Tetrandrine Suppresses Transient Receptor Potential Cation Channel Protein 6 Overexpression- Induced Podocyte Damage via Blockage of RhoA/ROCK1 Signaling

Abstract

Objective: Podocyte damage is common in many renal diseases characterized by proteinuria. Transient receptor potential cation channel protein 6 (TRPC6) plays an important role in renal function through its regulation of intracellular Ca²⁺ influx and RhoA/ROCK pathways. Chinese herb Stephania tetrandra, with the main active component being tetrandrine, has been used for the treatment of various kidney diseases for several years and has shown a positive effect. This study aimed at investigating the effect and mechanism of tetrandrine in podocyte damage induced by high expression of TRPC6.

Methods: Immortalized, differentiated murine podocytes, MPC5 were treated with valsartan (0-800 μM) and tetrandrine (0-40 μM) for 48 h. The maximum safe concentrations of valsartan and tetrandrine were selected using a cell viability assay. MPC5 podocytes stably expressing TRPC6 were constructed using a lentivirus packaging system, followed by treatment with valsartan, tetrandrine, and Y-27632 for 48 h and U73122 (10 μM) for 10 min. The RhoA/ROCK pathway and podocyte-specific proteins (nephrin and synaptopodin) levels were quantified. Podocyte apoptosis and intracellular Ca²⁺ concentration were measured.

Results: Maximum safe concentrations of 100 μM valsartan and 10 μM tetrandrine showed no observable toxicity in podocytes. MPC5 podocytes stably expressing TRPC6 had higher intracellular Ca²⁺ influx, apoptotic percentages, and expression of RhoA/ROCK proteins, but lower expression of nephrin and synaptopodin proteins. U73122 treatment for 10 min did not inhibit TRPC6, but suppressed RhoA/ROCK protein. Y-27632 decreased ROCK1 expression, but did not influence the expression of TRPC6 protein. Both 100 μM valsartan and 10 μM tetrandrine for 48 h significantly inhibited intracellular Ca²⁺ influx, apoptosis, and RhoA/ROCK pathway, and increased nephrin and synaptopodin proteins in podocytes stably expressing TRPC6.

Conclusion: Elevated TRPC6 expression can lead to podocyte injury by inducing intracellular Ca²⁺ influx and apoptosis of podocytes, and this effect may be mediated by activation of the RhoA/ROCK1 pathway. Tetrandrine can alleviate podocyte injury induced by TRPC6 expression through inhibition of the RhoA/ROCK pathway, suggesting a protective role in podocyte damage.

Keywords: RhoA/ROCK pathway; podocyte; tetrandrine; transient receptor potential cation channel protein 6.

Figure 1

Optimization of drug concentrations. (A) Cell viability of MPC5 podocytes under treatment with different concentrations of drugs detected by CCK8. (B) The LDH cytotoxicity assay in MPC5 podocytes treated with different drugs. **P<0.01 NC: containing blank lentivirus vector. blank: normal MPC5 podocyte.TRPC6 group: TRPC6-overexpressing.

Figure 2

Detection of TRPC6 mRNA and protein expression in podocyte transfection of lentivirus packaging plasmid, respectively. (A) detection of TRPC6 mRNA expression by RT-qPCR. (B) detection of TRPC6 protein levels by western-blot. NC: containing blank lentivirus vector. blank: normal MPC5 podocyte.TRPC6 group: TRPC6-overexpressing.

Figure 3

Tetrandrine inhibits TRPC6 overexpression-induced intracellular Ca²⁺ influx in MPC5 podocytes. Fluorescence image of fluo-3AM loaded cells (green) indicates intracellular Ca²⁺ influx. Differences were analyzed using one-way ANOVA. Significant differences with p < 0.05 are indicated by different letters. NC: containing blank lentivirus vector. blank:normal MPC5 podocyte. TRPC6 group: TRPC6-overexpressing.

Figure 4

Tetrandrine prevents TRPC6 overexpression-induced apoptosis in MPC5 podocytes. Apoptosis was quantified using flow cytometry. Differences were analyzed using one-way ANOVA. Different letters show significant differences with p < 0.05. NC: containing blank lentivirus vector. blank:normal MPC5 podocyte.TRPC6: TRPC6-overexpressing.

Figure 5

TRPC6 activates RhoA/ROCK1 signaling. (A) Analysis ofof TRPC6, RhoA and ROCK1 mRNA by RT-qPCR analysis. (B) Analysis of TRPC6, RhoA-GTP/RhoA and ROCK1, nephrin and synaptopodin protein levels by Western blot. Differences were analyzed using one-way ANOVA. Significant differences with p < 0.05 are indicated by different letters. NC: containing blank lentivirus vector. blank:normal MPC5 podocyte.TRPC6: TRPC6-overexpressing.

Figure 6

Analysis of TRPC6, ROCK1,and synaptopodin protein levels by Western blot in different groups when cells were treated with Y-27632. Differences were analyzed using one-way ANOVA. Significant differences with p < 0.05 are indicated by different letters. NC: containing blank lentivirus vector. blank: normal MPC5 podocyte. TRPC6: TRPC6-overexpressing.