Insulin-induced Na+ retention in the distal nephron may contribute to the development of oedema/hypertension in patients with type 2 diabetes. This response to insulin is usually attributed to phosphatidylinositol-3-kinase (PI3K)/serum and glucocorticoid-inducible kinase 1 (SGK1) but a role for protein kinase B (PKB) has been proposed. The present study therefore aimed to clarify the way in which insulin can evoke Na + retention. EXPERIMENTAL APPROACHWe examined the effects of nominally selective inhibitors of PI3K (wortmannin, PI103, GDC-0941), SGK1 (GSK650394A) and PKB (Akti-1/2) on Na + transport in hormone-deprived and insulin-stimulated cortical collecting duct (mpkCCD) cells, while PI3K, SGK1 and PKB activities were assayed by monitoring the phosphorylation of endogenous proteins. KEY RESULTSWortmannin substantially inhibited basal Na + transport whereas PI103 and GDC-0941 had only very small effects. However, these PI3K inhibitors all abolished insulin-induced Na + absorption and inactivated PI3K, SGK1 and PKB fully. GSK650394A and Akti-1/2 also inhibited insulin-evoked Na + absorption and while GSK650394A inhibited SGK1 without affecting PKB, Akti-1/2 inactivated both kinases. CONCLUSION AND IMPLICATIONSWhile studies undertaken using PI103 and GDC-0941 show that hormone-deprived cells can absorb Na + independently of PI3K, PI3K seems to be essential for insulin induced Na + transport. Akti-1/2 does not act as a selective inhibitor of PKB and data obtained using this compound must therefore be treated with caution. GSK650394A, on the other hand, selectively inhibits SGK1 and the finding that GSK650394A suppressed insulin-induced Na + absorption suggests that this response is dependent upon signalling via PI3K/SGK1.

There is good evidence for a causal link between excessive sympathetic drive to the kidney and hypertension. We hypothesized that sympathetic regulation of tubular Na(+) absorption may occur in the aldosterone-sensitive distal nephron, where the fine tuning of renal Na(+) excretion takes place. Here, the appropriate regulation of transepithelial Na(+) transport, mediated by the amiloride-sensitive epithelial Na(+) channel (ENaC), is critical for blood pressure control. To explore a possible effect of the sympathetic transmitter norepinephrine on ENaC-mediated Na(+) transport, we performed short-circuit current (Isc) measurements on confluent mCCDcl1 murine cortical collecting duct cells. Norepinephrine caused a complex Isc response with a sustained increase of amiloride-sensitive Isc by ∼44%. This effect was concentration dependent and mediated via basolateral α2-adrenoceptors. In cells pretreated with aldosterone, the stimulatory effect of norepinephrine was reduced. Finally, we demonstrated that noradrenergic nerve fibers are present in close proximity to ENaC-expressing cells in murine kidney slices. We conclude that the sustained stimulatory effect of locally elevated norepinephrine on ENaC-mediated Na(+) absorption may contribute to the hypertensive effect of increased renal sympathetic activity.

BACKGROUND AND PURPOSEAlthough the serum and glucocorticoid-inducible protein kinase 1 (SGK1) appears to be involved in controlling epithelial Na + absorption, its role in this physiologically important ion transport process is undefined. As SGK1 activity is dependent upon target of rapamycin complex 2 (TORC2)-catalysed phosphorylation of SGK1-Ser 422, we have explored the effects of inhibiting TORC2 and/or TORC1 upon the hormonal control of Na + absorption. EXPERIMENTAL APPROACHNa + absorption was quantified electrometrically in mouse cortical collecting duct cells (mpkCCD) grown to confluence on permeable membranes. Kinase activities were assessed by monitoring endogenous protein phosphorylation, with or without TORC1/2 inhibitors (TORIN1 and PP242) and the TORC1 inhibitor: rapamycin. KEY RESULTSInhibition of TORC1/2 (TORIN1, PP242) suppressed basal SGK1 activity, prevented insulin-and dexamethasone-induced SGK1 activation, and caused modest (10-20%) inhibition of basal Na + absorption and substantial (~80%) inhibition of insulin/dexamethasone-induced Na + transport. Inhibition of TORC1 did not impair SGK1 activation or insulin-induced Na + transport, but did inhibit (~80%) dexamethasone-induced Na + absorption. Arginine vasopressin stimulated Na + absorption via a TORC1/2-independent mechanism. CONCLUSION AND IMPLICATIONSTarget of rapamycin complex 2, but not TORC1, is important to SGK1 activation. Signalling via phosphoinositide-3-kinase/TORC2/SGK1 can explain insulin-induced Na + absorption. TORC2, but not TORC1, is also involved in glucocorticoid-induced SGK1 activation but its role is permissive. Glucocorticoid-induced Na + transport displayed a requirement for TORC1 activity. Therefore, TORC1 and TORC2 contribute to the regulation of Na + absorption. Pharmacological manipulation of TORC1/2 signalling may provide novel therapies for Na + -sensitive hypertension. AbbreviationsAPC, adenomatous polyposis; AVP, arginine vasopressin; CREB, cAMP response element binding protein; ENaC, epithelial sodium channel; GSK3, glycogen synthase kinase 3; IEq, equivalent short circuit current; IGF-1, insulin-like growth factor 1; mpkCCD, mouse cortical collecting duct cells; NDRG1, protein encoded by n-myc downstream regulated gene 1; Nedd-4/2, neural precursor cell expressed, developmentally down-regulated protein 4-2; P70-S6K, 70 kDa ribosomal S6 kinase; PDK1, phosphoinositide-dependent protein kinase 1; PI3K, phosphoinositide-3-kinase; PIP3, phosphatidylinositol 3,4,5-trisphosphate; PKA, adenine-nucleotide-dependent protein kinase; PKB, protein kinase B (also known as Akt); Rt, transepithelial resistance; SGK1, serum and glucocorticoid-inducible protein kinase 1; TORC1 and 2, target of rapamycin complex 1 and 2; Vt, transepithelial voltage BJP British Journal of Pharmacology

Serum and glucocorticoid‐inducible kinase 1 (SGK1) is a protein kinase that contributes to the hormonal control of renal Na+ retention by regulating the abundance of epithelial Na+ channels (ENaC) at the apical surface of the principal cells of the cortical collecting duct (CCD). Although glucocorticoids and insulin stimulate Na+ transport by activating SGK1, the responses follow different time courses suggesting that these hormones act by different mechanisms. We therefore explored the signaling pathways that allow dexamethasone and insulin to stimulate Na+ transport in mouse CCD cells (mpkCCD cl4). Dexamethasone evoked a progressive augmentation of electrogenic Na+ transport that became apparent after ~45 min latency and was associated with increases in SGK1 activity and abundance and with increased expression of SGK1 mRNA. Although the catalytic activity of SGK1 is maintained by phosphatidylinositol‐OH‐3‐kinase (PI3K), dexamethasone had no effect upon PI3K activity. Insulin also stimulated Na+ transport but this response occurred with no discernible latency. Moreover, although insulin also activated SGK1, it had no effect upon SGK1 protein or mRNA abundance. Insulin did, however, evoke a clear increase in cellular PI3K activity. Our data are consistent with earlier work, which shows that glucocorticoids regulate Na+ retention by inducing sgk1 gene expression, and also establish that this occurs independently of increased PI3K activity. Insulin, on the other hand, stimulates Na+ transport via a mechanism independent of sgk1 gene expression that involves PI3K activation. Although both hormones act via SGK1, our data show that they activate this kinase by distinct physiological mechanisms.

Background and purpose: Peroxisome proliferator-activated receptor g (PPARg) agonists, such as rosiglitazone and pioglitazone, sensitize cells to insulin, and are therefore used to treat type 2 diabetes. However, in some patients, these drugs induce oedema, and the present study tests the hypothesis that this side effect reflects serum and glucocorticoid-inducible kinase 1 (SGK1)-dependent enhancement of epithelia Na + absorption. Experimental approach: Na + absorbing epithelial cells (H441 cells, mpkCCD cells) on permeable membranes were mounted in Ussing chambers, and the effects of rosiglitazone (2 mM) and pioglitazone (10 mM) on transepithelial Na + absorption were quantified electrometrically. Changes in SGK1 activity were assessed by monitoring phosphorylation of residues within an endogenous protein. Key results: Both cell types absorbed Na+ via an electrogenic process that was enhanced by insulin. In mpkCCD cells, this stimulation of Na + transport was associated with increased activity of SGK1, whereas insulin regulated Na + transport in H441 cells through a mechanism that did not involve activation of this kinase. Rosiglitazone and pioglitazone had no discernible effect on transepithelial Na + absorption in unstimulated or insulin-stimulated cells and failed to alter cellular SGK1 activity. Conclusions and implications:Our results do not support the view that PPARg agonists stimulate epithelial Na + absorption or alter the control of cellular SGK1 activity. It is therefore likely that other mechanisms are involved in PPARg-mediated fluid retention, and a better understanding of these mechanisms may help with the identification of patients likely to develop oedema or heart failure when treated with these drugs.