Ruisheng Liu

Macula densa (MD)-mediated regulation of renal hemodynamics via tubuloglomerular feedback is regulated by interactions between factors such as superoxide (O₂⁻) and angiotensin II (ANG II). We have reported that NaCl-induced O₂⁻in the MD is produced by the NOX2 isoform of NADPH oxidase (NOX); however, the source of ANG II-induced O₂⁻in MD is unknown. Thus we determined the pathways by which ANG II increased O₂⁻in the MD by measuring O₂⁻in ANG II-treated MMDD1 cells, a MD-like cell line. ANG II caused MMDD1 O₂⁻levels to increase by more than twofold ( P < 0.01). This increase was blocked by losartan (AT₁receptor blocker) but not PD-123319 (AT₂receptor antagonist). Apocynin (a NOX inhibitor) decreased O₂⁻by 86% ( P < 0.01), whereas oxypurinol (a xanthine oxidase inhibitor) and NS-398 (a cyclooxygenase-2 inhibitor) had no significant effect. The NOX-dependent increase in O₂⁻was due to the NOX2 isoform; a short interfering (si)RNA against NOX2 blunted ANG II-induced increases in O₂⁻, whereas the NOX4/siRNA did not. Finally, we found that inhibiting the Rac1 subunit of NOX blunted ANG II-induced O₂⁻production in NOX4/siRNA-treated cells but did not further decrease it in NOX2/siRNA-treated cells. Our results indicate that ANG II stimulates O₂⁻production in the MD primarily via AT₁-dependent activation of NOX2. Rac1 is required for the full activation of NOX2. This pathway may be an important component of ANG II enhancement of tubuloglomerular feedback.

Superoxide (O₂⁻ ) enhances tubuloglomerular feedback (TGF) by scavenging nitric oxide at the macula densa (MD). The primary source of O₂⁻ in the MD during TGF is NADPH oxidase, which is activated by membrane depolarization. While Rac, a small GTP-binding protein, has been shown to enhance NADPH oxidase activity, its role in O₂⁻ generation by the MD is unknown. We hypothesized that depolarization of the MD leads to translocation of Rac to the apical membrane, and its activation, in turn, augments O₂⁻ generation during TGF. We tested this by measuring membrane potential and increased O₂⁻ levels during TGF responses in isolated, perfused tubules containing the intact MD plaque. Switching tubular NaCl from 10 to 80 mM, which induces TGF, depolarized membrane potential by 28.4 ± 4.5% from control ( P < 0.05) and O₂⁻ levels from 124 ± 19 to 361 ± 27 U/min. This NaCl-induced depolarization and O₂⁻ generation were blocked by a Cl⁻ channel blocker, 5-nitro-2(3-phenylpropylamino) benzoic acid (NPPB; 10⁻⁶ M). Inhibition of Rac blunted NaCl-induced O₂⁻ generation by 47%. When the NaCl content of the MD perfusate was increased from 10 to 80 mM, immunointensity of Rac on the apical side increased from 32 ± 3.1 to 46 ± 2.5% of the total immunofluorescence in the MD, indicating that high NaCl induces the translocation of Rac to the apical membrane. This NaCl-induced Rac translocation was blocked by a Cl⁻ channel blocker, NPPB, indicating that depolarization of the MD induced Rac translocation. In conclusion, we found that depolarization of the MD during TGF leads to translocation of Rac to the apical membrane, which enhances O₂⁻ generation by the MD.

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.

We hypothesized that elevated macula densa intracellular pH (pH_i) during tubuloglomerular feedback enhances O₂⁻production from NAD(P)H oxidase. Microdissected thick ascending limbs from rabbits with intact macula densa were cannulated and perfused with physiological saline. When luminal NaCl was switched from 10 to 80 mM, O₂⁻production increased from 0.53 ± 0.09 to 2.62 ± 0.54 U/min ( P < 0.01). To determine whether inhibiting the Na/H exchanger blocks O₂⁻production, we used dimethyl amiloride (DMA) to block Na/H exchange. In the presence of DMA, O₂⁻production induced by NaCl was blunted by 40%. To study the effect of pH_ion O₂⁻in intact macula densa cells, we measured O₂⁻while pH_iwas changed by adjusting luminal pH. When the macula densa was perfused with 80 mM NaCl and the pH of the perfusate was switched to 6.8, 7.4, and 8.0, O₂⁻production was significantly enhanced, but not at 10 mM NaCl. To ascertain the source of O₂⁻, we used the NAD(P)H oxidase inhibitor apocynin. In the presence of apocynin (10⁻⁵M), O₂⁻production induced by elevating pH_iwas blocked. Finally, we measured the optimum pH for O₂⁻production by the macula densa and found optimum extracellular pH is at 7.7 and optimum pH_iis ∼8 for O₂⁻production. We found that elevated pH_ienhances O₂⁻production from NAD(P)H oxidase induced by increasing luminal NaCl when the lumen is perfused with 80 mM NaCl, not 10 mM, and O₂⁻production is pH sensitive, with an optimum pH_iof 8.