INHIBITION OF NITRIC OXIDE GENERATION ENHANCES SUPEROXIDE PRODUCTION IN CULTURED HK2 and M1 CELLS

Abstract

An enhancement in superoxide (O2−) activity due to nitric oxide (NO) deficiency facilitates renal tubular sodium reabsorption leading to the condition of salt‐sensitivity. To examine the interaction between NO and O2− activity in the renal epithelial cells, cultured HK2 (proximal tubular) and M1 (distal tubular) cells were incubated with L‐NAME (1 mM) or vehicle for 2, 4 and 6 hrs in different wells (n=6 in each case) separately. O2− activity in incubated cells was determined by Lucigenin‐chemiluminescence assay. Compared to vehicle treated cells, L‐NAME treated cells showed increment in O2− activity within 2 hr of incubation in HK2 cells (973 ± 99 to 1691 ± 165 RLU/μg protein) and in M‐1 cells (1768 ± 244 to 3548 ± 622 RLU/μg protein). Such increases in O2− activity was prevented in cells co‐incubated with NO donor compound, DETA NONOate (1 mM). In separate sets, L‐NAME treated cells were co‐incubated with O2− scavenger, tempol (5 mM) or with NADPH oxidase inhibitor, apocynin (5 mM). O2− activity was minimal in cells co‐incubated with L‐NAME + tempol (189 ± 13 and 345 ± 24 RLU/μg protein) and L‐NAME + apocynin (242 ± 25 and 333 ± 27 RLU/μg protein) in HK2 and M1 cells respectively indicating that NADPH oxidase is the major source of O2− production in these cells. These data demonstrate that a reduction in NO generation leads to increases in O2− production induced by NADPH oxidase activity in renal epithelial cells.