Role of Tisp40/Smad2 Protein in High Glucose-induced Renal Interstitial Fibrosis

Abstract

Objective: To observe the effect of high glucose staining on human renal tubular epithelial cells (HKC) and to investigate the role of Tisp40/Smad2 protein in diabetes-induced renal fibrosis. Methods: Human renal tubular epithelial cells (HKC) were cultured in vitro and cells were incubated with a final concentration of 25, 50, and 100 mM high glucose for 24 h. The cell viability was detected by the CCK-8 assay; cells were treated with a final concentration of 50 mM high glucose for 6, 8, 12, 24, 48, and 72 h, and the degree of cell damage by high glucose at different time points was detected. Cells were treated with final concentrations of 25, 50, and 100 mM glucose for 24 h. Tisp40 protein expression was detected by enzyme-linked immunosorbent assay (ELISA) and Tisp40 mRNA expression was detected by polymerase chain reaction (RT-PCR). To observe the effect of high glucose on Tisp40/Smads signaling proteins, the Tisp40 cells were divided into six groups: (1) the normal control group; (2) the 25 mM high glucose group; (3) 50 mM high glucose group; (4) 100 mM high glucose group; (5) Tisp40-positive control group; (6) Tisp40-stimulated group (100 mM high glucose + 50 mM Tisp40), and the expression level of phosphorylated Smad2 protein (p-Smad2) was detected by immunoblotting (Western blot). Results: Compared with the control group, the survival rate of HKC cells decreased significantly with the increase of high glucose staining dose and time (P < 0.05) in a dose-dependent and time-dependent manner. ELISA results showed that the expression of Tisp40 protein was elevated to 44.3, 63.7, and 82.6 μg/ml after 24 h of high glucose action on HKC cells and the induction was in a dose-dependent manner. RT-PCR showed that the expression level of Tisp40 mRNA increased significantly with increasing glucose concentration, which was 2.75, 5.42 and 9.67 times higher than that of the control group, respectively, and the induction was in a dose-dependent manner (P < 0.05). The Western blot showed that the expression level of p-Smad2 protein increased significantly after 24h of glucose action in the cells at concentrations of 25, 50 and 100 mM glucose (P < 0.05), and the amount of p-Smad2 protein reached the maximum under the co-stimulation of 100 mM high glucose + 50 mM Tisp40 given to the cells (P < 0.05). Conclusion: Abnormal expression of Tisp40 and p-Smad2 was involved in the process of high glucose-induced HKC cell injury and Tisp40-induced high expression of Smads proteins played an important role in high glucose-induced renal fibrosis.