Synergic action of insulin and genistein on Na+/K+/2Cl − cotransporter in renal epithelium

Transepithelial Cl− secretion in polarized renal A6 cells is composed of two steps: (1) Cl− entry step across the basolateral membrane mediated by Na+/K+/2Cl− cotransporter (NKCC) and (2) Cl− releasing step across the apical membrane via cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel. We estimated CFTR Cl− channel activity and transcellular Cl− secretion by measuring 5-nitro 2-(3-phenylpropylamino)benzoate (NPPB, a blocker of CFTR Cl− channel)-sensitive transepithelial conductance (Gt) and short-circuit current (Isc), respectively. Pretreatment with 1 μM insulin for 24 h had no effects on NPPB-sensitive Gt or Isc. On the other hand, in A6 cells treated with carbobenzoxy-l-leucyl-leucyl-l-leucinal (MG132; 100 μM for 2 h) that inhibits endocytosis of proteins at the plasma membrane into the cytosolic space, insulin pretreatment increased the NPPB-sensitive Isc with no effects on NPPB-sensitive Gt. Genistein (100 μM) induced sustained increases in NPPB-sensitive Gt and Isc, which were diminished by brefeldin A (a blocker of protein translocation to Golgi apparatus from endoplasmic reticulum). Co-application of insulin and genistein synergically stimulated the NPPB-sensitive Isc without any effects on NPPB-sensitive Gt. These observations suggest that: (1) insertion and endocytosis of NKCC are stimulated by insulin, (2) the insulin-induced stimulation of NKCC insertion into the basolateral membrane is offset by the stimulatory action on NKCC endocytosis from the basolateral membrane, (3) genistein stimulates insertion of both CFTR Cl− channel into the apical membrane and NKCC into the basolateral membrane, and (4) insulin and genistein synergically stimulated NKCC insertion into the basolateral membrane.