MicroRNA-135a is involved in podocyte injury in a transient receptor potential channel 1-dependent manner

Abstract

Transient receptor potential (TRP) cation channels are essential for normal cellular physiology, and their abnormal expression may lead to a number of disorders, including podocytopathy. Therefore, it is crucial to understand the mechanisms underlying the regulation of TRP channels. In the present study, microRNA (miR)-135a was found to be upregulated in patients with focal segmental glomerulosclerosis and mice treated with adriamycin (ADR). In cultured podocytes, transforming growth factor (TGF)-β and ADR were found to promote miR‑135a expression. Conversely, TRP channel 1 (TRPC1) protein levels were markedly downregulated in podocytes from mice treated with ADR, as well as in cultured podocytes treated with ADR and TGF-β. Ectopic expression of miR-135a led to severe podocyte injury and disarray of the podocyte cytoskeleton, whereas podocyte-specific expression of TRPC1 was able to reverse the pathological effects of miR-135a in cultured podocytes. Moreover, using luciferase reporter assays and western blot analysis, TRPC1 was identified as a target gene of miR-135a. To the best of our knowledge, this is the first study to demonstrate the role of TRPC1 in the development of podocyte injury and disorders of the podocyte cytoskeleton, which may contribute to the development of novel therapeutics for podocyte injury-associated kidney diseases.

Figure 1

miR-135a is upregulated in podocyte injury. (A) Quantification by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) of miR-135a in the isolated glomeruli from 3 patients with focal segmental glomerulosclerosis (FSGS) and 3 patients with kidney rupture (control). (B) RT-qPCR analysis of miR-135a expression in the glomeruli isolated from BALB/c mice treated with adriamycin (ADR; 10.5 mg/kg) at different time points after the injection [n=3, means ± standard deviation (SD)]. (C and D) The miR-135a expression level was measured in the cultured mouse podocyte cell line 5 (MPC5) treated with ADR (C) at different doses and (D) for different periods of time. (E and F) Quantification of miR-135a in MPC5 cells treated with transforming growth factor (TGF)-β (E) at different doses and (F) for different periods of time. The data are expressed as mean + SD of three independent experiments. *P<0.05 and ^**P<0.01 indicate statistically significant and highly statistically significant differences, respectively, compared with the controls. NO1, patient number 1; NO2, patient number 2; NO3, patient number 3.

Figure 2

Ectopic expression of miR-135a led to severe podocyte injury. (A) The changes in the expression levels of nephrin, Wilms tumor 1 (WT1), E-cadherin, desmin, Snail and activated caspase-3 were determined by western blot analysis in the cultured MPC5 cells treated with miR-135a mimics. (B) Quantitative analysis of the results of western blot analysis. (C) Effects of miR-135a on cytoskeletal stabilization in podocytes. Scale bar, 10 μm. (D) MPC5 cell apoptosis was detected by flow cytometry following transfection of miR-135a mimics. ^*P<0.05 and ^**P<0.01 indicate statistically significant and highly statistically significant differences, respectively, compared with the control group.

Figure 3

Transient receptor potential channel 1 (TRPC1) expression was downregulated in podocyte injury. (A) Predicted miR-135a target TRPC1 3′ untranslated region (UTR) in human and mouse species. (B) The protein level of TRPC1 was downregulated in the glomeruli isolated from BALB/c mice treated with adriamycin (ADR) in a time-dependent manner. (C) Quantitative analysis of the results of (B). (D) TRPC1 expression was inhibited in cultured MPC5 cells treated with ADR (5 μg/ml, 24 h) and transforming growth factor (TGF)-β (5 ng/ml, 24 h). (E) Quantitative analysis of the results of (D). ^*P<0.05 and ^**P<0.01 indicate statistically significant and highly statistically significant differences, respectively, compared with the control group.

Figure 4

miR-135a directly targets transient receptor potential channel 1 (TRPC1) 3′ untranslated region (UTR). (A) Schematic diagram of TRPC1 3′UTR reporter construct. (B) Sequence alignment between miR-135a and mouse TRPC1 3′UTR, including wild-type (wt) and mutant (mut). (C) For dual Luciferase reporter assays, 293T cells were transfected with pCDNA3.1-Luciferase TRPC1-3′UTR reporter and miR-135a mimics. (D) Reverse transcription-quantitative polymerase chain reaction analysis was used to determine the effect of miR-135a on TRPC1 mRNA expression. (E) The effect of miR-135a on TRPC1 protein expression was determined by western blot analysis. (F) Quantitative analysis of the results of (E). ^*P<0.05 and ^**P<0.01 indicate statistically significant and highly statistically significant differences, respectively, compared with the controls.

Figure 5

Ectopic expression of transient receptor potential channel 1 (TRPC1) protects against podocyte damage from miR-135a. (A) Western blot analysis confirmed that the TRPC1 overexpression construct was available. (B) Western blot analysis was applied to determine the effects on the expression levels of nephrin, Wilms tumor 1 (WT1), E-cadherin, desmin, Snail and activated caspase-3 in MPC5 cells transfected with miR-135a and/or TRPC1 overexpression construct. (C) Quantitative analysis of the results of (B). (D) Stress fibre reduction in number and disarray, which was induced by miR-135a, was reversed by overexpression of TRPC1. Scale bar, 10 μm. (E) Ectopic expression of TRPC1 alleviated miR-135a-induced podocyte apoptosis.