Tetrandrine alleviates podocyte injury via calcium-dependent calpain-1 signaling blockade

Abstract

Background: Podocytes have become a crucial target for interventions in proteinuric kidney diseases. Many studies have reported that overexpression of transient receptor potential cation channel protein 6 (TRPC6) in podocyte injury upregulates intracellular Ca²⁺ influx and stimulates Ca²⁺-dependent protease calpain-1 signaling. The traditional Chinese drug, tetrandrine, a nonselective Ca²⁺ channel blocker, has long been used to treat chronic kidney disease. This research aimed to explore the possible mechanisms underlying the anti-proteinuric properties of tetrandrine.

Methods: We investigated the involvement of tetrandrine in Ca²⁺ dependent calpain-1 signaling in mouse podocytes and adriamycin-induced nephropathy rats. Cyclosporine A (CsA) and U73122 were used as positive controls. Cell viability, cytotoxicity, Ca²⁺ concentration, calpain activity, and mRNA and protein expression levels of calpain-1 signaling pathways were examined. The clinical and pathological changes were measured.

Results: Tetrandrine decreased intracellular Ca²⁺ influx in cultured TRPC6-overexpressing podocytes. In both in vitro and in vivo studies, the administration of tetrandrine downregulated calpain activity and the expression of calpain-1 and restored the expression of downstream Talin-1 and nephrin. Compared to CsA, tetrandrine treatment exhibited superior inhibitory effects on calpain activity and calpain-1 expression.

Conclusions: Tetrandrine has therapeutic potential in podocyte damage by blocking Ca²⁺-dependent activation of the calpain-1 signaling pathway. Tetrandrine reduced proteinuria, improved renal function, and alleviate renal pathological damage.

Keywords: Calpain-1; Podocyte injury; Tetrandrine; Transient receptor potential cation channel protein 6.

Fig. 1

Optimization of drug concentrations. MPC5 podocyte viability under treatment with different concentrations of tetrandrine (a) and CsA (b) detected by CCK8 assay. c The LDH cytotoxicity assay in MPC5 podocytes treated with tetrandrine, CsA and U73122. * P<0.05. NC: normal control. Lv-NC: blank lentivirus. OverTRP: TRPC6-overexpressing

Fig. 2

Construction of TRPC6 overexpressing cell model, and detection of Ca²⁺ influx among groups. The mRNA and protein levels of TRPC6 were determined by qRT-PCR (a) and western blotting (b), respectively. The intracellular Ca2⁺ influx was determined using Fluo-3 AM under confocal microscope (c) and semiquantitative analysis (d). Different letters represent statistically different values (P < 0.05), whereas the same letters indicate no significant difference

Fig. 3

Tetrandrine decreased Ca²⁺-dependent calpain-1 signaling in cultured podocytes. a Calpain and b calcineurin activity were measured. c Typical bands showing the protein levels of TRPC6, calpain-1, calcineurin, Talin-1 and nephrin, normalized to GAPDH. Detection of mRNA d-g and protein levels h-k of calpain-1, calcineurin, Talin-1 and nephrin, respectively. CsA and U73122 were used as positive controls. Different letters represent statistically different values (P < 0.05), whereas the same letters indicate no significant difference

Fig. 4

Tetrandrine-mediated inhibition of Ca²⁺/calpain-1 signaling in adriamycin-induced nephropathy rats. TRPC6 overexpressing rat model was established (a, b). c Calpain and (d) calcineurin activity was measured. e Typical bands showing the protein levels of TRPC6, calpain-1, calcineurin, Talin-1 and nephrin, normalized to GAPDH. The mRNA (f-i) and protein expressions (j-m) of calpain-1, Talin-1, calcineurin and nephrin, respectively. CsA was used as the positive control. Different letters represent statistically different values (P < 0.05), whereas the same letters indicate no significant difference

Fig. 5

Protective effects of drugs on renal function in adriamycin-induced nephropathy rats. 24 h-proteinuria (a), plasma albumin (b), serum creatinine (c), and blood urea nitrogen (d) were measured and compared. Different letters represent statistically different values (P < 0.05), whereas the same letters indicate no significant difference

Fig. 6

Morphological characteristics of kidney in rats. Typical morphological characteristics of glomerulus under light microscopy (HE staining, magnification: 400×) and electron microscopic evaluation of podocyte structure among different groups of rats (magnification: 20000×)