Inhibition of the TRPC5 ion channel protects the kidney filter

Abstract

An intact kidney filter is vital to retention of essential proteins in the blood and removal of waste from the body. Damage to the filtration barrier results in albumin loss in the urine, a hallmark of cardiovascular disease and kidney failure. Here we found that the ion channel TRPC5 mediates filtration barrier injury. Using Trpc5-KO mice, a small-molecule inhibitor of TRPC5, Ca2+ imaging in isolated kidney glomeruli, and live imagining of podocyte actin dynamics, we determined that loss of TRPC5 or its inhibition abrogates podocyte cytoskeletal remodeling. Inhibition or loss of TRPC5 prevented activation of the small GTP-binding protein Rac1 and stabilized synaptopodin. Importantly, genetic deletion or pharmacologic inhibition of TRPC5 protected mice from albuminuria. These data reveal that the Ca2+-permeable channel TRPC5 is an important determinant of albuminuria and identify TRPC5 inhibition as a therapeutic strategy for the prevention or treatment of proteinuric kidney disease.

Figure 1. Genetic Trpc5 deletion is protective in 2 models of filter barrier damage.

(A) TRPC5 colocalized with synaptopodin. (B) TEM showed that WT and Trpc5-KO mice had an intact filtration barrier, demonstrated by normally arranged FPs and intervening slit diaphragms. After LPS injection, WT mice developed characteristic FPE, while Trpc5-KO mice maintained an intact filtration barrier. (C) PBS did not induce albuminuria in WT or Trpc5-KO mice. WT mice developed significant albuminuria after LPS injection, whereas Trpc5-KO mice were protected from LPS-induced albuminuria (n = 8–12 per group). (D) Western blot from isolated mouse glomeruli showed intact synaptopodin (Synpo) abundance in PBS-injected animals. LPS-injected WT mice showed synaptopodin degradation, including the appearance of the canonical 75-kDa degradation fragment (asterisk). In contrast, Trpc5-KO glomeruli were protected from synaptopodin degradation. GAPDH served as loading control. (E) WT and Trpc5-KO mice perfused with HBSS had a normal filtration barrier. Whereas WT mice perfused with PS developed FPE, Trpc5-KO mice were protected from PS-induced FPE. (F) HBSS-perfused WT and Trpc5-KO animals showed comparable FP numbers (1.8 ± 0.08 and 1.8 ± 0.01, respectively). In contrast, PS perfusion caused significant FPE in WT mice, whereas Trpc5-KO mice were protected to near-normal FP numbers (1.2 ± 0.08 and 1.7 ± 0.07, respectively; n = 6 mice and 90–150 images per group). Original magnification, ×400 (A), ×15,000 (B and E). ***P < 0.001, ANOVA.

Figure 2. LPS- and PS-induced in situ podocyte Ca²⁺ transients are reduced in isolated glomeruli from Trpc5-KO mice.

(A) Approach for imaging Ca²⁺ in podocytes in situ on intact, acutely isolated mouse glomeruli. Podocytes — located on the exterior surface of the isolated glomerulus, as shown — were efficiently loaded with Fura-2 and displayed measurable changes in Ca²⁺ in response to various stimuli. (B) LPS mediated Ca²⁺ influx (arrows) in glomeruli of WT mice, but the response was attenuated in Trpc5-KO glomeruli. (C) Quantification of peak transient amplitude (at Δt = 1 min) revealed a significantly greater response in WT (n = 24) versus Trpc5-KO (n = 10) glomeruli, attributed to TRPC5-mediated Ca²⁺ influx. (D) PS mediated Ca²⁺ influx (arrows) in WT glomeruli, but the response was attenuated in Trpc5-KO glomeruli. (E) Quantification of peak transient amplitude revealed a significantly greater response in WT versus Trpc5-KO glomeruli (n = 19 per group), attributed to TRPC5-mediated Ca²⁺ influx. Original magnification, ×400 (A, B, and D). Boxed regions are shown enlarged in B and D (enlarged ×9 and ×3, respectively). **P < 0.01, ***P < 0.001, Student’s t test.

Figure 3. ML204 unmasks the contribution of TRPC5 to podocyte Ca²⁺ dynamics.

(A) Representative traces of single-channel recordings in the outside-out configuration in HEK cells expressing TRPC5-GFP confirmed that LPS increased TRPC5 single-channel activity by increasing P_o. This was blocked by 3 μM ML204 in a reversible manner. o, open channel; c, closed channel. (B) PS activated Ca²⁺ transients in podocytes (n = 15 cells), whose peak amplitude was efficiently reduced by bath perfusion of 3 μM ML204 (n = 40 cells). (C) PS-mediated Ca²⁺ influx (arrows) in WT glomeruli was attenuated by 3 μM ML204. (D) Quantification of Ca²⁺ responses revealed a significantly greater response in PS versus PS+ML204 glomeruli (n = 9–10 per group), attributed to TRPC5-mediated Ca²⁺ influx. Scale bar: 50 μm (C). *P < 0.02, ***P < 0.001, Student’s t test.

Figure 4. TRPC5 inhibition protects podocytes from cytoskeletal remodeling.

(A) Treatment of podocytes with 300 μg/ml PS resulted in disrupted actin (red) and loss of stress fibers. Under these conditions, podocytes were largely devoid of synaptopodin (green). ML204 treatment blocked synaptopodin loss and cytoskeletal disruption. (B) Quantification of cytoskeletal remodeling (defined as no visible actin fibers) showed that ML204 rescued podocytes in a dose-dependent manner (n = 90 images/condition). (C) Loss of synaptopodin abundance and cytoskeletal remodeling by 300 μg/ml PS was prevented by TRPC5 shRNA–mediated Trpc5 gene silencing. Scr, scrambled shRNA control. (D) Quantification of cytoskeletal remodeling showed that TRPC5 shRNA–mediated Trpc5 depletion rescued podocytes, in contrast to scrambled control. n = 90 images/condition (10 images × 3 repeats × 3 independent trials). (E) Cell lysates from podocytes treated with PS or PS+ML204 showed that synaptopodin abundance, attenuated by 300 μg/ml PS compared with PBS-treated controls, was rescued by 10 or 30 μM ML204 in a dose-dependent manner. (F) Quantification of 4 Western blots from 4 independent experiments showed that 30 μM ML204 rescued synaptopodin abundance for PS-mediated degradation. Treatment with 10 μM ML204 trended in the same direction, but did not achieve statistical significance. (G) Rac1 activity (Rac1-GTP) increased in podocytes treated with 300 μg/ml PS compared with PBS-treated controls; 30 μM ML204 abrogated this activation. (H) Quantification of 3 Western blots from 3 independent experiments showed that 30 μM ML204 significantly blocked PS-mediated Rac1 activation. Original magnification, ×400 (A and C). *P < 0.05, ****P < 0.00001, ANOVA.

Figure 5. Real-time imaging revealed the dynamics of TRPC5 inhibition on PS-induced actin remodeling.

LifeAct technology and confocal microscopy were used to assay the timing and severity of cytoskeletal remodeling in the presence of PS. PS induced increased ruffling activity and lamellipodia formation within 13 minutes of its initial application. By 28 minutes, actin stress fibers were disrupted and no longer extended across the span of the cell’s cytoplasm, as they did before PS application. This cytoskeletal remodeling resulted in the involution of the cell onto itself after 34 minutes, with the appearance of an intensely fluorescent structure at the center of a previously healthy podocyte. This PS-mediated actin remodeling was blocked by 10 μM ML204, resulting in the preservation of stress fibers and the cell’s cytoskeleton throughout the same time frame. Representative images are shown (see Supplemental Video 1). Original magnification, ×400.

Figure 6. Inhibition of muscarinic receptor–mediated TRPC5 activity prevents podocyte damage.

(A) The muscarinic receptor agonist Cch induces a significant increase in Ca²⁺ transients in podocytes located on acutely isolated glomeruli from WT mice but not from Trpc5-KO mice. (B) Quantification of averaged peak amplitude at Δt = 1 min (n = 11 glomeruli per group). (C) Peak amplitude of Cch-induced Ca²⁺ transients in podocytes on acutely isolated glomeruli was attenuated by 3 μM ML204. (D) Quantification of averaged peak amplitude at Δt = 1 min (n = 12 glomeruli [Cch], 10 glomeruli [Cch+ML204]). (E) Loss of stress fibers induced by 100 μM Cch was blocked by ML204. (F) Significant rescue of stress fiber formation by ML204 (n = 90 images/condition). Scale bar: 50 μm (C). Original magnification, ×400 (E). **P < 0.01, ***P < 0.001, Student’s t test (B and D); ****P < 0.00001, ANOVA (F).

Figure 7. TRPC5 inhibition protects against PS- or LPS-induced filter barrier disruption.

(A) PS induced FPE. Coperfusion of 10 μM ML204 in PS-perfused mice preserved FPs, similar to HBSS controls. (B) ML204 treatment conferred protection from FPE (HBSS, 1.84 ± 0.08 FPs/μm GBM; PS, 1.16 ± 0.08 FPs/μm GBM; PS+ML204, 1.78 ± 0.2 FPs/μm GBM) (n = 6–7 animals and 90–150 images per group). (C) LPS injection led to FPE. Treatment of LPS-injected mice with 20 mg/kg/d ML204 i.p. mitigated FPE. (D) FP numbers in ML204-treated animals (PBS, 2.21 ± 0.07 FPs/μm GBM; LPS, 1.67 ± 0.10 FPs/μm GBM; LPS+ML204, 2.04 ± 0.04 FPs/μm GBM) (n = 3–4 animals and 60 images per group). (E) PBS or PBS+ML204 injection did not induce albuminuria in WT mice. After LPS injection, WT mice developed significant albuminuria, which was significantly reduced after ML204 treatment (n = 5–12 animals/group). Original magnification, ×15,000 (A and C). *P < 0.05, ***P < 0.001, ****P < 0.0001, ANOVA.

Figure 8. Model for (dys)regulation of kidney filter function by TRPC5.

Top-down view of podocyte interdigitating FPs composed of parallel actin bundles. TRPC5 activation in response to injury leads to Rac1 activation (Rac1-GTP) and synaptopodin degradation. These events promote podocyte cytoskeletal remodeling. Loss of filter integrity ensues, leading to albuminuria, a potentially reversible condition. TRPC5 inhibition therefore emerges as a therapeutic strategy for the stabilization of the human filter barrier.