Gq signaling causes glomerular injury by activating TRPC6

Abstract

Familial forms of focal segmental glomerulosclerosis (FSGS) have been linked to gain-of-function mutations in the gene encoding the transient receptor potential channel C6 (TRPC6). GPCRs coupled to Gq signaling activate TRPC6, suggesting that Gq-dependent TRPC6 activation underlies glomerular diseases. Here, we developed a murine model in which a constitutively active Gq α subunit (Gq(Q209L), referred to herein as GqQ>L) is specifically expressed in podocytes and examined the effects of this mutation in response to puromycin aminonucleoside (PAN) nephrosis. We found that compared with control animals, animals expressing GqQ>L exhibited robust albuminuria, structural features of FSGS, and reduced numbers of glomerular podocytes. Gq activation stimulated calcineurin (CN) activity, resulting in CN-dependent upregulation of TRPC6 in murine kidneys. Deletion of TRPC6 in GqQ>L-expressing mice prevented FSGS development and inhibited both tubular damage and podocyte loss induced by PAN nephrosis. Similarly, administration of the CN inhibitor FK506 reduced proteinuria and tubular injury but had more modest effects on glomerular pathology and podocyte numbers in animals with constitutive Gq activation. Moreover, these Gq-dependent effects on podocyte injury were generalizable to diabetic kidney disease, as expression of GqQ>L promoted albuminuria, mesangial expansion, and increased glomerular basement membrane width in diabetic mice. Together, these results suggest that targeting Gq/TRPC6 signaling may have therapeutic benefits for the treatment of glomerular diseases.

Figure 7. Effect of GqQ>L on diabetic kidney disease.

(A) Albuminuria was enhanced at 16 and 20 weeks of age in Akita mice expressing GqQ>L (Gq Akita mice). (B and C) Mesangial expansion was increased in Gq Akita mice. Scale bars: 10 μm. (D and E) Nodular, subepithelial thickening of the GBM was seen in Gq Akita mice, which was associated with an increase in average GBM width. Focal areas of FP flattening were also observed (arrows). (F) Total collagen content was increased in both groups of Akita mice. (G and H) Trpc6 and Rcan1 mRNA levels were increased in glomerular preparations from WT Akita mice. Cox2 mRNA was increased in Gq Akita mice. In contrast, mRNA levels for Trpc6 and Rcan1 were decreased in Gq Akita mice compared with levels in WT Akita mice. For the albuminuria and histologic studies, 18 WT Akita mice and 8 Gq Akita mice were used. For the collagen studies, 16 WT Akita mice, 7 Gq Akita mice, and 15 nondiabetic age- and sex-matched controls were used. Three WT Akita mice, 2 Gq Akita mice, and 3 WT controls were used for the TEM studies. For qRT-PCR, samples from 13 WT controls, 16 WT Akita mice, and 7 Gq Akita mice were used. Red blood cells are labeled in the capillary loops. *P < 0.05 or †P < 0.01 versus the indicated groups using Fisher’s exact test for mesangial expansion data and ANOVA, followed by Bonferroni’s post-hoc analysis, for the other studies.

Figure 6. Effect of CN inhibition on PAN nephrosis in GqQ>L mice.

(A) FK506 inhibited albuminuria induced by PAN in GqQ>L mice. (B and C) The development of FSGS was similar in GqQ>L mice treated with either vehicle or FK506. Scale bars: 10 μm. (D and E) FK506 significantly inhibited tubule dilation and casts. Scale bars: 40 μm. (F and G) FK506 significantly preserved SYN expression. (H) PAN nephrosis significantly reduced podocyte numbers in GqQ>L mice treated with vehicle compared with those in an untreated non-Tg control group. FK506 caused a modest and nonsignificant increase in podocyte numbers. (I) FK506 tended to reduce expression of the CN-responsive genes Trpc6 and Rcan1. (J) The increase in Cox2 mRNA caused by induction of GqQ>L (Figure 4I) was not affected by FK506. For the albuminuria studies, 16 vehicle-treated mice and 21 mice treated with FK506 were used. Sixteen vehicle-treated mice and 8 mice treated with FK506 were used for the histologic studies. For the immunoblot studies, 4 vehicle-treated mice and 4 mice treated with FK506 were used. To assess podocyte numbers, 11 untreated controls, 13 GqQ>L mice treated with vehicle, and 8 GqQ>L mice treated with FK506 were used. For qRT-PCR, mRNA samples from 14 vehicle-treated mice and 7 mice treated with FK506 were used. *P < 0.05, †P < 0.025, or ‡P < 0.01 versus the indicated groups using Fisher’s exact test for histologic data, a 2-tailed t test for albuminuria and immunoblotting data, and ANOVA, followed by Bonferroni’s post-hoc analysis, for the other studies.

Figure 5. Effect of Trpc6 KO on glomerular ultrastructure.

(A) FP effacement was observed in all groups of PAN-treated mice (arrows), but was qualitatively more severe in Trpc6^+/+ GqQ>L mice treated with PAN. Microvillous changes were observed in a few Trpc6^+/+ GqQ>L mice (asterisks). Scale bars: 1 μm. (B) The number of filtration slits was significantly reduced in Trpc6^+/+ GqQ>L mice compared with that in either WT controls or Trpc6-KO GqQ>L mice. Red blood cells (RBC) are labeled in the capillary loops. For the experiments, 2–3 mice were studied in each group. **P < 0.05 versus the indicated groups using ANOVA, followed by Bonferroni’s post-hoc analysis.

Figure 4. Effect of Trpc6-KO on PAN nephrosis.

(A) Albuminuria was increased in Trpc6^+/+ GqQ>L mice. KO of Trpc6 attenuated the increase in albuminuria. (B and D) FSGS was increased in Trpc6^+/+ GqQ>L mice. KO of Trpc6 prevented the development of FSGS. Scale bars: 10 μm. (C and E) Tubule dilation and casts were reduced by KO of Trpc6. Scale bars: 40 μm. (F) Treatment with PAN reduced podocyte numbers in Trpc6^+/+ GqQ>L mice. Podocyte numbers in Trpc6-KO mice were similar to those in WT controls. (G and H) KO of Trpc6 preserved expression of SYN and WT1 in GqQ>L mice. (I and J) Cox2 mRNA was increased in Trpc6^+/+ GqQ>L mice, and this increase was prevented by KO of Trpc6. A similar pattern was observed for Rcan1 and Trpc6 mRNA levels. The albuminuria experiments were performed in 15 WT controls, 20 Trpc6^+/+ GqQ>L mice, and 26 Trpc6-KO GqQ>L mice. For the histology studies, 8 WT controls, 22 Trpc6^+/+ GqQ>L mice, and 16 Trpc6-KO GqQ>L mice were used. To assess podocyte numbers, 9 WT controls, 9 Trpc6^+/+ GqQ>L mice, and 9 Trpc6-KO GqQ>L mice were used. For immunoblotting experiments, 4 controls, 4 Trpc6^+/+ GqQ>L mice, and 10 Trpc6-KO GqQ>L mice were used. For qRT-PCR, mRNA samples from 12 WT controls, 17 Trpc6^+/+ GqQ>L mice, and 10 Trpc6-KO GqQ>L mice were used. *P < 0.05, †P < 0.01, or ‡P < 0.005 versus the indicated groups using a χ² analysis for histologic data and ANOVA, followed by Bonferroni’s post-hoc analysis, for the other studies. KO GqQ>L, Trpc6-KO GqQ>L mice; WT GqQ>L, Trpc6^+/+ GqG>L mice.

Figure 3. Induction of GqQ>L in podocytes enhances TRPC6 expression in vivo.

(A) Treatment with DOX enhanced Trpc6 mRNA levels in glomerular preparations from GqQ>L mice compared with levels detected in controls. (B) FK506 inhibited expression of Trpc6 mRNA in glomerular preparations from GqQ>L mice treated with DOX. (C and D) TRPC6 expression was similar in controls treated with DOX and in GqQ>L mice treated with sucrose. Treatment with DOX enhanced TRPC6 expression in GqQ>L mice compared with expression in either controls treated with DOX or in GqQ>L mice treated with sucrose. Induction of TRPC6 protein in GqQ>L mice treated with DOX was blocked by FK506. Actin was used as a loading control, and HEK293 cells transfected with a TRPC6 construct were used as a positive control for the immunoblotting studies. Because of the high levels of TRPC6 expression in the HEK293 cells, small amounts of the HEK293 cell lysate were used, and the actin loading control was not visible at this length of exposure. For the qRT-PCR analysis, 11 controls treated with DOX, 4 GqQ>L mice treated with DOX, 8 GqQ>L mice treated with DOX and vehicle, and 4 GqQ>L mice treated with DOX and FK506 were used. For the immunoblotting studies, 4 controls treated with DOX, 4 GqQ>L mice treated with sucrose, 4 GqQ>L mice treated with DOX, and 4 GqQ>L mice treated with DOX and FK506 were used. *P < 0.025 or ^†P < 0.01 versus the indicated groups using a 2-tailed t test for qRT-PCR data and ANOVA, followed by a Bonferroni’s post-hoc test, for densitometric data.

Figure 2. Activation of an NFAT reporter construct in GqQ>L mice.

(A) In GqQ>L mice expressing the NFAT reporter construct (triple-Tg mice), induction of GqQ>L activated the reporter construct in kidney cortex compared with both other-Tg (double- and single-Tg) and non-TG mice, with lesser activation in the other tissues examined. (B) Treatment with DOX caused robust activation of the NFAT reporter construct in glomerular preparations from triple-Tg mice compared with that seen in sucrose-treated triple-Tg mice. For the experiments, 3 mice were studied in each group. *P < 0.05 or †P < 0.01 versus the indicated groups using ANOVA, followed by a Bonferroni’s post-hoc test.

Figure 1. Effect of GqQ>L induction on PAN nephrosis.

(A) PAN caused a significant increase in albuminuria in mice expressing GqQ>L. There was significantly less albuminuria induced by PAN in control mice (CTRL) than in GqQ>L mice. (B and C) Treatment with PAN caused a significant increase in the percentage of mice that developed FSGS. Cystic structures were seen in a few glomeruli in GqQ>L mice (B, lower left panel), which likely indicates the accumulation of lipids or proteins in the cytoplasm of epithelial cells. Scale bars: 10 μm. (D) Treatment with PAN induced tubule dilation and casts in GqQ>L mice. Scale bars: 40 μm. (E) Podocyte numbers were significantly reduced by treatment with PAN in GqQ>L mice compared with numbers in both control mice treated with PAN and untreated control mice. (F and G) Treatment with PAN caused a significant decrease in both SYN and WT1 expression in GqQ>L mice. For albuminuria and the histologic studies, 17 control and 14 GqQ>L mice were used. Four controls and 5 GqQ>L mice were used for the immunoblotting experiments. To assess podocyte numbers, samples from 11 untreated controls, 9 controls treated with PAN, and 13 GqQ>L mice treated with PAN were used. *P < 0.05, †P < 0.01, and ‡P < 0.005 versus the indicated groups by Fisher’s exact test for histologic data and by ANOVA, followed by Bonferroni’s post-hoc analysis, for the other studies.