GTPase-Rac enhances depolarization-induced superoxide production by the macula densa during tubuloglomerular feedback

Abstract

Superoxide (O(2)(-) ) enhances tubuloglomerular feedback (TGF) by scavenging nitric oxide at the macula densa (MD). The primary source of O(2)(-) 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 O(2)(-) 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 O(2)(-) generation during TGF. We tested this by measuring membrane potential and increased O(2)(-) 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 + or - 4.5% from control (P < 0.05) and O(2)(-) levels from 124 + or - 19 to 361 + or - 27 U/min. This NaCl-induced depolarization and O(2)(-) 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 O(2)(-) 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 + or - 3.1 to 46 + or - 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 O(2)(-) generation by the MD.

Fig. 1.

Increases in luminal NaCl induce macula densa (MD) depolarization and superoxide (O₂⁻) generation. A: when the luminal NaCl was switched from 10 to 80 mM, a maneuver that initiates tubuloglomerular feedback (TGF), membrane potential was depolarized by 28.4 ± 4.5% from the control. Repeating of the switch induced similar changes in membrane potential (*P < 0.05, 10 vs. 80 mM NaCl; n = 6). B: increasing luminal NaCl from 10 to 80 mM significantly enhanced O₂⁻ generation by the MD. Repetitive switching of luminal NaCl induced similar O₂⁻ generation (*P < 0.01, 10 vs. 80 mM NaCl; n = 5).

Fig. 2.

Valinomycin+KCl induces MD depolarization and O₂⁻ generation. A: when valinomycin (10⁻⁶ M), a K-selective ionophore, and 25 mM KCl were added in tubule perfusate without changing luminal NaCl concentration, MD membrane potential depolarized significantly (*P < 0.05; n = 6). B: when valinomycin (10⁻⁶ M) and 25 mM KCl were added in tubule perfusate without changing luminal NaCl concentration, O₂⁻ significantly increased (*P < 0.01, n = 5).

Fig. 3.

Cl⁻ channel inhibition prevents MD depolarization and O₂⁻ generation. A: when the luminal NaCl solution was switched from 10 to 80 mM, membrane potential depolarized significantly. The luminal NaCl was then switched to 10 mM, and a Cl⁻ channel blocker, 5-nitro-2(3-phenylpropylamino) benzoic acid (NPPB; 10⁻⁶ M) was added to the lumen for 30 min. When luminal NaCl was increased to 80 mM again, the NaCl-induced depolarization was blocked (*P < 0.05, 80 mM in control vs. 80 mM in NPPB; n = 5). B: inhibition of Cl⁻ channels with NPPB (10⁻⁶ M) blocked NaCl-induced O₂⁻ generation (*P < 0.01, 80 mM in control vs. 80 mM in NPPB; n = 6).

Fig. 4.

Representative experiment of NaCl-induces Rac translocation in the MD. A: when tubule was perfused with 10 mM NaCl, Rac was evenly distributed in the MD cells. B: when tubule was perfused with 80 mM NaCl, immunointensity of Rac on apical side increased. C and D: images of light microscopy of A and B, respectively. G, glomerulus; cTAL, cortical thick ascending limb.

Fig. 5.

Depolarization induces Rac translocation in the MD. When the MD was perfused with 10 mM NaCl, immunointensity of Rac among apical, middle, and basolateral sides were not significantly different. When the MD was perfused with 80 mM NaCl, immunointensity of Rac on the apical side was increased from 32 ± 3.1 to 46 ± 2.5% of the total immunofluorescence in the MD, and the middle was decreased from 34 ± 7.9 to 23 ± 1.8% (P < 0.05 vs. 10 mM NaCl, n = 5). In the presence of a Cl⁻ channel blocker to prevent depolarization NPPB (10⁻⁶ M), when the MD was perfused with 80 mM NaCl, translocation of Rac was blocked (*P < 0.01, 10 vs. 80 mM NaCl; #P < 0.05, 10 vs. 80 mM NaCl; n = 5).

Fig. 6.

The effect of Rac1 inhibition on O₂⁻ generation by the MD in response to NaCl- and valinomycin+KCl-induced depolarization. Switching from the low- to high-NaCl perfusate caused O₂⁻ to increase by 228 ± 23 U/min (from 121 ± 15 to 349 ± 33 U/min). This increase was blunted by 47% after the MD was treated with the Rac1 inhibitor, NSC23766 (*P < 0.05 vs. control, n = 5). When valinomycin (10⁻⁶ M) and 25 mM KCl were added in tubule perfusate without changing luminal NaCl concentration, O₂⁻ increased by 199 ± 14 U/min (from 115 ± 13 to 314 ± 14 units/min). The Rac inhibitor also blunted valinomycin+KCl-induced increases in O₂⁻ by 31% (#P < 0.05 vs. without Rac inhibition, n = 5).