Characterization of the regulation of renal Na+/H+ exchanger NHE3 by insulin

Insulin receptors are widely distributed in the kidney and affect multiple aspects of renal function. In the proximal tubule, insulin regulates volume and acid-base regulation through stimulation of the Na(+)/H(+) exchanger NHE3. This paper characterizes the signaling pathway by which insulin stimulates NHE3 in a cell culture model [opossum kidney (OK) cell]. Insulin has two distinct phases of action on NHE3. Chronic insulin (24 h) activates NHE3 through the classic phosphatidylinositol 3-kinase-serum- and glucocorticoid-dependent kinase 1 (PI3K-SGK1) pathway as insulin stimulates SGK1 phosphorylation and the insulin effect can be blocked by the PI3K inhibitor wortmannin or a dominant-negative SGK1. We showed that SGK1 transcript and protein are expressed in rat proximal tubule and OK cells. We previously showed that glucocorticoids augment the effect of insulin on NHE3 (Klisic J, Hu MC, Nief V, Reyes L, Fuster D, Moe OW, Ambuhl PM. Am J Physiol Renal Physiol 283: F532-F539, 2002). Part of this can be mediated via induction of SGK1 by glucocorticoids, and indeed the insulin effect on NHE3 can also be amplified by overexpression of SGK1. We next addressed the acute effect of insulin (1-2 h) on NHE3 by systematically examining the candidate signaling cascades and activation mechanisms of NHE3. We ruled out the PI3K-SGK1-Akt and TC10 pathways, increased surface NHE3, NHE3 phosphorylation, NHE3 association with calcineurin homologous protein 1 or megalin as mechanisms of acute activation of NHE3 by insulin. In summary, insulin stimulates NHE3 acutely via yet undefined pathways and mechanisms. The chronic effect of insulin is mediated by the classic PI3K-SGK1 route.

Insulin activates Na(+)/H(+) exchanger 3: biphasic response and glucocorticoid dependence.

Insulin is an important regulator of renal salt and water excretion, and hyperinsulinemia has been implicated to play a role in hypertension. One of the target proteins of insulin action in the kidney is Na(+)/H(+) exchanger 3 (NHE3), a principal Na(+) transporter responsible for salt absorption in the mammalian proximal tubule. The molecular mechanisms involved in activation of NHE3 by insulin have not been studied so far. In opossum kidney (OK) cells, insulin increased Na(+)/H(+) exchange activity in a time- and concentration-dependent manner. This effect is due to activation of NHE3 as it persisted after pharmacological inhibition of NHE1 and NHE2. In the early phase of stimulation (2-12 h), NHE3 activity was increased without changes in NHE3 protein and mRNA. At 24 h, enhanced NHE3 activity was accompanied by an increase in total and cell surface NHE3 protein and NHE3 mRNA abundance. All the effects of insulin on NHE3 activity, protein, and mRNA were amplified in the presence of hydrocortisone. These results suggest that insulin stimulates renal tubular NHE3 activity via a biphasic mechanism involving posttranslational factors and an increase in NHE3 gene expression and the effects are dependent on the permissive action of hydrocortisone.