GAS5 promotes podocyte injury in sepsis by inhibiting PTEN expression

Y. Fang, J.-F. Hu, Z.-H. Wang, S.-G. Zhang, R.-F. Zhang, L.-M. Sun, H.-W. Cui, F. Yang

Intensive Care Unit, The First Affiliated Hospital, Zhengzhou University, Zhengzhou, China. rigw72@163.com

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• Nephrology

OBJECTIVE: To investigate the potential role of long noncoding RNA (lncRNA) growth arrest specific transcript 5 (GAS5) in sepsis-induced podocyte injury and its underlying mechanism.

MATERIALS AND METHODS: The sepsis model was established by lipopolysaccharide (LPS) induction in podocytes. The expression levels of Nephrin and GAS5 were detected by quantitative Real-time polymerase chain reaction (qRT-PCR) after LPS induction in podocytes for 12 h, 24 h and 36 h, respectively. Western blot was used to detect the expression level of Nephrin in sepsis-induced podocytes. The mRNA expressions of GAS5 and Nephrin in podocytes were detected after transfection of GAS5 siRNA. Albumin influx in podocytes after GAS5 knockdown was detected by Transwell assay. Western blot was used to detect the protein expression of Snail in sepsis after GAS5 knockdown. The target gene of GAS5 was predicted by bioinformatics analysis. QRT-PCR and Western blot were used to detect the protein and mRNA levels of PTEN (phosphatase and tensin homolog deleted on chromosome ten). Nephrin expression and the albumin inflow after PTEN knockdown were then measured. The expression of PI3K/AKT/GSK3β was also detected after GAS5 was downregulated while PTEN was upregulated.

RESULTS: LPS stimulation downregulated the mRNA expression of Nephrin in podocytes and achieved the lowest level at 24 h. The protein expression change of Nephrin was consistent with its mRNA expression. In the septic state, the albumin influx of podocytes remarkably increased, but the function of podocyte barrier was weakened. Besides, GAS5 expression decreased in a time-dependent manner in LPS-induced podocytes. After GAS5 knockdown by siRNA, Nephrin expression and the function of podocyte barrier were significantly reduced. Snail expression was also upregulated in septic state, and GAS5 knockdown increased the expressions of phosphorylated Snail and PI3K/AKT/GSK3β. After knockdown of GAS5, the mRNA and protein levels of PTEN significantly decreased, which was contract to the expression of Snail. However, overexpression of PTEN could reverse the promotive effect of GAS5 on PI3K/AKT activation.

CONCLUSIONS: GAS5 expression decreased in sepsis-induced podocyte injury, and GAS5 was involved in regulating sepsis-induced podocyte injury by reducing PTEN expression.

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