Objectives: Insulin is recognized to increase renal salt reabsorption in the distal nephron and hyperinsulinemic states have been shown to be associated with increased expression of the renal NaCl cotransporter (NCC). However, the effect of insulin on NCC functional activity has not been reported.
Methods: Using a heterologous expression system of Xenopus laevis oocytes, a mouse distal convoluted cell line, mDCT15 cells, endogenously expressing NCC, and an ex-vivo kidney perfusion technique, we assessed the effect of insulin on the activity and phosphorylation of NCC. The signaling pathway involved was analyzed.
Results: In Xenopus oocytes insulin increases the activity of NCC together with its phosphorylation at threonine residue 58. Activation of NCC by insulin was also observed in mDCT15 cells. Additionally, insulin increased the NCC phosphorylation in kidney under the ex-vivo perfusion technique. In oocytes and mDCT15 cells, insulin effect on NCC was prevented with inhibitors of phosphatidylinositol 3-kinase (PI3K), mTORC2, and AKT1 kinases, but not by inhibitors of MAP or mTORC1 kinases, suggesting that PI3K-mTORC2-AKT1 is the intracellular pathway required. Additionally, activation of NCC by insulin was not affected by wild-type or mutant versions of with no lysine kinase 1, with no lysine kinase 4, or serum glucocorticoid kinase 1, but it was no longer observed in the presence of wild-type or the dominant negative, catalytically inactive with no lysine kinase 3, implicating this kinase in the process.
Conclusion: Insulin induces activation and phosphorylation of NCC. This effect could play an important role in arterial hypertension associated with hyperinsulinemic states, such as obesity, metabolic syndrome, or type 2 diabetes mellitus.
aMolecular Physiology Unit, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México and Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Tlalpan, Mexico City, Mexico
bDepartment of Medicine, University of Chicago, Chicago, Illinois
cDepartment of Nephrology, Instituto Nacional de Cardiología Ignacio Chávez, Tlalpan, Mexico City, Mexico
dDivision of Nephrology, Department of Medicine, Emory University, and Veteran's Administration Medical Center, Atlanta, Georgia, USA
Correspondence to Gerardo Gamba, Molecular Physiology Unit, Vasco de Quiroga No. 15, Tlalpan 14000, Mexico City, Mexico. Tel: +52 55 5513 3868; e-mail: firstname.lastname@example.org
Abbreviations: DCT, distal convoluted tubule; ENaC, epithelial Na+ channel; MAPK, mitogen-activated protein kinase; mTOR, mammalian target of rapamycin; NCC, Na+Cl− cotransporter; PI3K, phosphatidylinositol 3-kinase; ROMK, renal outer medulla potassium channel; SGK1, serum glucocorticoid kinase 1; WNK1, with no lysine kinase 1; WNK3, with no lysine kinase 3; WNK4, with no lysine kinase 4
Received 19 July, 2012
Revised 3 October, 2012
Accepted 20 October, 2012
Part of this work was presented during the 2010 Annual Meeting of the American Society of Nephrology in Denver, CO and published as an abstract (J Am Soc Nephrol 2010; 21:64A).
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