The small GTPase regulatory protein Rac1 drives podocyte injury independent of cationic channel protein TRPC5

Abstract

Transient receptor potential canonical channels (TRPCs) are non-selective cationic channels that play a role in signal transduction, especially in G -protein-mediated signaling cascades. TRPC5 is expressed predominantly in the brain but also in the kidney. However, its role in kidney physiology and pathophysiology is controversial. Some studies have suggested that TRPC5 drives podocyte injury and proteinuria, particularly after small GTPase Rac1 activation to induce the trafficking of TRPC5 to the plasma membrane. Other studies using TRPC5 gain-of-function transgenic mice have questioned the pathogenic role of TRPC5 in podocytes. Here, we show that TRPC5 over-expression or inhibition does not ameliorate proteinuria induced by the expression of constitutively active Rac1 in podocytes. Additionally, single-cell patch-clamp studies did not detect functional TRPC5 channels in primary cultures of podocytes. Thus, we conclude that TRPC5 plays a role redundant to that of TRPC6 in podocytes and is unlikely to be a useful therapeutic target for podocytopathies.

Keywords: Rac1; TRPC5; calcium; focal segmental glomerulosclerosis.

Figure 1:. TRPC5 activity does not influence Rac1-induced proteinuria.

(A) A model adapted from Chung and Shaw. The model depicts the endless feedback loop between TRPC5 and Rac1. The sequence of events includes: (1) Injury due to external or internal factors; (2) Activation of Rac1; (3) Movement of TRPC5 to the cell surface due to the presence of Rac1; (4) Ca²⁺ influx; (5) Ca²⁺-dependent Rac1 activation; (6) Rac1 signaling to other mediator molecules; and (7) Podocyte damage. (B) Albumin (mg) to creatinine (g) ratio (ACR) is shown on the y-axis with the progression of proteinuria in various groups of mice as indicated. The black color plot indicates Rac1^Dtg mice, the red color shows Rac1^Dtg–TRPC5^WT and the green color indicates Rac1^Dtg–TRPC5^DN mice. Error bars denote SEM. Statistical significance between groups was assessed by the Kruskal-Wallis test (*, p< 0.05; **, p<0.01; NS, p>0.05). (C) ACR measurements of mice injected with DMSO (black bars), ML204 low dose (LD) (red), ML204 high dose (HD) (Orange), AC1903 LD (dark blue), and AC1903 HD (green). Low doses of ML204 and AC1903 were 0.3 and 0.75 mg/kg, and high doses were 1 mg/kg and 1.5 mg/kg, respectively. Error bars denote SEM. No significant difference was detected by the Kruskal-Wallis test. (D) Representative traces of unitary currents demonstrating activity from Rac1^Dtg, Rac1^Dtg–TRPC5^WT, and Rac1^Dtg–TRPC5^DN podocytes, respectively. Membrane potentials are shown. C: closed state. (E) Current-voltage (I/V) relationships for identified channels. Two types of channels were detected in cells from mice overexpressing TRPC5 (n=8), with unitary slope conductances of 38±1 pS, and 25±1 pS. Only the 25 pS channels were detected in mice overexpressing TRPC5^DN (n=9) and Rac1 (n=7).

Figure 2:. TRPC5 channels have no effect on podocyte foot process (FP) morphology.

Representative TEM images of podocyte FPs from control double transgenic (Dtg) Rac1^Dtg–TRPC5^WTmice (A) and Rac1^Dtg–TRPC5^DN (B) that were fed a normal diet. DOX induction of Rac1 resulted in visible foot process effacement in both (C) Rac1^Dtg–TRPC5^WT and (D) Rac1^Dtg–TRPC5^DN. Treatment with the TRPC5 inhibitor AC1903 did not improve FP morphology in either (E) Rac1^Dtg–TRPC5^WT or (F) Rac1^Dtg–TRPC5^DN mice. Scale bar: 1 μm. Foot slit density measurement methodology (G) for the quantification of foot process lengths (H). Statistical significance between groups was assessed by one-way ANOVA test (*, p< 0.05; ****, p<0.0001; NS, p>0.05).