Inhibition of mTORC2 promotes natriuresis in Dahl salt-sensitive rats via the decrease of NCC and ENaC activity.

We have previously observed that prolonged administration of rapamycin, an inhibitor targeting the mammalian target of rapamycin 1 (mTORC1), partially reduced hypertension and alleviated kidney inflammation in Dahl salt-sensitive (SS) rats. In contrast, treatment with PP242, an inhibitor affecting both mTORC1/mTORC2, not only completely prevented hypertension but also provided substantial protection against kidney injury. Notably, PP242 exhibited potent natriuretic effects that were not evident with rapamycin. The primary objective of this study was to pinpoint the specific tubular sites responsible for the natriuretic effect of PP242 in SS rats subjected to either 0.4% NaCl (NS) or 4.0% NaCl (HS) diet. Acute effects of PP242 on natriuretic, diuretic, and kaliuretic responses were determined in unanesthetized SS rats utilizing benzamil, furosemide, or hydrochlorothiazide (inhibitors of ENaC, NKCC2, or NCC, respectively) either administered alone or in combination. The findings indicate that the natriuretic effects of PP242 in SS rats stem predominantly from the inhibition of NCC and a reduction of ENaC open probability. Molecular analysis revealed that mTORC2 regulates NCC activity through protein phosphorylation and ENaC activity through proteolytic cleavage in vivo. Evidence also indicated that PP242 also prevents the loss of K+ associated with the inhibition of NCC. These findings suggest that PP242 may represent an improved therapeutic approach for antihypertensive intervention, potentially controlling blood pressure and mitigating kidney injury in salt-sensitive human subjects.

Brown-Norway chromosome 1 mitigates the upregulation of proinflammatory pathways in mTAL cells and subsequent age-related CKD in Dahl SS/JrHsdMcwi rats.

Chronic kidney disease (CKD) has a strong genetic component; however, the underlying pathways are not well understood. Dahl salt-sensitive (SS)/Jr rats spontaneously develop CKD with age and are used to investigate the genetic determinants of CKD. However, there are currently several genetically diverse Dahl SS rats maintained at various institutions and the extent to which some exhibit age-related CKD is unclear. We assessed glomerulosclerosis (GS) and tubulointerstitial fibrosis (TIF) in 3- and 6-mo-old male and female SS/JrHsdMcwi, BN/NHsd/Mcwi [Brown-Norway (BN)], and consomic SS-Chr 1BN/Mcwi (SS.BN1) rats, in which chromosome 1 from the BN rat was introgressed into the genome of the SS/JrHsdMcwi rat. Rats were fed a 0.4% NaCl diet. GS (31 ± 3% vs. 7 ± 1%) and TIF (2.3 ± 0.2 vs. 0.5 ± 0.1) were significantly greater in 6-mo-old compared with 3-mo-old SS/JrHsdMcwi rats, and CKD was exacerbated in males. GS was minimal in 6- and 3-mo-old BN (3.9 ± 0.6% vs. 1.2 ± 0.4%) and SS.BN1 (2.4 ± 0.5% vs. 1.0 ± 0.3%) rats, and neither exhibited TIF. In SS/JrHsdMcwi and SS.BN1 rats, mean arterial blood pressure was significantly greater in 6-mo-old compared with 3-mo-old SS/JrHsdMcwi (162 ± 4 vs. 131 ± 2 mmHg) but not SS.BN1 (115 ± 2 vs. 116 ± 1 mmHg) rats. In 6-mo-old SS/JrHsdMcwi rats, blood pressure was significantly greater in females. RNA-sequencing analysis revealed that inflammatory pathways were upregulated in isolated medullary thick ascending tubules in 7-wk-old SS/JrHsdMcwi rats, before the development of tubule pathology, compared with SS.BN1 rats. In summary, SS/JrHsdMcwi rats exhibit robust age-related progression of medullary thick ascending limb abnormalities, CKD, and hypertension, and gene(s) on chromosome 1 have a major pathogenic role in such changes.NEW & NOTEWORTHY This study shows that the robust age-related progression of kidney disease in Dahl SS/JrHsdMcw rats maintained on a normal-salt diet is abolished in consomic SS.BN1 rats. Evidence that medullary thick ascending limb segments of SS/JrHsdMcw rats are structurally abnormal and enriched in proinflammatory pathways before the development of protein casts provides new insights into the pathogenesis of kidney disease in this model.

Hydrogen peroxide (H2O2) mediated activation of mTORC2 increases intracellular Na+ concentration in the renal medullary thick ascending limb of Henle.

Hydrogen peroxide (H2O2) production in the renal outer medulla is an important determinant of renal medullary blood flow and blood pressure (BP) salt-sensitivity in Dahl salt-sensitive (SS) rats. The mechanisms and pathways responsible for these actions are poorly understood. Recently, we have discovered that the mTOR complex 2 (mTORC2) plays a critical role in BP salt-sensitivity of SS rats by regulating Na+ homeostasis. PP242, an inhibitor of mTORC1/2 pathways exhibits potent natriuretic actions and completely prevented salt-induced hypertension in SS rats. In the present study, we have found that chronic infusion of H2O2 into the single remaining kidney of Sprague Dawley (SD) rats (3 days) stimulated the functional marker (pAKTSer473/AKT) of mTORC2 activity measured by Western Blot analysis. No changes in mTORC1 activity in OM were observed as determined by pS6Ser235/236/S6. Using fluorescent microscopy and the Na+ sensitive dye Sodium Green, we have shown that H2O2 (100 µM added in the bath) increased intracellular sodium concentration ([Na+]i) in renal medullary thick ascending limbs (mTALs) isolated from SD rats. These responses were almost completely abolished by pretreatment of mTAL with 10 µM PP242, indicating that mTORC1/2 pathways were involved in the H2O2 induced increase of [Na+]i. mTAL cell volume remained unchanged (± 1%) by H2O2 as determined by 3D reconstruction confocal laser scanning microscopy techniques. Consistent with the microscopy data, Western Blot analysis of proteins obtained from freshly isolated mTAL treated with 100 µM H2O2 exhibited increased activity/phosphorylation of AKT (pAKTSer473/AKT) that was inhibited by PP242. This was associated with increased protein activity of the apical membrane cotransporter Na+-K+-2Cl- (NKCC2) and the Na/H exchanger (NHE-3). Na+-K+-ATPase activity was increased as reflected an increase in the ratio of pNa+-K+-ATPaseSer16 to total Na+-K+-ATPase. Overall, the results indicate that H2O2 mediated activation of mTORC2 plays a key role in transducing the observed increases of cytosolic [Na+]i despite associated increases of basolateral pump activity.

NOX4/H2O2/mTORC1 Pathway in Salt-Induced Hypertension and Kidney Injury.

We have reported that a high-salt (4.0% NaCl) dietary intake activates mTORC1 and inhibition of this pathway with rapamycin blunts the chronic phase of salt-induced hypertension and renal injury in Dahl salt-sensitive (SS) rats. In SS rats, high-salt intake is known to increase the renal production of H2O2 by NOX4, the most abundant NOX isoform in the kidney, and the global knockout of NOX4 blunts salt-sensitivity in these rats. Here, we explored the hypothesis that elevations of H2O2 by NOX4 in high-salt fed SS rat stimulate mTORC1 for the full development of salt-induced hypertension and renal injury. Our in vitro studies found that H2O2 activates mTORC1 independent of PI3K/AKT and AMPK pathways. To determine the in vivo relevance of NOX4/H2O2/mTORC1 in the salt-induced hypertension, SS-Nox4 knockout (SSNox4-/-) rats were daily administrated with vehicle/rapamycin fed a high-salt diet for 21 days. Rapamycin treatment of SSNox4-/- rats had shown no augmented effect on the salt-induced hypertension nor upon indices of renal injury. Significant reductions of renal T lymphocyte and macrophage together with inhibition of cell proliferation were observed in rapamycin treated rats suggesting a role of mTORC1 independent of NOX4 in the proliferation of immune cell. Given the direct activation of mTORC1 by H2O2 and absence of any further protection from salt-induced hypertension in rapamycin-treated SSNox4-/- rats, we conclude that NOX4-H2O2 is a major upstream activator of mTORC1 that contributes importantly to salt-induced hypertension and renal injury in the SS rat model.

The early effects of uninephrectomy on rat kidney metabolic state using optical imaging.

Uninephrectomy (UNX) is known to result in structural and metabolic changes to the remaining kidney, although it is uncertain if this alters the mitochondrial redox state and how soon such changes may occur. A custom-designed fluorescence cryo-imaging technique was used to quantitatively assess the effect of UNX by measuring the levels of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) in the remaining kidney. Kidneys were snap-frozen 3 days following UNX, and the intrinsic fluorescence of NADH and FAD were optically acquired. The 3D images were created to characterize the NADH/FAD redox ratios (RR) of the right kidneys, which underwent UNX and the remaining kidneys 3 days following UNX. Both the NADPH-oxidases (Nox2 and Nox4) and the mitochondria are the main sources of reactive oxygen species (ROS) production in tubular epithelial cells. Responses to the UNX were obtained in kidneys of normal Sprague Dawley (SD) rats, Dahl salt-sensitive (SS) rats and SS rats in which NADPH-oxidase isoform 4 (Nox4) was knocked out (SSNox4-/- ). The results found that each of the strains exhibited similar increase in kidney weights averaging 17% after 3 days of UNX. SD and SSNox4-/- rats both exhibited global reductions of the RR (P < .05) with a similar tendency observed in SS rats (P < .08), indicating increased ROS production. The unexpected reduction of the RR in the remnant kidneys of SSNox4-/- rats indicates that mechanisms independent of H2 O2 produced from Nox4 may be responsible for this global increase of ROS. We propose that the reduced RR was largely a consequence of enhanced mitochondrial bioenergetics due to increased tubular workload of the remaining kidney. The data indicate that mitochondria become the dominant source of increased ROS following UNX and could represent an important hypertrophic signaling mechanism.

Voltage gated proton channels modulate mitochondrial reactive oxygen species production by complex I in renal medullary thick ascending limb.

Hv1 is a voltage-gated proton channel highly expressed in immune cells where, it acts to maintain NAD(P)H oxidase activity during the respiratory burst. We have recently reported that Hv1 is expressed in cells of the medullary thick ascending limb (mTAL) of the kidney and is critical to augment reactive oxygen species (ROS) production by this segment. While Hv1 is associated with NOX2 mediated ROS production in immune cells, the source of the Hv1 dependent ROS in mTAL remains unknown. In the current study, the rate of ROS formation was quantified in freshly isolated mTAL using dihydroethidium and ethidium fluorescence. Hv1 dependent ROS production was stimulated by increasing bath osmolality and ammonium chloride (NH4Cl) loading. Loss of either p67phox or NOX4 did not abolish the formation of ROS in mTAL. Hv1 was localized to mitochondria within mTAL, and the mitochondrial superoxide scavenger mitoTEMPOL reduced ROS formation. Rotenone significantly increased ROS formation and decreased mitochondrial membrane potential in mTAL from wild-type rats, while treatment with this inhibitor decreased ROS formation and increased mitochondrial membrane potential in mTAL from Hv1-/- mutant rats. These data indicate that NADPH oxidase is not the primary source of Hv1 dependent ROS production in mTAL. Rather Hv1 localizes to the mitochondria in mTAL and modulates the formation of ROS by complex I. These data provide a potential explanation for the effects of Hv1 on ROS production in cells independent of its contribution to maintenance of cell membrane potential and intracellular pH.

Therapeutic Suppression of mTOR (Mammalian Target of Rapamycin) Signaling Prevents and Reverses Salt-Induced Hypertension and Kidney Injury in Dahl Salt-Sensitive Rats.

mTOR (mammalian target of rapamycin) signaling has emerged as a key regulator in a wide range of cellular processes ranging from cell proliferation, immune responses, and electrolyte homeostasis. mTOR consists of 2 distinct protein complexes, mTORC1 (mTOR complex 1) and mTORC2 (mTOR complex 2) with distinct downstream signaling events. mTORC1 has been implicated in pathological conditions, such as cancer and type 2 diabetes mellitus in humans, and inhibition of this pathway with rapamycin has been shown to attenuate salt-induced hypertension in Dahl salt-sensitive rats. Several studies have found that the mTORC2 pathway is involved in the regulation of renal tubular sodium and potassium transport, but its role in hypertension has remained largely unexplored. In the present study, we, therefore, determined the effect of mTORC2 inhibition with compound PP242 on salt-induced hypertension and renal injury in salt-sensitive rats. We found that PP242 not only completely prevented but also reversed salt-induced hypertension and kidney injury in salt-sensitive rats. PP242 exhibited potent natriuretic actions, and chronic administration tended to produce a negative Na+ balance even during high-salt feeding. The results indicate that mTORC2 and the related downstream associated pathways play an important role in regulation of sodium balance and arterial pressure regulation in salt-sensitive rats. Therapeutic suppression of the mTORC2 pathway represents a novel pathway for the potential treatment of hypertension.

Role of Nox4 and p67phox subunit of Nox2 in ROS production in response to increased tubular flow in the mTAL of Dahl salt-sensitive rats.

Nox4 and Nox2 are the most abundant NADPH oxidases (Nox) in the kidney and have been shown to contribute to hypertension, renal oxidative stress, and injury in Dahl salt-sensitive (SS) hypertensive rats. The present study focused on the role of Nox4 and p67phox/Nox2 in the generation of H2O2 and O2 (·-) in the renal medullary thick ascending limb of Henle (mTAL) of SS rats in response to increasing luminal flow (from 5 to 20 nl/min). Nox4 and p67phox/Nox2 genes were found to be expressed in the mTAL of SS rats. Responses of SS rats were compared with those of SS rats with knockout of Nox4 (SS(Nox4-/-)) or functional mutation of p67phox (SS(p67phox-/-)). Nox4 was the dominant source of increased intracellular H2O2 production in response to increased luminal flow as determined using the fluorescent dye peroxyfluor 6-AM (PF6-AM). The rate of mitochondrial H2O2 production [as determined by mitochondria peroxy yellow 1 (mitoPY1)] was also significantly reduced in SS(Nox4-/-) compared with SS rats, but not in SS(p67phox-/-) rats. In contrast, intracellular superoxide (O2 (·-)) production (the ratio of ethidium to dihydroethidium) in the mTAL of SS(Nox4-/-) rats was nearly identical to that of SS rats in response to luminal flow, indicating that Nox4 made no measurable contribution. mTAL O2 (·-) production was reduced in SS(p67phox-/-) compared with SS rats at the lower luminal flow of 5 nl/min and progressively increased when perfusion was changed to 20 nl/min. We conclude that increased mTAL luminal flow results in increases in intracellular and mitochondrial H2O2, which are dependent on the presence of Nox4, and that p67phox/Nox2 accounts solely for increases in O2 (·-) production.

Evidence of the Importance of Nox4 in Production of Hypertension in Dahl Salt-Sensitive Rats.

This study reports the consequences of knocking out NADPH (nicotinamide adenine dinucleotide phosphate) oxidase 4 (Nox4) on the development of hypertension and kidney injury in the Dahl salt-sensitive (SS) rat. Zinc finger nuclease injection of single-cell SS embryos was used to create an 8 base-pair frame-shift deletion of Nox4, resulting in a loss of the ≈68 kDa band in Western blot analysis of renal cortical tissue of the knock out of Nox4 in the SS rat (SS(Nox4-/-)) rats. SS(Nox4-/-) rats exhibited a significant reduction of salt-induced hypertension compared with SS rats after 21 days of 4.0% NaCl diet (134±5 versus 151±3 mm Hg in SS) and a significant reduction of albuminuria, tubular casts, and glomerular injury. Optical fluorescence 3-dimensional cryoimaging revealed significantly higher redox ratios (NADH/FAD [reduced nicotinamide adenine dinucleotide/flavin adenine dinucleotide]) in the kidneys of SS(Nox4-/-) rats even when fed the 0.4% NaCl diet, indicating greater levels of mitochondrial electron transport chain metabolic activity and reduced oxidative stress compared with SS rats. Before the development of hypertension, RNA expression levels of Nox subunits Nox2, p67(phox), and p22(phox) were found to be significantly lower (P<0.05) in SS(Nox4-/-) compared with SS rats in the renal cortex. Thus, the mutation of Nox4 seems to modify transcription of several genes in ways that contribute to the protective effects observed in the SS(Nox4-/-) rats. We conclude that the reduced renal injury and attenuated blood pressure response to high salt in the SS(Nox4-/-) rat could be the result of multiple pathways, including gene transcription, mitochondrial energetics, oxidative stress, and protein matrix production impacted by the knock out of Nox4.

Reactive oxygen species as important determinants of medullary flow, sodium excretion, and hypertension.

The physiological evidence linking the production of superoxide, hydrogen peroxide, and nitric oxide in the renal medullary thick ascending limb of Henle (mTAL) to regulation of medullary blood flow, sodium homeostasis, and long-term control of blood pressure is summarized in this review. Data obtained largely from rats indicate that experimentally induced elevations of either superoxide or hydrogen peroxide in the renal medulla result in reduction of medullary blood flow, enhanced Na(+) reabsorption, and hypertension. A shift in the redox balance between nitric oxide and reactive oxygen species (ROS) is found to occur naturally in the Dahl salt-sensitive (SS) rat model, where selective reduction of ROS production in the renal medulla reduces salt-induced hypertension. Excess medullary production of ROS in SS rats emanates from the medullary thick ascending limbs of Henle [from both the mitochondria and membrane NAD(P)H oxidases] in response to increased delivery and reabsorption of excess sodium and water. There is evidence that ROS and perhaps other mediators such as ATP diffuse from the mTAL to surrounding vasa recta capillaries, resulting in medullary ischemia, which thereby contributes to hypertension.

Mitochondrial proteomic analysis reveals deficiencies in oxygen utilization in medullary thick ascending limb of Henle in the Dahl salt-sensitive rat.

The renal medullary thick ascending limb (mTAL) of the Dahl salt-sensitive (SS) rat is the site of enhanced NaCl reabsorption and excess superoxide production. In the present studies we isolated mitochondria from mTAL of SS and salt-resistant control strain SS.13(BN) rats on 0.4 and 8% salt diet for 7 days and performed a proteomic analysis. Purity of mTAL and mitochondria isolations exceeded 93.6 and 55%, respectively. Using LC/MS spectral analysis techniques we identified 96 mitochondrial proteins in four biological mTAL mitochondria samples, run in duplicate, as defined by proteins with a false discovery rate <5% and scan count ≥2. Seven of these 96 proteins, including IDH2, ACADM, SCOT, Hsp60, ATPA, EFTu, and VDAC2 were differentially expressed between the two rat strains. Oxygen consumption and high-resolution respirometry analyses showed that mTAL cells and the mitochondria in the outer medulla of SS rats fed high-salt diet exhibited lower rates of oxygen utilization compared with those from SS.13(BN) rats. These studies advance the conventional proteomic paradigm of focusing exclusively upon whole tissue homogenates to a focus upon a single cell type and specific subcellular organelle. The results reveal the importance of a largely unexplored role for deficiencies of mTAL mitochondrial metabolism and oxygen utilization in salt-induced hypertension and renal medullary oxidative stress.

c-Src inactivation reduces renal epithelial cell-matrix adhesion, proliferation, and cyst formation.

c-Src is a non-receptor tyrosine kinase whose activity is induced by phosphorylation at Y418 and translocation from the cytoplasm to the cell membrane. Increased activity of c-Src has been associated with cell proliferation, matrix adhesion, motility, and apoptosis in tumors. Immunohistochemistry suggested that activated (pY(418))-Src activity is increased in cyst-lining autosomal dominant polycystic kidney disease (ADPKD) epithelial cells in human and mouse ADPKD. Western blot analysis showed that SKI-606 (Wyeth) is a specific inhibitor of pY(418)-Src without demonstrable effects on epidermal growth factor receptor or ErbB2 activity in renal epithelia. In vitro studies on mouse inner medullary collecting duct (mIMCD) cells and human ADPKD cyst-lining epithelial cells showed that SKI-606 inhibited epithelial cell proliferation over a 24-h time frame. In addition, SKI-606 treatment caused a striking statistically significant decrease in adhesion of mIMCD and human ADPKD to extracellular collagen matrix. Retained viability of unattached cells was consistent with a primary effect on epithelial cell anchorage dependence mediated by the loss of extracellular matrix (ECM)-attachment due to α(2)ß(1)-integrin function. SKI-606-mediated attenuation of the human ADPKD hyperproliferative and hyper-ECM-adhesive epithelial cell phenotype in vitro was paralleled by retardation of the renal cystic phenotype of Pkd1 orthologous ADPKD heterozygous mice in vivo. This suggests that SKI-606 has dual effects on cystic epithelial cell proliferation and ECM adhesion and may have therapeutic potential for ADPKD patients.

Epidermal growth factor-mediated proliferation and sodium transport in normal and PKD epithelial cells.

Members of the epidermal growth factor (EGF) family bind to ErbB (EGFR) family receptors which play an important role in the regulation of various fundamental cell processes including cell proliferation and differentiation. The normal rodent kidney has been shown to express at least three members of the ErbB receptor family and is a major site of EGF ligand synthesis. Polycystic kidney disease (PKD) is a group of diseases caused by mutations in single genes and is characterized by enlarged kidneys due to the formation of multiple cysts in both kidneys. Tubule cells proliferate, causing segmental dilation, in association with the abnormal deposition of several proteins. One of the first abnormalities described in cell biological studies of PKD pathogenesis was the abnormal mislocalization of the EGFR in cyst lining epithelial cells. The kidney collecting duct (CD) is predominantly an absorptive epithelium where electrogenic Na(+) entry is mediated by the epithelial Na(+) channel (ENaC). ENaC-mediated sodium absorption represents an important ion transport pathway in the CD that might be involved in the development of PKD. A role for EGF in the regulation of ENaC-mediated sodium absorption has been proposed. However, several investigations have reported contradictory results indicating opposite effects of EGF and its related factors on ENaC activity and sodium transport. Recent advances in understanding how proteins in the EGF family regulate the proliferation and sodium transport in normal and PKD epithelial cells are discussed here. This article is part of a Special Issue entitled: Polycystic Kidney Disease.

EGF and its related growth factors mediate sodium transport in mpkCCDc14 cells via ErbB2 (neu/HER-2) receptor.

Amiloride-sensitive sodium entry, via the epithelial sodium channel (ENaC), is the rate-limiting step for Na(+) absorption. Epidermal growth factor (EGF) is involved in the regulation of Na(+) transport and ENaC activity. However it is still controversial exactly how EGF regulates ENaC and Na(+) absorption. The aim of the present study was to characterize the EGF regulation of Na(+) transport in cultured mouse renal collecting duct principal mpkCCD(c14) cells, a highly differentiated cell line which retains many characteristics of the cortical collecting duct (CCD). EGF dose dependently regulates basal transepithelial Na(+) transport in two phases: an acute phase (<4 h) and a chronic phase (>8 h). Similar effects were observed with TGF-alpha, HB-EGF, and amphiregulin which also belong to the EGF-related peptide growth factor family. Inhibition of MEK1/2 by PD98059 or U0126 increased acute effects and disrupted chronic effects of EGF on Na(+) reabsorption. Inhibition of PI3-kinase with LY294002 abolished acute effect of EGF. As assessed by Western blotting, ErbB2 is the most predominant member of the ErbB family detected in mpkCCD(c14) cells. Immunohistochemistry analysis revealed localization of ErbB2 in the CCD in Sprague-Dawley rat kidneys. Both acute and long-term effects of EGF were abolished when cells were treated with tyrphostin AG-825 and ErbB2 inhibitor II, chemically dissimilar selective inhibitors of the ErbB2 receptor. Thus, we conclude that EGF and its related growth factors are important for maintaining transepithelial Na(+) transport and that EGF biphasically modulates sodium transport in mpkCCD(c14) cells via the ErbB2 receptor.

Function and modulation of delta-containing GABA(A) receptors.

alphabetadelta-Containing GABA(A) receptors are (1) localized to extra- and perisynaptic membranes, (2) exhibit a high sensitivity to GABA, (3) show little desensitization, and (4) are believed to be one of the primary mediators of tonic inhibition in the central nervous system. This type of signaling appears to play a key role in controlling cell excitability. This review article briefly summarizes recent knowledge on tonic GABA-mediated inhibition. We will also consider the mechanism of action of many clinically important drugs such as anxiolytics, anticonvulsants, and sedative/hypnotics and their effects on delta-containing GABA receptor activation. We will conclude that alphabetadelta-containing GABA(A) receptors exhibit a relatively low efficacy that can be potentiated by endogenous modulators and anxiolytic agents. This scenario enables these particular GABA receptor combinations, upon neurosteroid exposure for example, to impart a profound effect on excitability in the central nervous system.

Rho small GTPases activate the epithelial Na(+) channel.

Small G proteins in the Rho family are known to regulate diverse cellular processes, including cytoskeletal organization and cell cycling, and more recently, ion channel activity and activity of phosphatidylinositol 4-phosphate 5-kinase (PI(4)P 5-K). The present study investigates regulation of the epithelial Na(+) channel (ENaC) by Rho GTPases. We demonstrate here that RhoA and Rac1 markedly increase ENaC activity. Activation by RhoA was suppressed by the C3 exoenzyme. Inhibition of the downstream RhoA effector Rho kinase, which is necessary for RhoA activation of PI(4)P 5-K, abolished ENaC activation. Similar to RhoA, overexpression of PI(4)P 5-K increased ENaC activity suggesting that production of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) in response to RhoA-Rho kinase signaling stimulates ENaC. Supporting this idea, inhibition of phosphatidylinositol 4-kinase, but not the RhoA effector phosphatidylinositol 3-kinase and MAPK cascades, markedly attenuated RhoA-dependent activation of ENaC. RhoA increased ENaC activity by increasing the plasma membrane levels of this channel. We conclude that RhoA activates ENaC via Rho kinase and subsequently activates PI(4)P 5-K with concomitant increases in PI(4,5)P(2) levels promoting channel insertion into the plasma membrane.