Effect of Insulin on the Regulation of Na/K/2Cl Cotransporter(NKCC2) in HEK293 Cells

Abstract

Insulin is one of the regulators involved in Na+ reabsorption in kidney. The Na+ channel and Na/K/2Cl cotransporter (NKCC2) system are primarily considered as the possible routes for Na+ movement into the cell. The purpose of this study is to understand the regulation of NKCC2 as a candidate for Na+ movement across the kidney cell in response to insulin. In order to clarify the role of the insulin in human embryonic kidney cell line, HEK293, the cells were exposed to the different circumstances (isotonic, hypertonic), and then cell volume, expression of NKCC2 mRNA, intracellular Ca2+ and functional activity of the cotransporter using pH dye, and the intracellular Ca2+ level were analyzed in each condition. The cell volume of HEK293 which was shrunken by hypertonic stress recovered to nearly original state by the addition of 1 μM insulin. It is obvious that insulin prevented cell shrinkage by operating the regulatory volume increase (RVI) machinery of the cell. However, insulin-induced RVI was blocked by the treatment of 10 μM bumetanide. Furthermore, NKCC2 mRNA was expressed remarkably in the presence of 1 μM insulin. Such results might suggest that the volume regulation of HEK293 cell was, at least, in part mediated through bumetanide sensitive NKCC. On the other hand, the HEK293 cell showed the typical change in intracellular Ca2+ concentration in response to carbachol as seen in other normal cells. However, insulin did not induce the change of intracellular calcium level of the HEK293 cell. Taken all together, these findings suggest that the insulin stimulates the sodium transport of HEK293 cell through the upregulation of NKCC2 under the hypertonic condition.