The roles of autophagy in the treatment of diabetic nephropathy with rapamycin.

Abstract

Background: Rapamycin is known to induce autophagy, promote cell survival and inhibit the progression of diabetic nephropathy (DN).

Objectives: The aim of this study was to examine the role of autophagy in the treatment of DN with rapamycin to provide the basis for the DN treatment with rapamycin.

Material and methods: Human mesangial cells (HMC) were cultured in a constant temperature incubator with 5% CO2, at 37°C and saturated humidity. Cells were divided into 5 groups and the 5-ethynyl-2-deoxyuridine (EdU) cell proliferation assay was used to determine cell proliferation. Flow cytometry was used to determine cell apoptosis, while GFP-RFP-LC3 showed autophagy flow. Western blot was employed to detect the expression of autophagy-related proteins LC3-II/LC3-I and P62. Enzyme-linked immunosorbent assay (ELISA) was used to determine the contents of type IV collagen fiber (Col4), hyaluronic acid (HA) and laminin (LA) in the extracellular matrix (ECM).

Results: Cell proliferation was the lowest in the hyperglycemic group. Additionally, the hyperglycemic group displayed the lowest number of autolysosomes compared to other groups. In contrast, the rapamycin group exhibited the highest number of autolysosomes. The LC3-II/LC3-I ratio was also the lowest in the hyperglycemic group, measuring 0.53 (0.50-0.58), while the expression level of P62 was significantly higher in that group at 0.98 (0.95-1.01) compared to other groups. Upon the introduction of rapamycin, the LC3-II/LC3-I ratio was significantly increased at 2.21 (1.95-2.21), and P62 was significantly decreased 0.38 (0.38-0.39) compared to the hyperglycemic group. Both changes were statistically significant, with p-values of 0.034 and 0.010, respectively. Enzyme-linked immunosorbent assay was employed to detect Col4, HA and LA content. The study findings demonstrated significantly higher levels of glucose in the hyperglycemic group in comparison to other groups. In contrast, the rapamycin group exhibited significantly lower levels of glucose than the hyperglycemic group, yet the difference was not statistically significant.

Conclusions: Hyperglycemic can inhibit the autophagic activity of HMC, promote cell apoptosis, enhance ECM accumulation, and facilitate the DN progression. In contrast, rapamycin can elicit autophagy, decrease mesangial matrix proliferation, and therefore impede DN progression.