Insulin treatment enhances AT1receptor function in OK cells

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Abstract
Increased renal sodium retention is considered a major risk factor contributing to hypertension associated with chronic hyperinsulinemia and obesity. However, the molecular mechanism involved is not understood. The present study investigates the effect of insulin treatment on AT1receptor expression and ANG II-induced stimulation of Na/H exchanger (NHE) and Na-K-ATPase (NKA) in opossum kidney (OK) cells, a proximal tubule cell line. The presence of the AT1receptors in OK cells was confirmed by the specific binding of125I-sar-ANG II and by detecting ∼43-kDa protein on Western blot analysis with AT1receptor antibody and blocking peptide as well as by expression of AT1receptor mRNA as determined by RT-PCR. Insulin treatment (100 nM for 24 h) caused an increase in125I-sar-ANG II binding, AT1receptor protein content, and mRNA levels. The whole cell lysate and membrane showed similar insulin-induced increase in the AT1receptor protein expression, which was blocked by genistein (100 nM), a tyrosine kinase inhibitor, and cycloheximide (1.5 μg/ml), a protein synthesis inhibitor. Determination of ethyl isopropyl amiloride-sensitive22Na+uptake, a measure of the NHE activity, revealed that ANG II (1–100 pM)-induced stimulation of NHE in insulin-treated cells was significantly greater than in the control cells. Similarly, ANG II (1–100 pM)-induced stimulation of ouabain-sensitive86Rb+uptake, a measure of NKA activity in insulin-treated cells, was significantly greater than in the control cells. ANG II stimulation of both the transporters was blocked by AT1receptor antagonist losartan, suggesting the involvement of AT1receptors. Thus chronic insulin treatment causes upregulation of AT1receptors, which evoked ANG II-induced stimulation of NHE and NKA. We propose that insulin-induced increase in the renal AT1receptor function serves as a mechanism responsible for the increased renal sodium reabsorption and thus may contribute to development of hypertension in conditions associated with hyperinsulinemia.