Isoforms and Functions of NAD(P)H Oxidase at the Macula Densa

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Abstract
Macula densa cells produce superoxide (O 2 − ) 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 O 2 − 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 O 2 − production. We used small-interfering RNA to knock down NOX2 or NOX4 in MMDD1 cells and measured O 2 − 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 O 2 − concentrations from 0.75±0.28 to 1.48±0.46 U/min per 10 5 cells in LS and HS, respectively ( P 2 − (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 O 2 − levels. O 2 − 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 2 − 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 2 − generation.

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