This study aimed at comparing the effects of metformin on tubulointerstitial fibrosis (TIF) in different stages of diabetic nephropathy (DN) in vivo and evaluating the mechanism in high glucose (HG)-treated renal tubular epithelial cells (RTECs) in vitro. Sprague–Dawley (SD) rats were used to establish a model of DN, and the changes of biochemical indicators and body weight were measured. The degree of renal fibrosis was quantified using histological analysis, immunohistochemistry, and immunoblot. The underlying relationship between autophagy and DN, and the cellular regulatory mechanism of metformin on epithelial-to-mesenchymal transition (EMT) were investigated. Metformin markedly improved renal function and histological restoration of renal tissues, especially in the early stages of DN, with a significant increase in autophagy and a decrease in the expression of fibrotic biomarkers (fibronectin and collagen I) in renal tissue. Under hyperglycemic conditions, renal tubular epithelial cells inactivated p-AMPK and activated partial EMT. Metformin-induced AMPK significantly ameliorated renal autophagic function, inhibited the partial EMT of RTECs, and attenuated TIF, all of which effectively prevented or delayed the onset of DN. This evidence provides theoretical and experimental basis for the following research on the potential clinical application of metformin in the treatment of diabetic TIF.

Our previous study demonstrated that metformin plays an anti-fibrotic role in addition to its hypoglycemic effect. Worryingly, it often requires more than 5 times the hypoglycemic dose to achieve a satisfactory anti-fibrotic effect, which greatly increases the risk of systemic acidosis caused by metformin overdose. Low-molecular-weight chitosan (LMWC) has natural kidney-targeting properties and good biocompatibility and degradability. Thus, we synthesized a novel carrier metformin-grafted chitosan (CS-MET) based on an imine reaction between oxidized chitosan and metformin. Then, GFP was recruited to form GFP-loaded CS-MET nanoparticles (CS-MET/GFP NPs) with controllable particle size. We hypothesized that CS-MET/GFP NPs would enrich in the kidney and be absorbed by HK-2 cells via megalin-mediated endocytosis by intravenous injection, which may avoid systemic acidosis caused by metformin overdose. Subsequently, the nanoparticle ruptures and releases metformin to exert its anti-apoptotic, anti-inflammatory, and anti-fibrotic effects. Our results showed that CS-MET/GFP NPs have great transfection efficiency and could enter HK-2 cells mainly through megalin-mediated endocytosis. Compared to the free metformin, CS-MET/GFP NPs showed similar anti-apoptotic ability but better therapeutic effects on cellular inflammation and fibrosis in vitro. On the other hand, CS-MET/GFP NPs showed great kidney-targeting ability and superior anti-apoptotic, anti-inflammatory, and anti-fibrotic effects in vivo.

The aim of the study was to compare the role of metformin on tubulointerstitial fibrosis (TIF) in different stages of diabetic nephropathy (DN) in vivo and evaluate its mechanism in high-glucose-treated Renal tubular epithelial cells (RTECs) in vitro. Sprague-Dawley (SD) rats were used to establish model of DN, then the changes of biochemical indicators and body weight were measured. The degree of renal fibrosis was quantified via histological analysis, immunohistochemistry, and immunoblot. The underlying relationship between autophagy and DN was analyzed and the cellular regulatory mechanism of metformin on epithelial-to-mesenchymal transition (EMT) was detected. Metformin markedly improved renal function and showed histological restoration of renal tissues especially in the early stage of DN, with a significant improvement of autophagy and a low expression of fibrotic biomarkers (Fibronectin and Collagen I) in renal tissue. RTECs under hyperglycemic conditions exhibited inactivation of p-AMPK and activation of EMT. But the promotion of AMPK activated by metformin significantly improved renal autophagic function, inhibited the EMT of RTECs, attenuated TIF, so as to effectively prevent or delay the course of DN. This evidence provided theoretical and experimental basis for the following research on the potential clinical usefulness of metformin for the treatment of diabetic TIF.