Chronic activation of vasopressin-2 receptors induces hypertension in Liddle mice by promoting Na+ and water retention.

The renin-angiotensin-aldosterone and arginine vasopressin-V2 receptor-aquaporin-2 (AQP2) systems converge on the epithelial Na+ channel (ENaC) to regulate blood pressure and plasma tonicity. Although it is established that V2 receptors initiate renal water reabsorption through AQP2, whether V2 receptors can also induce renal Na+ retention through ENaC and raise blood pressure remains an open question. We hypothesized that a specific increase in V2 receptor-mediated ENaC activity can lead to high blood pressure. Our approach was to test effects of chronic activation of V2 receptors in Liddle mice, a genetic mouse model of high ENaC activity, and compare differences in ENaC activity, urine Na+ excretion, and blood pressure with control mice. We found that ENaC activity was elevated in Liddle mice and could not be stimulated further by administration of desmopressin (dDAVP), a V2 receptor-specific agonist. In contrast, Liddle mice showed higher levels of expression of AQP2 and aquaporin-3, but they could still respond to dDAVP infusion by increasing phospho-AQP2 expression. With dDAVP infusion, Liddle mice excreted smaller urine volume and less urine Na+ and developed higher blood pressure compared with control mice; this hypertension was attenuated with administration of the ENaC inhibitor benzamil. We conclude that V2 receptors contribute to hypertension in the Liddle mouse model by promoting primary Na+ and concomitant water retention.NEW & NOTEWORTHY Liddle syndrome is a classic model for hypertension from high epithelial Na+ channel (ENaC) activity. In the Liddle mouse model, vasopressin-2 receptors stimulate both ENaC and aquaporin-2, which increases Na+ and water retention to such an extent that hypertension ensues. Liddle mice will preserve plasma tonicity at the expense of a higher blood pressure; these data highlight the inherent limitation in which the kidney must use ENaC as a pathway to regulate both plasma tonicity and blood pressure.

NOXA1-dependent NADPH oxidase 1 signaling mediates angiotensin II activation of the epithelial sodium channel.

The activity of the epithelial Na+ channel (ENaC) in principal cells of the distal nephron fine-tunes renal Na+ excretion. The renin-angiotensin-aldosterone system modulates ENaC activity to control blood pressure, in part, by influencing Na+ excretion. NADPH oxidase activator 1-dependent NADPH oxidase 1 (NOXA1/NOX1) signaling may play a key role in angiotensin II (ANG II)-dependent activation of ENaC. The present study aimed to explore the role of NOXA1/NOX1 signaling in ANG II-dependent activation of ENaC in renal principal cells. Patch-clamp electrophysiology and principal cell-specific Noxa1 knockout (PC-Noxa1 KO) mice were used to determine the role of NOXA1/NOX1 signaling in ANG II-dependent activation of ENaC. The activity of ENaC in the luminal plasma membrane of principal cells was quantified in freshly isolated split-opened tubules using voltage-clamp electrophysiology. ANG II significantly increased ENaC activity. This effect was robust and observed in response to both acute (40 min) and more chronic (48-72 h) ANG II treatment of isolated tubules and mice, respectively. Inhibition of ANG II type 1 receptors with losartan abolished ANG II-dependent stimulation of ENaC. Similarly, treatment with ML171, a specific inhibitor of NOX1, abolished stimulation of ENaC by ANG II. Treatment with ANG II failed to increase ENaC activity in principal cells in tubules isolated from the PC-Noxa1 KO mouse. Tubules from wild-type littermate controls, though, retained their ability to respond to ANG II with an increase in ENaC activity. These results indicate that NOXA1/NOX1 signaling mediates ANG II stimulation of ENaC in renal principal cells. As such, NOXA1/NOX1 signaling in the distal nephron plays a central role in Na+ homeostasis and control of blood pressure, particularly as it relates to regulation by the renin-ANG II axis.NEW & NOTEWORTHY Activity of the epithelial Na+ channel (ENaC) in the distal nephron fine-tunes renal Na+ excretion. Angiotensin II (ANG II) has been reported to enhance ENaC activity. Emerging evidence suggests that NADPH oxidase (NOX) signaling plays an important role in the stimulation of ENaC by ANG II in principal cells. The present findings indicate that NOX activator 1/NOX1 signaling mediates ANG II stimulation of ENaC in renal principal cells.

High salt intake induces collecting duct HDAC1-dependent NO signaling.

We reported that high salt (HS) intake stimulates renal collecting duct (CD) endothelin (ET) type B receptor (ETBR)/nitric oxide (NO) synthase 1ß (NOS1ß)-dependent NO production inhibiting the epithelial sodium channel (ENaC) promoting natriuresis. However, the mechanism underlying the HS-induced increase of NO production is unclear. Histone deacetylase 1 (HDAC1) responds to increased fluid flow, as can occur in the CD during HS intake. The renal inner medulla (IM), in particular the IMCD, has the highest NOS1 activity within the kidney. Hence, we hypothesized that HS intake provokes HDAC1 activation of NO production in the IM. HS intake for 1 wk significantly increased HDAC1 abundance in the IM. Ex vivo treatment of dissociated IM from HS-fed mice with a selective HDAC1 inhibitor (MS-275) decreased NO production with no change in ET-1 peptide or mRNA levels. We further investigated the role of the ET-1/ETBR/NOS1ß signaling pathway with chronic ETBR blockade (A-192621). Although NO was decreased and ET-1 levels were elevated in the dissociated IM from HS-fed mice treated with A-192621, ex vivo MS-275 did not further change NO or ET-1 levels suggesting that HDAC1-mediated NO production is regulated at the level or downstream of ETBR activation. In split-open CDs from HS-fed mice, patch clamp analysis revealed significantly higher ENaC activity after MS-275 pretreatment, which was abrogated by an exogenous NO donor. Moreover, flow-induced increases in mIMCD-3 cell NO production were blunted by HDAC1 or calcium inhibition. Taken together, these findings indicate that HS intake induces HDAC1-dependent activation of the ETBR/NO pathway contributing to the natriuretic response.

Renal Na+ excretion consequent to pharmacogenetic activation of Gq-DREADD in principal cells.

Stimulation of metabotropic Gq-coupled purinergic P2Y2 receptors decreases activity of the epithelial Na+ channel (ENaC) in renal principal cells of the distal nephron. The physiological consequences of P2Y2 receptor signaling disruption in the P2Y2 receptor knockout mouse are decreased Na+ excretion and increased arterial blood pressure. However, because of the global nature of this knockout model, the quantitative contribution of ENaC and distal nephron compared with that of upstream renal vascular and tubular elements to changes in urinary excretion and arterial blood pressure is obscure. Moreover, it is uncertain whether stimulation of P2Y2 receptor inhibition of ENaC is sufficient to drive renal (urinary) Na+ excretion (UNaV). Here, using a pharmacogenetic approach and selective agonism of the P2Y2 receptor, we test the sufficiency of targeted stimulation of Gq signaling in principal cells of the distal nephron and P2Y2 receptors to increase UNaV. Selective stimulation of the P2Y2 receptor with the ligand MRS2768 decreased ENaC activity in freshly isolated tubules (as assessed by patch-clamp electrophysiology) and increased UNaV (as assessed in metabolic cages). Similarly, selective agonism of hM3Dq-designer receptors exclusively activated by designer drugs (DREADD) restrictively expressed in principal cells of the distal nephron with clozapine- N-oxide decreased ENaC activity and, consequently, increased UNaV. Clozapine- N-oxide, when applied to control littermates, failed to affect ENaC and UNaV. This study represents the first use of pharmacogenetic (DREADD) technology in the renal tubule and demonstrated that selective activation of the P2Y2 receptor and Gq signaling in principal cells is sufficient to promote renal salt excretion.

Physiological regulation of the epithelial Na+ channel by casein kinase II.

epithelial Na+ channel, ENaC, is the final arbiter of sodium excretion in the kidneys. As such, discretionary control of ENaC by hormones is critical to the fine-tuning of electrolyte and water excretion and, consequently, blood pressure. Casein kinase 2 (CK2) phosphorylates ENaC. Phosphorylation by CK2 is necessary for normal ENaC activity. We tested the physiological importance of CK2 regulation of ENaC as the degree to which ENaC activity is dependent on CK2 phosphorylation in the living organism is unknown. This was addressed using patch-clamp analysis of ENaC in completely split-open collecting ducts and whole animal physiological studies of sodium excretion in mice. We also used ENaC-harboring CK2 phosphorylation site mutations to elaborate the mechanism. We found that ENaC activity in ex vivo preparations of murine collecting duct had a significant decrease in activity in response to selective antagonism of CK2. In whole animal experiments selective antagonism of CK2 caused a natriuresis similar to benzamil, but not additive to benzamil, suggesting an ENaC-dependent mechanism. Regulation of ENaC by CK2 was abolished by mutation of the canonical CK2 phosphorylation sites in beta and gamma ENaC. Together, these results demonstrate that the appropriate regulation of ENaC by CK2 is necessary for the normal physiological role played by this key renal ion channel in the fine-tuning of sodium excretion.

Collecting duct principal, but not intercalated, cell prorenin receptor regulates renal sodium and water excretion.

The collecting duct is the predominant nephron site of prorenin and prorenin receptor (PRR) expression. We previously demonstrated that the collecting duct PRR regulates epithelial Na+ channel (ENaC) activity and water transport; however, which cell type is involved remains unclear. Herein, we examined the effects of principal cell (PC) or intercalated cell (IC) PRR deletion on renal Na+ and water handling. PC or IC PRR knockout (KO) mice were obtained by crossing floxed PRR mice with mice harboring Cre recombinase under the control of the AQP2 or B1 subunit of the H+ ATPase promoters, respectively. PC KO mice had reduced renal medullary ENaC-α abundance and increased urinary Na+ losses on a low-Na+ diet compared with controls. Conversely, IC KO mice had no apparent differences in Na+ balance or ENaC abundance compared with controls. Acute treatment with prorenin increased ENaC channel number and open probability in acutely isolated cortical collecting ducts from control and IC PRR KO, but not PC PRR KO, mice. Furthermore, compared with controls, PC KO, but not IC KO mice, had increased urine volume, reduced urine osmolality, and reduced abundance of renal medullary AQP2. Taken together, these findings indicate that PC, but not IC, PRR modulates ENaC activity, urinary Na+ excretion, and water transport.

Toxicity of iron oxide nanoparticles: Size and coating effects.

Toxicological research of novel nanomaterials is a major developmental step of their clinical approval. Since iron oxide magnetic nanoparticles have a great potential in cancer treatment and diagnostics, the investigation of their toxic properties is very topical. In this paper we synthesized bovine serum albumin-coated iron oxide nanoparticles with different sizes and their polyethylene glycol derivative. To prove high biocompatibility of obtained nanoparticles the number of in vitro toxicological tests on human fibroblasts and U251 glioblastoma cells was performed. It was shown that albumin nanoparticles' coating provides a stable and biocompatible shell and prevents cytotoxicity of magnetite core. On long exposure times (48 hours), cytotoxicity of iron oxide nanoparticles takes place due to free radical production, but this toxic effect may be neutralized by using polyethylene glycol modification.

ENaC activity in the cortical collecting duct of HKα1 H+,K+-ATPase knockout mice is uncoupled from Na+ intake.

Modulation of the epithelial Na+ channel (ENaC) activity in the collecting duct (CD) is an important mechanism for normal Na+ homeostasis. ENaC activity is inversely related to dietary Na+ intake, in part due to inhibitory paracrine purinergic regulation. Evidence suggests that H+,K+-ATPase activity in the CD also influences Na+ excretion. We hypothesized that renal H+,K+-ATPases affect Na+ reabsorption by the CD by modulating ENaC activity. ENaC activity in HKα1 H+,K+-ATPase knockout (HKα1-/-) mice was uncoupled from Na+ intake. ENaC activity on a high-Na+ diet was greater in the HKα1-/- mice than in WT mice. Moreover, dietary Na+ content did not modulate ENaC activity in the HKα1-/- mice as it did in WT mice. Purinergic regulation of ENaC was abnormal in HKα1-/- mice. In contrast to WT mice, where urinary [ATP] was proportional to dietary Na+ intake, urinary [ATP] did not increase in response to a high-Na+ diet in the HKα1-/- mice and was significantly lower than in the WT mice. HKα1-/- mice fed a high-Na+ diet had greater Na+ retention than WT mice and had an impaired dipsogenic response. These results suggest an important role for the HKα1 subunit in the regulation of purinergic signaling in the CD. They are also consistent with HKα1-containing H+,K+-ATPases as important components for the proper regulation of Na+ balance and the dipsogenic response to a high-salt diet. Such observations suggest a previously unrecognized element in Na+ regulation in the CD.

Loop diuretics are K+-sparing in the presence of a low-Na+, high-K+ diet.

Under most conditions, loop diuretics are K+-wasting, requiring potassium supplementation. In this issue, Wang and colleagues demonstrate that in mice fed a low-Na+, high-K+ diet, loop diuretics, in contrast, are K+-sparing. This observation suggests that possible elevations in plasma K+ should be monitored when using a loop diuretic with a low-Na+, high-K+ diet, particularly when in combination with a potassium supplement.

Renal tubular epithelial cell prorenin receptor regulates blood pressure and sodium transport.

The physiological significance of the renal tubular prorenin receptor (PRR) has been difficult to elucidate due to developmental abnormalities associated with global or renal-specific PRR knockout (KO). We recently developed an inducible renal tubule-wide PRR KO using the Pax8/LC1 transgenes and demonstrated that disruption of renal tubular PRR at 1 mo of age caused no renal histological abnormalities. Here, we examined the role of renal tubular PRR in blood pressure (BP) regulation and Na(+) excretion and investigated the signaling mechanisms by which PRR regulates Na(+) balance. No detectable differences in BP were observed between control and PRR KO mice fed normal- or low-Na(+) diets. However, compared with controls, PRR KO mice had elevated plasma renin concentration and lower cumulative Na(+) balance with normal- and low-Na(+) intake. PRR KO mice had an attenuated hypertensive response and reduced Na(+) retention following angiotensin II (ANG II) infusion. Furthermore, PRR KO mice had significantly lower epithelial Na(+) channel (ENaC-α) expression. Treatment with mouse prorenin increased, while PRR antagonism decreased, ENaC activity in isolated split-open collecting ducts (CD). The prorenin effect was prevented by protein kinase A and Akt inhibition, but unaffected by blockade of AT1, ERK1/2, or p38 MAPK pathways. Taken together, these data indicate that renal tubular PRR, likely via direct prorenin/renin stimulation of PKA/Akt-dependent pathways, stimulates CD ENaC activity. Absence of renal tubular PRR promotes Na(+) wasting and reduces the hypertensive response to ANG II.

Binding of human myeloperoxidase to red blood cells: Molecular targets and biophysical consequences at the plasma membrane level.

Myeloperoxidase (MPO) is an oxidant-producing enzyme that can also bind to cellular surface proteins. We found that band 3 protein and glycophorins A and B were the key MPO-binding targets of human red blood cells (RBCs). The interaction of MPO with RBC proteins was mostly electrostatic in nature because it was inhibited by desialation, exogenic sialic acid, high ionic strength, and extreme pH. In addition, MPO failed to interfere with the lectin-induced agglutination of RBCs, suggesting a minor role of glycan-recognizing mechanisms in MPO binding. Multiple biophysical properties of RBCs were altered in the presence of native (i.e., not hypochlorous acid-damaged) MPO. These changes included transmembrane potential, availability of intracellular Ca(2+), and lipid organization in the plasma membrane. MPO-treated erythrocytes became larger in size, structurally more rigid, and hypersensitive to acidic and osmotic hemolysis. Furthermore, we found a significant correlation between the plasma MPO concentration and RBC rigidity index in type-2 diabetes patients with coronary heart disease. These findings suggest that MPO functions as a mediator of novel regulatory mechanism in microcirculation, indicating the influence of MPO-induced abnormalities on RBC deformability under pathological stress conditions.

NOS1-dependent negative feedback regulation of the epithelial sodium channel in the collecting duct.

With an increase in urine flow there is a significant increase in shear stress against the renal epithelium including the inner medullary collecting duct, resulting in an increase in nitric oxide (NO) production. The mechanisms of the shear stress-mediated increases in NO are undetermined. Previous studies found that shear stress increases epithelial sodium channel (ENaC) open probability and endothelin (ET)-1 production in an ENaC-dependent mechanism in the collecting duct (CD). Given that ET-1 stimulates NO production in the CD, we hypothesized that shear stress-induced NO production is downstream of shear stress-induced ENaC activation and ET-1 production in a negative feedback loop. We determined that nitric oxide synthase 1 (NOS1) and NOS3 contribute to shear stress-mediated NO production in the CD, that is attenuated by low doses of the ENaC inhibitors amiloride and benzamil. Moreover, ETB receptor blockade significantly blunted the shear stress-mediated NO production. We further elucidated whether mice lacking NOS1 in the collecting duct (CDNOS1KO) have an impaired renal ET-1 system in the CD. Although urinary ET-1 production and inner medullary ET receptor expression were similar between flox control and CDNOS1KO mice, acute ET-1 treatment significantly reduced ENaC open probability in CDs from flox mice but not CDNOS1KO mice compared with basal. Basal ENaC activity in CDs was similar between the genotypes. We conclude that during acute shear stress across the CD, ENaC acts in a negative feedback loop to stimulate NO production in an ETB/NOS1-dependent manner resulting in a decrease in ENaC open probability and promoting natriuresis.

Activation of ENaC by AVP contributes to the urinary concentrating mechanism and dilution of plasma.

Arginine vasopressin (AVP) activates the epithelial Na(+) channel (ENaC). The physiological significance of this activation is unknown. The present study tested if activation of ENaC contributes to AVP-sensitive urinary concentration. Consumption of a 3% NaCl solution induced hypernatremia and plasma hypertonicity in mice. Plasma AVP concentration and urine osmolality increased in hypernatremic mice in an attempt to compensate for increases in plasma tonicity. ENaC activity was elevated in mice that consumed 3% NaCl solution compared with mice that consumed a diet enriched in Na(+) with ad libitum tap water; the latter diet does not cause hypernatremia. To determine whether the increase in ENaC activity in mice that consumed 3% NaCl solution served to compensate for hypernatremia, mice were treated with the ENaC inhibitor benzamil. Coadministration of benzamil with 3% NaCl solution decreased urinary osmolality and increased urine flow so that urinary Na(+) excretion increased with no effect on urinary Na(+) concentration. This decrease in urinary concentration further increased plasma Na(+) concentration, osmolality, and AVP concentration in these already hypernatremic mice. Benzamil similarly compromised urinary concentration in water-deprived mice and in mice treated with desmopressin. These results demonstrate that stimulation of ENaC by AVP plays a critical role in water homeostasis by facilitating urinary concentration, which can compensate for hypernatremia or exacerbate hyponatremia. The present findings are consistent with ENaC in addition to serving as a final effector of the renin-angiotensin-aldosterone system and blood pressure homeostasis, also playing a key role in water homeostasis by regulating urine concentration and dilution of plasma.

Aldosterone-independent regulation of the epithelial Na+ channel (ENaC) by vasopressin in adrenalectomized mice.

The epithelial Na(+) channel (ENaC) in the aldosterone-sensitive distal nephron (ASDN) is under negative-feedback regulation by the renin-angiotensin-aldosterone system in protection of sodium balance and blood pressure. We test here whether aldosterone is necessary and sufficient for ENaC expression and activity in the ASDN. Surprisingly, ENaC expression and activity are robust in adrenalectomized (Adx) mice. Exogenous mineralocorticoid increases ENaC activity equally well in control and Adx mice. Plasma [AVP] is significantly elevated in Adx vs. control mice. Vasopressin (AVP) stimulates ENaC. Inhibition of the V(2) AVP receptor represses ENaC activity in Adx mice. The absence of aldosterone combined with elevated AVP release compromises normal feedback regulation of ENaC in Adx mice in response to changes in sodium intake. These results demonstrate that aldosterone is sufficient but not necessary for ENaC activity in the ASDN. Aldosterone-independent stimulation by AVP shifts the role of ENaC in the ASDN from protecting Na(+) balance to promoting water reabsorption. This stimulation of ENaC likely contributes to the hyponatremia of adrenal insufficiency.

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.

Adenylyl cyclase VI mediates vasopressin-stimulated ENaC activity.

Vasopressin modulates sodium reabsorption in the collecting duct through adenylyl cyclase-stimulated cyclic AMP, which exists as multiple isoforms; the specific isoform involved in vasopressin-stimulated sodium transport is unknown. To assess this, we studied mice deficient in adenylyl cyclase type VI specifically in the principal cells of the collecting duct. Knockout mice had increased urine volume and reduced urine sodium concentration, but regardless of the level of sodium intake, they did not exhibit significant alterations in urinary sodium excretion, arterial pressure, or pulse rate. Plasma renin concentration was elevated in knockout mice, however, suggesting a compensatory response. Valsartan significantly reduced arterial pressure in knockout mice but not in controls. Knockout mice had decreased renal cortical mRNA content of all three epithelial sodium channel (ENaC) isoforms, and total cell sodium channel isoforms α and γ were reduced in these animals. Patch-clamp analysis of split-open cortical collecting ducts revealed no difference in baseline activity of sodium channels, but knockout mice had abolished vasopressin-stimulated ENaC open probability and apical membrane channel number. In summary, these data suggest that adenylyl cyclase VI mediates vasopressin-stimulated ENaC activity in the kidney.

Synergistic Antitumor Activity of Talazoparib and Temozolomide in Malignant Rhabdoid Tumors.

Malignant rhabdoid tumors (MRTs) are among the most aggressive and treatment-resistant malignancies affecting infants, originating in the kidney, brain, liver, and soft tissues. The 5-year event-free survival rate for these cancers is a mere 20%. In nearly all cases of MRT, the SMARCB1 gene (occasionally SMARCA4)-a pivotal component of the SWI/SNF chromatin remodeling complex-is homozygously deleted, although the precise etiology of these tumors remains unknown. While young patients with localized MRT generally show improved outcomes, especially those who are older and have early-stage disease, the overall prognosis remains poor despite optimal standard treatments. This highlights the urgent need for more effective treatment strategies. We investigated the antitumor activity of a PARP1 inhibitor (talazoparib, TLZ) combined with a DNA alkylating agent (temozolomide, TMZ) in MRT xenograft models. PARP1 is a widely targeted molecule in cancer treatment and, beyond its role in DNA repair, it participates in transcriptional regulation by recruiting chromatin remodeling complexes to modulate DNA accessibility for RNA polymerases. To widen the therapeutic window of the drug combination, we employed PEGylated TLZ (PEG~TLZ), which has been reported to reduce systemic toxicity through slow drug release. Remarkably, our findings indicate that five out of six MRT xenografts exhibited an objective response to PEG~TLZ+TMZ therapy. Significantly, the loss of SMARCB1 was found to confer a protective effect, correlating with higher expression levels of DNA damage and repair proteins in SMARCB1-deficient MRT cells. Additionally, we identified MGMT as a potential biomarker indicative of in vivo MRT response to PEG~TLZ+TMZ therapy. Moreover, our analysis revealed alterations in signaling pathways associated with the observed antitumor efficacy. This study presents a novel and efficacious therapeutic approach for MRT, along with a promising candidate biomarker for predicting tumor response.

Flow-sensitive K+-coupled ATP secretion modulates activity of the epithelial Na+ channel in the distal nephron.

The epithelial Na(+) channel (ENaC) in the aldosterone-sensitive distal nephron (ASDN) is under tonic inhibition by a local purinergic signaling system responding to changes in dietary sodium intake. Normal BK(Ca) channel function is required for flow-sensitive ATP secretion in the ASDN. We tested here whether ATP secreted through connexin channels in a coupled manner with K(+) efflux through BK(Ca) channels is required for inhibitory purinergic regulation of ENaC in response to increases in sodium intake. Inhibition of connexin channels relieves purinergic inhibition of ENaC. Deletion of the BK-ß4 regulatory subunit, which is required for normal BK(Ca) channel function and flow-sensitive ATP secretion in the ASDN, suppresses increases in urinary ATP in response to increases in sodium intake. As a consequence, ENaC activity, particularly in the presence of high sodium intake, is inappropriately elevated in BK-ß4 null mice. ENaC in BK-ß4 null mice, however, responds normally to exogenous ATP, indicating that increases in activity do not result from end-organ resistance but rather from lowered urinary ATP. Consistent with this, disruption of purinergic regulation increases ENaC activity in wild type but not BK-ß4 null mice. Consequently, sodium excretion is impaired in BK-ß4 null mice. These results demonstrate that the ATP secreted in the ASDN in a BK(Ca) channel-dependent manner is physiologically available for purinergic inhibition of ENaC in response to changes in sodium homeostasis. Impaired sodium excretion resulting form loss of normal purinergic regulation of ENaC in BK-ß4 null mice likely contributes to their elevated blood pressure.

P2Y2 receptor decreases blood pressure by inhibiting ENaC.

Stimulating the Gq-coupled P2Y2 receptor (P2ry2) lowers blood pressure. Global knockout of P2ry2 increases blood pressure. Vascular and renal mechanisms are believed to participate in P2ry2 effects on blood pressure. To isolate the role of the kidneys in P2ry2 effects on blood pressure and to reveal the molecular and cellular mechanisms of this action, we test here the necessity of the P2ry2 and the sufficiency of Gq-dependent signaling in renal principal cells to the regulation of the epithelial Na+ channel (ENaC), sodium excretion, and blood pressure. Activating P2ry2 in littermate controls but not principal cell-specific P2ry2-knockout mice decreased the activity of ENaC in renal tubules. Moreover, deletion of P2ry2 in principal cells abolished increases in sodium excretion in response to stimulation of P2ry2 and compromised the normal ability to excrete a sodium load. Consequently, principal cell-specific knockout of P2ry2 prevented decreases in blood pressure in response to P2ry2 stimulation in the deoxycorticosterone acetate-salt (DOCA-salt) model of hypertension. In wild-type littermate controls, such stimulation decreased blood pressure in this model of hypertension by promoting a natriuresis. Pharmacogenetic activation of Gq exclusively in principal cells using targeted expression of Gq-designer receptors exclusively activated by designer drugs and clozapine N-oxide decreased the activity of ENaC in renal tubules, promoting a natriuresis that lowered elevated blood pressure in the DOCA-salt model of hypertension. These findings demonstrate that the kidneys play a major role in decreasing blood pressure in response to P2ry2 activation and that inhibition of ENaC activity in response to P2ry2-mediated Gq signaling lowered blood pressure by increasing renal sodium excretion.

Collecting duct-specific endothelin B receptor knockout increases ENaC activity.

Collecting duct (CD)-derived endothelin-1 (ET-1) acting via endothelin B (ETB) receptors promotes Na(+) excretion. Compromise of ET-1 signaling or ETB receptors in the CD cause sodium retention and increase blood pressure. Activity of the epithelial Na(+) channel (ENaC) is limiting for Na(+) reabsorption in the CD. To test for ETB receptor regulation of ENaC, we combined patch-clamp electrophysiology with CD-specific knockout (KO) of endothelin receptors. We also tested how ET-1 signaling via specific endothelin receptors influences ENaC activity under differing dietary Na(+) regimens. ET-1 significantly decreased ENaC open probability in CD isolated from wild-type (WT) and CD ETA KO mice but not CD ETB KO and CD ETA/B KO mice. ENaC activity in WT and CD ETA but not CD ETB and CD ETA/B KO mice was inversely related to dietary Na(+) intake. ENaC activity in CD ETB and CD ETA/B KO mice tended to be elevated under all dietary Na(+) regimens compared with WT and CD ETA KO mice, reaching significance with high (2%) Na(+) feeding. These results show that the bulk of ET-1 inhibition of ENaC activity is mediated by the ETB receptor. In addition, they could explain the Na(+) retention and elevated blood pressure observed in CD ET-1 KO, CD ETB KO, and CD ETA/B KO mice consistent with ENaC regulation by ET-1 via ETB receptors contributing to the antihypertensive and natriuretic effects of the local endothelin system in the mammalian CD.