Isoforms and functions of NAD(P)H oxidase at the macula densa

Macula densa cells produce superoxide (O2-) during tubuloglomerular feedback primarily via NAD(P)H oxidase (NOX). The purpose of the present study was to determine NOXs expressed by the macula densa and the role of each one in NaCl-induced O2- production. To identify which isoforms are expressed, we applied single-cell RT-PCR to macula densa cells isolated by laser capture microdissection and to MMDD1 cells (a macula densa-like cell line). The captured cells expressed neuronal NOS (marker of macula densa), NOX2, and NOX4 but not NOX1. Expression of the NOXs and neuronal NOS was essentially identical in the MMDD1 cells. Thus, we used MMDD1 cells to investigate which isoform is responsible for NaCl-induced O2- production. We used small-interfering RNA to knock down NOX2 or NOX4 in MMDD1 cells and measured O2- exposed to low-salt solution (LS; 70 mmol/L of NaCl) or high-salt solution (HS; 140 mmol/L of NaCl). Exposing control cells (scrambled small-interfering RNA) to HS increased O2- concentrations from 0.75+/-0.28 to 1.48+/-0.46 U/min per 10(5) cells in LS and HS, respectively (P<0.001). Inhibiting NOX2 blocked the HS-induced increase in O2- (0.62+/-0.39 versus 0.76+/-0.31 U/min per 10(5) cells in LS and HS groups, respectively). Blocking NOX4 did not affect HS-induced O2- levels. O2- levels in the control cells during LS and HS were 0.80+/-0.30 and 1.56+/-0.49 U/min per 10(5) cells, respectively (P<0.001); whereas O2- levels in NOX4-small-interfering RNA-treated cells during LS and HS were 0.40+/-0.25 and 1.26+/-0.51 U/min per 10(5) cells, respectively (P<0.001). We conclude that, whereas macula densa cells express the NOX2 and NOX4 isoforms, NOX2 is primarily responsible for NaCl-induced O2- generation.