mTORC2 critically regulates renal potassium handling

The mTOR pathway orchestrates cellular homeostasis. The rapamycin-sensitive mTOR complex (mTORC1) in the kidney has been widely studied; however, mTORC2 function in renal tubules is poorly characterized. Here, we generated mice lacking mTORC2 in the distal tubule (Rictorfl/fl Ksp-Cre mice), which were viable and had no obvious phenotype, except for a 2.5-fold increase in plasma aldosterone. Challenged with a low-Na+ diet, these mice adequately reduced Na+ excretion; however, Rictorfl/fl Ksp-Cre mice rapidly developed hyperkalemia on a high-K+ diet, despite a 10-fold increase in serum aldosterone levels, implying that mTORC2 regulates kaliuresis. Phosphorylation of serum- and glucocorticoid-inducible kinase 1 (SGK1) and PKC-α was absent in Rictorfl/fl Ksp-Cre mice, indicating a functional block in K+ secretion activation via ROMK channels. Indeed, patch-clamp experiments on split-open tubular segments from the transition zone of the late connecting tubule and early cortical collecting duct demonstrated that Ba2+-sensitive apical K+ currents were barely detectable in the majority of Rictorfl/fl Ksp-Cre mice. Conversely, epithelial sodium channel (ENaC) activity was largely preserved, suggesting that the reduced ability to maintain K+ homeostasis is the result of impaired apical K+ conductance and not a reduced electrical driving force for K+ secretion. Thus, these data unravel a vital and nonredundant role of mTORC2 for distal tubular K+ handling

Regulation of distal tubule sodium transport: mechanisms and roles in homeostasis and pathophysiology.

Regulated Na+ transport in the distal nephron is of fundamental importance to fluid and electrolyte homeostasis. Further upstream, Na+ is the principal driver of secondary active transport of numerous organic and inorganic solutes. In the distal nephron, Na+ continues to play a central role in controlling the body levels and concentrations of a more select group of ions, including K+, Ca++, Mg++, Cl-, and HCO3-, as well as water. Also, of paramount importance are transport mechanisms aimed at controlling the total level of Na+ itself in the body, as well as its concentrations in intracellular and extracellular compartments. Over the last several decades, the transporters involved in moving Na+ in the distal nephron, and directly or indirectly coupling its movement to that of other ions have been identified, and their interrelationships brought into focus. Just as importantly, the signaling systems and their components-kinases, ubiquitin ligases, phosphatases, transcription factors, and others-have also been identified and many of their actions elucidated. This review will touch on selected aspects of ion transport regulation, and its impact on fluid and electrolyte homeostasis. A particular focus will be on emerging evidence for site-specific regulation of the epithelial sodium channel (ENaC) and its role in both Na+ and K+ homeostasis. In this context, the critical regulatory roles of aldosterone, the mineralocorticoid receptor (MR), and the kinases SGK1 and mTORC2 will be highlighted. This includes a discussion of the newly established concept that local K+ concentrations are involved in the reciprocal regulation of Na+-Cl- cotransporter (NCC) and ENaC activity to adjust renal K+ secretion to dietary intake

Protein Kinase B Alpha (PKBα) Stimulates the Epithelial Sodium Channel (ENaC) Heterologously Expressed in Xenopus laevis Oocytes by Two Distinct Mechanisms

Kinases contribute to the regulation of the epithelial sodium channel (ENaC) in a complex manner. For example, SGK1 (serum- and glucocorticoid-inducible kinase type 1) enhances ENaC surface expression by phosphorylating Nedd4-2, thereby preventing ENaC retrieval and degradation. An additional mechanism of ENaC activation by SGK1 involves an SGK consensus motif (616RSRYWS621) in the C-terminus of the channel’s -subunit. This consensus motif may also be a target for ENaC regulation by protein kinase B (PKB) known to be activated by insulin and growth factors. Therefore, we investigated a possible role of PKBin the regulation of rat ENaC heterologously expressed in Xenopus laevis oocytes. We found that recombinant PKBincluded in the pipette solution increased ENaC currents in outsideout patches by about 4-fold within 15-20 min. Replacing the serine residue S621 of the SGK consensus motif by an alanine (S621A) abolished this stimulatory effect. In co-expression experiments active PKBbut not catalytically inactive PKBsignificantly increased ENaC whole-cell currents and surface expression by more than 50 % within 24 hours of coexpression. Interestingly, this stimulatory effect was preserved in oocytes expressing ENaC with the S621A mutation. We conclude that the acute stimulatory effect of PKBinvolves a specific kinase consensus motif in the C-terminus of the channel’s -subunit. In contrast, the increase in channel surface expression caused by co-expression of PKBdoes not depend on this site in the channel and is probably mediated by an effect on channel trafficking

Bile acids inhibit human purinergic receptor P2X4 in a heterologous expression system

We recently demonstrated that bile acids, especially tauro-deoxycholic acid (t-DCA), modify the function of the acid-sensing ion channel ASIC1a and other members of the epithelial sodium channel (ENaC)/degenerin (DEG) ion channel family. Surprisingly, ASIC1 shares a high degree of structural similarity with the purinergic receptor P2X4, a nonselective cation channel transiently activated by ATP. P2X4 is abundantly expressed in the apical membrane of bile duct epithelial cells and is therefore exposed to bile acids under physiological conditions. Here, we hypothesize that P2X4 may also be modulated by bile acids and investigate whether t-DCA and other common bile acids affect human P2X4 heterologously expressed in Xenopus laevis oocytes. We find that application of either t-DCA or unconjugated deoxycholic acid (DCA; 250 mu M) causes a strong reduction (similar to 70%) of ATP-activated P2X4-mediated whole-cell currents. The inhibitory effect of 250 mu M taurochenodeoxycholic acid is less pronounced (similar to 30%), and 250 mu M chenodeoxycholic acid, cholic acid, or tauro-cholic acid did not significantly alter P2X4-mediated currents. t-DCA inhibits P2X4 in a concentration-dependent manner by reducing the efficacy of ATP without significantly changing its affinity. Single-channel patch-clamp recordings provide evidence that t-DCA inhibits P2X4 by stabilizing the channel's closed state. Using site-directed mutagenesis, we identifiy several amino acid residues within the transmembrane domains of P2X4 that are critically involved in mediating the inhibitory effect of t-DCA on P2X4. Importantly, a W46A mutation converts the inhibitory effect of t-DCA into a stimulatory effect. We conclude that t-DCA directly interacts with P2X4 and decreases ATP-activated P2X4 currents by stabilizing the closed conformation of the channel

Aldosterone-dependent and -independent regulation of the epithelial sodium channel (ENaC) in mouse distal nephron

Aldosterone is thought to be the main hormone to stimulate the epithelial sodium channel (ENaC) in the aldosterone-sensitive distal nephron (ASDN) comprising the late distal convoluted tubule (DCT2), the connecting tubule (CNT) and the entire collecting duct (CD). There is immunohistochemical evidence for an axial gradient of ENaC expression along the ASDN with highest expression in the DCT2 and CNT. However, most of our knowledge about renal ENaC function stems from studies in the cortical collecting duct (CCD). Here we investigated ENaC function in the transition zone of DCT2/CNT or CNT/CCD microdissected from mice maintained on different sodium diets to vary plasma aldosterone levels. Single-channel recordings demonstrated amiloride-sensitive Na(+) channels in DCT2/CNT with biophysical properties typical for ENaC previously described in CNT/CCD. In animals maintained on a standard salt diet, the average ENaC-mediated whole cell current (ΔI(ami)) was higher in DCT2/CNT than in CNT/CCD. A low salt diet increased ΔI(ami) in CNT/CCD but had little effect on ΔI(ami) in DCT2/CNT. To investigate whether aldosterone is necessary for ENaC activity in the DCT2/CNT, we used aldosterone synthase knockout (AS(-/-)) mice that lack aldosterone. In CNT/CCD of AS(-/-) mice, ΔI(ami) was lower than that in wild-type (WT) animals and was not stimulated by a low salt diet. In contrast, in DCT2/CNT of AS(-/-) mice, ΔI(ami) was similar to that in DCT2/CNT of WT animals both on a standard and on a low salt diet. We conclude that ENaC function in the DCT2/CNT is largely independent of aldosterone which is in contrast to its known aldosterone sensitivity in CNT/CCD

Mechanisms of renal control of potassium homeostasis in complete aldosterone deficiency

Aldosterone-independent mechanisms may contribute to K(+) homeostasis. We studied aldosterone synthase knockout (AS(-/-)) mice to define renal control mechanisms of K(+) homeostasis in complete aldosterone deficiency. AS(-/-) mice were normokalemic and tolerated a physiologic dietary K(+) load (2% K(+), 2 days) without signs of illness, except some degree of polyuria. With supraphysiologic K(+) intake (5% K(+)), AS(-/-) mice decompensated and became hyperkalemic. High-K(+) diets induced upregulation of the renal outer medullary K(+) channel in AS(-/-) mice, whereas upregulation of the epithelial sodium channel (ENaC) sufficient to increase the electrochemical driving force for K(+) excretion was detected only with a 2% K(+) diet. Phosphorylation of the thiazide-sensitive NaCl cotransporter was consistently lower in AS(-/-) mice than in AS(+/+) mice and was downregulated in mice of both genotypes in response to increased K(+) intake. Inhibition of the angiotensin II type 1 receptor reduced renal creatinine clearance and apical ENaC localization, and caused severe hyperkalemia in AS(-/-) mice. In contrast with the kidney, the distal colon of AS(-/-) mice did not respond to dietary K(+) loading, as indicated by Ussing-type chamber experiments. Thus, renal adaptation to a physiologic, but not supraphysiologic, K(+) load can be achieved in aldosterone deficiency by aldosterone-independent activation of the renal outer medullary K(+) channel and ENaC, to which angiotensin II may contribute. Enhanced urinary flow and reduced activity of the thiazide-sensitive NaCl cotransporter may support renal adaptation by activation of flow-dependent K(+) secretion and increased intratubular availability of Na(+) that can be reabsorbed in exchange for K(+) secreted