MON-003 Relationship between urinary podocytes and urinary protein fraction evaluated by SDS-PAGE

Abstract

In vitro, the conditionally immortalized mouse podocytes (MPC5) were used to investigate the effects of Yi Qi Qing Re Gao-containing serum (YQ-S) on actin cytoskeleton reorganization. Podocytes injury were evaluated by released lactic dehydrogenase (LDH) activity assay. Podocytes apoptosis were determined by Annexin V assay. Wound healing assay and transwell migration assay were used to assess podocyte motility. Immunofluorescence was used to demonstrate the formation and distribution of stress fiber. Western Blot was performed to detect the expression of key proteins of RhoA/ROCK signaling pathway. Results: YQQRG significantly decreased urinary protein level, lowered serum lipid level, elevated serum albumin, and ameliorated renal function in Wistar rats, similar to the effects of CsA. Renal pathological lesions, especially podocytes effacement were obviously attenuated. The experiments in vivo and in vitro both displayed that YQQRG could downregulate the mRNA and protein expressions of a-actinin-4, synaptopodin, vimentin, desmin, and nestin in renal tissues and podocytes. Besides, YQ-S alleviated PAN-induced podocytes injury and apoptosis, and reduced podocyte motility and migration. Disruption of F-actin and disorganization of cytoskeleton were largely recovered by YQ-S. Furthermore, YQ-S significantly antagonized PAN-induced downregulation of the protein expression of RhoA and ROCK, decreased the phosphorylation of MLC, LIMK and cofilin, and increased F-actin/Gactin levels. Addition of Y-27632 (a Rho-kinase inhibitor) could weaken the effects of YQ-S on F-actin reorganization and expressions of ROCK, p-MLC, and p-LIMK. Conclusions: YQQRG alleviates actin cytoskeleton reorganization and attenuates podocytes injury in PAN model in vivo and in vitro, and its mechanism may involve the upregulation of RhoA/ROCK signaling pathway. These findings suggest that YQQRG might be a promising natural drug for the treatment of kidney diseases.