Abstract
Podocyte injury is closely associated with the onset of proteinuria in many kidney diseases. A major mechanism of podocyte injury is the reorganization of actin cytoskeleton, which leads to increased podocyte motility and foot process effacement. Yi Qi Qing Re Gao (YQQRG), a traditional Chinese herbal formula, has been used clinically to treat chronic nephritis for decades with dramatic anti-proteinuria effects. In this study, we investigated the effects of YQQRG on actin cytoskeleton reorganization induced by puromycin aminonucleoside (PAN) and explored the possible mechanisms. Wistar rats were randomly divided into sham group, PAN group, PAN + YQQRG group, and PAN + Cyclosprin A (CsA) group. The PAN model was established by a single intravenous injection of PAN (40mg/kg body weight). Serum biochemical parameters and 24h urinary protein were assessed on days 5, 9, and 14, respectively. Renal tissues were harvested for observations of pathologic alterations and ultrastructural changes. Quantitative expressions of regulatory proteins of cytoskeleton were measured using Western-blot and real-time polymerase chain reaction. 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. 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 α-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/G-actin 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. 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.
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
