that the molecule adopts due to the presence of a cyclopropyl and a 5-quinolinyl substituent on the central pyrazole ring of the molecule.