Prostaglandin E2 stimulates the epithelial sodium channel (ENaC) in cultured mouse cortical collecting duct cells in an autocrine manner

Funding: This study was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation), project number 387509280, SFB 1350), the Alexander von Humboldt Foundation (3.3-GRO/1143730 STP), the Interdisziplin ̈ares Zentrum für KlinischeForschung of Friedrich-Alexander University (IZKF, TP-A33), and the Bayerische Forschungsstiftung (PDOK-74-10).Prostaglandin E2 (PGE2) is the most abundant prostanoid in the kidney, affecting a wide range of renal functions. Conflicting data have been reported regarding the effects of PGE2 on tubular water and ion transport. The amiloride-sensitive epithelial sodium channel (ENaC) is rate limiting for transepithelial sodium transport in the aldosterone-sensitive distal nephron. The aim of the present study was to explore a potential role of PGE2 in regulating ENaC in cortical collecting duct (CCD) cells. Short-circuit current (ISC) measurements were performed using the murine mCCDcl1 cell line known to express characteristic properties of CCD principal cells and to be responsive to physiological concentrations of aldosterone and vasopressin. PGE2 stimulated amiloride-sensitive ISC via basolateral prostaglandin E receptors type 4 (EP4) with an EC50 of ∼7.1 nM. The rapid stimulatory effect of PGE2 on ISC resembled that of vasopressin. A maximum response was reached within minutes, coinciding with an increased abundance of β-ENaC at the apical plasma membrane and elevated cytosolic cAMP levels. The effects of PGE2 and vasopressin were nonadditive, indicating similar signaling cascades. Exposing mCCDcl1 cells to aldosterone caused a much slower (∼2 h) increase of the amiloride-sensitive ISC. Interestingly, the rapid effect of PGE2 was preserved even after aldosterone stimulation. Furthermore, application of arachidonic acid also increased the amiloride-sensitive ISC involving basolateral EP4 receptors. Exposure to arachidonic acid resulted in elevated PGE2 in the basolateral medium in a cyclooxygenase 1 (COX-1)-dependent manner. These data suggest that in the cortical collecting duct, locally produced and secreted PGE2 can stimulate ENaC-mediated transepithelial sodium transport.Publisher PDFPeer reviewe

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

Activation of the Epithelial Sodium Channel (ENaC) by Aldosterone in mCCDcl1 Mouse Renal Cortical Collecting Duct Cells is Dependent on SGK1 and Can be Prevented by Inhibiting Endogenous Proteases:Experimental Biology 2017

The epithelial sodium channel (ENaC) is a heteromeric channel composed of three subunits (α, β, γ) critically important for renal sodium homeostasis and the long term control of arterial blood pressure. In the cortical collecting duct aldosterone is the main hormonal regulator of ENaC activity, and SGK1 (serum and glucocorticoid-regulated kinase 1) is thought to play a pivotal role in mediating ENaC stimulation by aldosterone. Proteolytic cleavage by the Golgi-associated convertase furin at three putative furin sites (two in α- and one in γENaC) is thought to be important for ENaC maturation in the biosynthetic pathway before the channel reaches the plasma membrane. The final step in proteolytic ENaC activation probably takes place at the plasma membrane where γENaC is cleaved by membrane-bound proteases and/or extracellular proteases in a region distal to the furin cleavage site. In this study we investigated whether ENaC activation by aldosterone is dependent on SGK1 and requires proteolytic channel activation. A highly differentiated murine principal cell line (mCCDcl1) derived from microdissected cortical collecting duct was used, and cells were grown on permeable supports. ENaC-mediated sodium transport was assessed by recording the amiloride-sensitive equivalent short circuit current (ISC-Ami) in Ussing chambers for up to 6 h. Exposure of the mCCDcl1 cells to 3 nM aldosterone for 2.5 h stimulated ISC-Ami about 2.5-fold. Subsequent application of chymotrypsin (24 or 240 μg/ml) had no additional stimulatory effect. The stimulatory response to aldosterone was completely abolished by the SGK1 inhibitor GSK650394 (10 μM). In untreated control cells apical application of chymotrypsin also showed no stimulatory effect on ISC-Ami. Therefore, we hypothesized that in mCCDcl1 cells ENaC is fully activated by endogenous proteases in the presence and absence of aldosterone. To inhibit these endogenous proteases, we used an intracellularly acting convertase inhibitor (furin inhibitor-1) and extracellularly acting serine protease inhibitors (aprotinin or nafamostat). A combination of aprotinin (30 μg/ml)/nafamostat (1 μM) and furin inhibitor-1 (40 μM) essentially abolished baseline ISC-Ami. Subsequent apical application of chymotrypsin stimulated ISC-Ami back to baseline levels. Importantly, pre-treatment of the cells with furin inhibitor-1 in combination with aprotinin/nafamostat essentially prevented ENaC stimulation by aldosterone. Subsequent chymotrypsin rapidly and completely rescued the stimulatory effect of aldosterone in mCCDcl1 cells treated with protease inhibitors. We conclude that stimulation of ENaC activity by aldosterone is not only dependent on SGK1 but also requires proteolytic channel activation. It is tempting to speculate that SGK1 may contribute to the regulation of endogenous proteases necessary for channel activation. Endogenous proteases relevant for proteolytic ENaC activation under physiological and pathopyhsiological conditions remain to be elucidated.Support or Funding InformationThis work was supported by The Alexander von Humboldt Foundation (MKM) and Bayerische Forschungsstiftung (CK)

Inhibitors of the proteasome stimulate the epithelial sodium channel (ENaC) through SGK1 and mimic the effect of aldosterone

The epithelial sodium channel (ENaC) marks the tightly regulated, rate-limiting step of sodium re-absorption in the aldosterone-sensitive distal nephron (ASDN). Stimulation of ENaC activity by aldosterone involves the serum and glucocorticoid-induced kinase 1 (SGK1) and is mediated via complex mechanisms including inhibition of channel retrieval. Retrieved channels may be recycled or degraded, e.g. by the proteasomal pathway. The aim of the present study was to investigate whether inhibitors of the proteasome affect ENaC activity and surface expression, and to explore a possible involvement of SGK1. Short circuit current (ISC) measurements were performed on confluent mCCDcl1 murine cortical collecting duct cells to investigate the effect of two distinct proteasomal inhibitors, MG132 and bortezomib, on amiloride-sensitive ENaC-mediated ISC. Both inhibitors robustly stimulated amiloride-sensitive ISC. The time course and magnitude of the stimulatory effect of the proteasomal inhibitors on ISC were similar to those of aldosterone. Both, MG132 and aldosterone, significantly increased the abundance of β-ENaC at the cell surface. SGK1 activity was assessed by monitoring the phosphorylation of a downstream target, NDRG1, and was found to be increased by MG132. Importantly, inhibiting SGK1 activity prevented not only the stimulatory effect of aldosterone but also that of proteasomal inhibition. In conclusion, these data suggest that ENaC stimulation following proteasomal inhibition is due to an accumulation of active SGK1 resulting in increased expression of ENaC at the cell surface. Thus, inhibition of the proteasome mimics SGK1-dependent stimulation of ENaC by aldosterone.</p

Prostaglandin E₂ stimulates the epithelial sodium channel (ENaC) in cultured mouse cortical collecting duct cells in an autocrine manner

Prostaglandin E2 (PGE2) is the most abundant prostanoid in the kidney, affecting a wide range of renal functions. Conflicting data have been reported regarding the effects of PGE2 on tubular water and ion transport. The amiloride-sensitive epithelial sodium channel (ENaC) is rate limiting for transepithelial sodium transport in the aldosterone-sensitive distal nephron. The aim of the present study was to explore a potential role of PGE2 in regulating ENaC in cortical collecting duct (CCD) cells. Short-circuit current (ISC) measurements were performed using the murine mCCDcl1 cell line known to express characteristic properties of CCD principal cells and to be responsive to physiological concentrations of aldosterone and vasopressin. PGE2 stimulated amiloride-sensitive ISC via basolateral prostaglandin E receptors type 4 (EP4) with an EC50 of ∼7.1 nM. The rapid stimulatory effect of PGE2 on ISC resembled that of vasopressin. A maximum response was reached within minutes, coinciding with an increased abundance of β-ENaC at the apical plasma membrane and elevated cytosolic cAMP levels. The effects of PGE2 and vasopressin were nonadditive, indicating similar signaling cascades. Exposing mCCDcl1 cells to aldosterone caused a much slower (∼2 h) increase of the amiloride-sensitive ISC. Interestingly, the rapid effect of PGE2 was preserved even after aldosterone stimulation. Furthermore, application of arachidonic acid also increased the amiloride-sensitive ISC involving basolateral EP4 receptors. Exposure to arachidonic acid resulted in elevated PGE2 in the basolateral medium in a cyclooxygenase 1 (COX-1)-dependent manner. These data suggest that in the cortical collecting duct, locally produced and secreted PGE2 can stimulate ENaC-mediated transepithelial sodium transport