Search

CKIP-1 participates in the activation of Nrf2 signaling pathway by Cx43 and the regulation of diabetic renal fibrosis Under normal conditions, CKIP-1 was combined with Cx43 via Cx43 CT domain on the membrane of GMCs, which induced the interaction between CKIP-1 and Nrf2 and the nuclear accumulation of Nrf2, leading the activation of Nrf2 anti-oxidative stress signaling pathway. When the cells were stimulated by high glucose, the expression of Cx43 was inhibited, following with the down-regulation of CKIP-1 protein level. Moreover, the interaction between CKIP-1 and Nrf2 was weaken whereas the binding of Nrf2 to its inhibitory protein Keap1 was enhanced, as a result of which the activity of Nrf2 signaling pathway was inhibited with the overproduction of intracellular superoxide. Increased oxidative stress further promoted the expression of FN eventually exacerbating diabetic renal fibrosis. • CKIP-1 participated in the activation of Nrf2 signaling pathway by Cx43 and the regulation of diabetic renal fibrosis. • Cx43 interacted with CKIP-1, and the interaction was weakened by high glucose treatment. • Cx43 regulated the expression of CKIP-1 and the interaction of CKIP-1 with Nrf2 via Cx43 carboxyl terminus domain. We previously reported that both Cx43 and CKIP-1 attenuated diabetic renal fibrosis via the activation of Nrf2 signaling pathway. However, whether CKIP-1, a scaffold protein, participates in regulating the activation of Nrf2 signaling pathway by Cx43 remains to be elucidated. In this study, the effect of adenovirus-mediated Cx43 overexpression on renal fibrosis in CKIP-1−/− diabetic mice was investigated. We found that overexpression of Cx43 could significantly alleviate renal fibrosis by activating the Nrf2 pathway in diabetic mice, but have no obvious effect in CKIP-1−/− diabetic mice. Cx43 overexpressed plasmid and CKIP-1 small interfering RNA were simultaneously transfected into glomerular mesangial cells and the result demonstrated that the effect of activation of Nrf2 signaling pathway by Cx43 was blocked by CKIP-1 depletion. The interaction between Cx43 and CKIP-1 was analyzed by immunofluorescence and immunoprecipitation assays. We found that Cx43 interacted with CKIP-1, and the interaction was weakened by high glucose treatment. Moreover, Cx43 regulated the expression of CKIP-1 and the interaction of CKIP-1 with Nrf2 via Cx43 carboxyl terminus (CT) domain, thereby activating Nrf2 signaling pathway. According to the results, we preliminary infer that CKIP-1 acts downstream to CX43 on the activation of Nrf2 signaling pathway to protect from renal fibrosis in diabetes, the mechanism of which might be related to the interaction of CKIP-1 with Nrf2 through Cx43 CT. Our study provides further experimental basis for targeting the Cx43-CKIP-1-Nrf2 axis to resist diabetic renal fibrosis. [ABSTRACT FROM AUTHOR]

• Gentiopicroside reversed the downregulation of TGR5 and the upregulation of ECM and inflammatory factors in HG-induced GMCs. • Gentiopicroside inhibited the activation of the NF-κB signalling pathway by preventing the phosphorylation of IκBα. • The depletion of TGR5 blocked the inhibition of the NF-κB signalling pathway by GPS. • Gentiopicroside enhanced the interaction between β-arrestin2 and IκBα via TGR5 activation. Our previous studies indicated that the G-protein-coupled bile acid receptor, Gpbar1 (TGR5), inhibits inflammation by inhibiting the NF-κB signalling pathway, eventually attenuating diabetic nephropathy (DN). Gentiopicroside (GPS), the main active secoiridoid glycoside of Gentiana manshurica Kitagawa , has been demonstrated to inhibit inflammation in various diseases via inhibiting the inflammatory signalling pathways. However, whether GPS inhibits the NF-κB signalling pathway by activating TGR5 and regulates the pathological progression of diabetic renal fibrosis requires further investigation. In this study, we found that GPS significantly reversed the downregulation of TGR5 and inhibited the overproduction of fibronectin (FN), transforming growth factor β1 (TGF-β1), intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1) in glomerular mesangial cells (GMCs) exposed to high glucose (HG). Additionally, GPS prevented the phosphorylation and degradation of IκBα, and subsequently inhibited the activation of the NF-κB signalling pathway. Further investigation found that GPS enhanced the stabilization of IκBα by promoting the interaction of β-arrestin2 with IκBα via TGR5 activation, which contributed to the inhibition of NF-κB signalling pathway. Importantly, the depletion of TGR5 blocked the inhibition of the NF-κB signalling pathway and reversed the downregulation of FN, ICAM-1, VCAM-1 and TGF-β1 by GPS in HG-induced GMCs. Moreover, GPS increased the TGR5 protein levels and promoted the interaction between IκBα and β-arrestin2, thereby inhibiting the reduction of IκBα and blocked NF-κB p65 nuclear translocation in the kidneys of STZ-induced diabetic mice. Collectively, these data suggested that GPS regulates the TGR5-β-arrestin2-NF-κB signalling pathway to prevent inflammation in the kidneys of diabetic mice, and ultimately ameliorates the pathological progression of diabetic renal fibrosis. [ABSTRACT FROM AUTHOR]