Abstract:

Objective

To investigate the effect of high glucose on MST1 (mammalian sterile 20-like kinase 1)expression in primary glomerular podocytes.

Methods

Primary podocytes cultured from healthy SD rat kidneys after gradient centrifugation were divided into normal group (cultured with glucose of 5 mmol/L) and high glucose group (cultured with glucose of 25 mmol/L). The cell properties were verified with flow cytometry and immunofluorescence. The time points for observation were after 12, 24, 48, and 72 hours of cell culture. And immunofluorescence and Western blotting method were used for detecting MST1 and caspase-3 expression. Multiple-group comparison was performed with single factor analysis of variance,while two groups were compared with Q test.

Results

(1) It was shown by flow cytometry that 93.18% of the podocytes cultured expressed nephrin protein, suggesting that stable culture of primary podocytes was successful. Besides, indirect immunofluorescence staining showed that the podocytes stably expressed nephrin and WT-1 proteins. (2) Immunofluorescence staining showed a small amount of MST1 expression in podocytes of the normal group mainly locating in the cytoplasm, while the expression of MST1 increased in the high glucose group, and more nuclear expression began to appear after 48 h of treatment. (3) Western blotting showed that the levels of caspase-3 and MST1 were significantly higher in the high glucose group than in the normal group (F =84. 989, 312. 407, P ＜0. 001). Furthermore, after 48 h of treatment,cleaved form of MST1 was observed in the high glucose group, which also increased with the time of treatment.

Conclusions

The dysfunction of MST1 pathway may be involved in the pathogenesis of diabetic nephropathy.

Key words: Podocyte, Protein kinase, High glucose, Diabetic nephropathy