Increased glomerular filtration rate and impaired contractile function of mesangial cells in TRPC6 knockout mice

Abstract

The present study was conducted to determine if TRPC6 regulates glomerular filtration rate (GFR) and the contractile function of glomerular mesangial cells (MCs). GFR was assessed in conscious TRPC6 wild type and knockout mice, and in anesthetized rats with and without in vivo knockdown of TRPC6 in kidneys. We found that GFR was significantly greater, and serum creatinine level was significantly lower in TRPC6 deficient mice. Consistently, local knockdown of TRPC6 in kidney using TRPC6 specific shRNA construct significantly attenuated Ang II-induced GFR decline in rats. Furthermore, Ang II-stimulated contraction and Ca²⁺ entry were significantly suppressed in primary MCs isolated from TRPC6 deficient mice, and the Ca²⁺ response could be rescued by re-introducing TRPC6. Moreover, inhibition of reverse mode of Na⁺-Ca²⁺ exchange by KB-R7943 significantly reduced Ca²⁺ entry response in TRPC6-expressing, but not in TRPC6-knocked down MCs. Ca²⁺ entry response was also significantly attenuated in Na⁺ free solution. Single knockdown of TRPC6 and TRPC1 resulted in a comparable suppression on Ca²⁺ entry with double knockdown of both. These results suggest that TRPC6 may regulate GFR by modulating MC contractile function through multiple Ca²⁺ signaling pathways.

Figure 1

GFR measurements in conscious TRPC6 WT and KO mice. (A and B): representative plasma clearance kinetics of FITC-inulin in a WT (A) and KO (B) mouse. (C) Summarized GFR in WT and KO mice. (D) Concentration of serum creatinine in WT and KO mice.

Figure 2

Influence of TRPC6 knockout on arterial blood pressure and urinary albumin excretion. (A) Mean arterial blood pressure (MAP) in WT and KO mice measured by radiotelemetry. (B) Urinary albumin excretion in WT and TRPC6 KO mice. NS indicates no significant difference, KO vs. WT. “n” indicates the number of mice in each group.

Figure 3

mRNA expression of TRPC1, TRPC3, and TRPC6 in the renal cortex of WT and KO mice. (A) A representative image or TRPC6 mRNA bands. An original and full-length gel showing TRPC1, TRPC3 and TRPC6 mRNA bands is presented in Fig. 1S of Supplementary Information. (B–D) Summary data of quantitative real time RT-PCR. Actin was used as a control.

Figure 4

Effect of in vivo knockdown of TRPC6 in kidney on Ang II-induced GFR response in rats. Rats were delivered with EGFP plasmid (Control) or EGFP-tagged shRNA construct against rat TRPC6 (rT6-shRNA-EGFP) into the left kidney. GFR was measured 4 days after treatment. (A) GFR in rats treated with EGFP alone and rT6-shRNA-EGFP before (Pre-Ang II) and after (Post-Ang II) Ang II infusion. *Denotes P < 0.05, comparison between the groups as indicated. (B) Representative Western blot, showing TRPC6 protein expression in the renal cortical tissues of the left kidney (transfected kidney) from EGFP alone or rT6-shRNA-EGFP treated rats. α-tubulin was used as a loading control. (C) Immunofluorescence staining, showing distribution of rT6-shRNA-EGFP (green) in kidney. Glomerular MCs were labeled with OX-7, shown by red signals. Positively transfected MCs are indicated by yellow fluorescence in the panels of Overlap. The right bottom panel is an enlarged image of the region indicated by a rectangle in the panel of Overlap.

Figure 5

Identification of mouse MCs. (A) Monolayer of spindle shaped MCs. (B) Staining MCs for α-smooth muscle actin (red). (C) Staining MCs for desmin (red). In both B and C, cell nuclei were stained with DAPI (blue). Magnification: × 100.

Figure 6

Ang II-induced contraction of primary mouse MCs isolated from TRPC6 WT and KO mice. (A) Representative morphology of MCs before and 30 min after 1 μM Ang II stimulation. The changes in cell size by Ang II are illustrated on the right panel (Overlay), by overlapping the images of the same cell before and after Ang II treatment using Photoshop software. The cells before Ang II treatment were white and after treatment black. The right panels are enlarged regions indicated by the dashed rectangles in the left and middle panels. (B) Summary data in 23 normal MCs from 8 WT mice and 31 TRPC6 deficient MCs from 10 KO mice, showing the Ang II-induced decreases in the surface area of MC with and without TRPC6. **Denotes P < 0.01, KO vs. WT.

Figure 7

Ang II-induced Ca²⁺ response in primary mouse MCs. (A and B) Representative traces, showing [Ca²⁺]_i in response to 1 μM Ang II in the MC isolated from a WT (A) and TRPC6^−/− (B) mouse. [Ca²⁺]_B indicates the Ca²⁺ concentration in bathing solution. Application of Ang II is indicated by a horizontal bar at the top. (C) Summarized Ca²⁺ entry response to 1 μM Ang II in WT, TRPC6^−/− mouse MCs, and TRPC6^−/− mouse MCs introduced with rat trpc6. Δ[Ca²⁺]_i was calculated by subtracting [Ca²⁺]_i before re-addition of 2 mM Ca²⁺ to the bath from the peak [Ca²⁺]_i after re-addition. The numbers inside the parentheses represent the number of cells analyzed from 6 WT and 6 KO mice. *P < 0.05, compared to both WT and TRPC6^−/− + TRPC6.

Figure 8

Endothelin-1-induced Ca²⁺ response in primary mouse MCs. (A and B) Representative traces, showing [Ca²⁺]_i in response to 100 nM endothelin-1 (ET-1) in the MC isolated from a WT (A) and TRPC6^−/− (B) mouse. [Ca²⁺]_B indicates the Ca²⁺ concentration in bathing solution. Application of ET-1 is indicated by a horizontal bar at the top. (C) Summarized Ca²⁺ entry response from experiments presented in A and B. Δ[Ca²⁺]_i was calculated by subtracting [Ca²⁺]_i before re-addition of 2 mM Ca²⁺ to the bath from the peak [Ca²⁺]_i after re-addition. The numbers inside the parentheses represent the number of cells analyzed from 6 WT and 6 KO mice. *P < 0.05, compared to both WT. (D) The basal [Ca²⁺]_i (before administration of Ang II in Fig. 6 and ET-1 in Fig. 7A–C) in TRPC6−/− and WT mouse MCs. “n” indicates the number of cells analyzed in each group.

Figure 9

Ang II-induced Ca²⁺ response in human MCs with different treatments. (A) Ca²⁺ entry response (∆[Ca²⁺]_i) in MCs transiently transfected with pSHAG (Vector) or rat TRPC6 expression plasmid (TRPC6), or shRNA construct against human TRPC6 (hTRPC6-shRNA) with and without treatment with KB-R7943 (10 μM). KB-R7943 was added to the bathing solution before re-addition of Ca²⁺. *Denotes P < 0.05, compared to Vector; †Denotes P < 0.05, compared to TRPC6. (B) Ca²⁺ entry response in MCs bathed in physiological saline solution (PSS) and Na⁺ free solution. **Denotes P < 0.01, PSS vs. Na⁺ free. (C) Ca²⁺ entry response in MCs with stable transfection of pSHAG (Vector) or shRNA construct against human TRPC1 (hTRPC1-shRNA) or hTRPC6-shRNA or both hTRPC1-shRNA and hTRPC6-shRNA. *Denotes P < 0.05, compared to Vector. In all graphs, “n” indicates the number of cells analyzed. The data shown in A to B represent at least 4 independent experiments.