Effects of knockout receptor activator of NF-κB (RANK) in podocytes on proteinuria in mice

Abstract

Objective To investigate the role of RANK in injured podocyte and its effects on proteinuria. Methods PCR analysis was carried out to identify the genotypes of podocyte-specific RANK knockout mice. Six (6-8 weeks old) female RANK-/- mice were chosen as KO group. Six (6-8 weeks old) littermates and six (6-8 weeks old) C57/B6j mice were chosen as WT group and C57 group respectively. Each group was injected with LPS to create podocyte-injured proteinuria model. 24h urine was collected before and after injection. Urinary albumin, urine creatinine and UACR were detected by automatic biochemical analyzer. The mice were sacrificed after 48 h. Cortical kidney tissue samples were stained by PAS and collected for transmission electron microscopy to detect the foot process effacement. The number of podocytes was examined at different time points by immunohistochemistry. The expression of intergrin-β1, integrin-β3 and uPAR was detected by immunofluorescence and westernblot. Results (1) Comparing KO group, WT group and C57 group, it was found that there was no difference in weight, morphology, function of kidney and ACR. (2) After LPS injection, proteinuria and ACR were increased in three groups, but less significantly in KO group (23.70±9.90), compared to those in WT group (107.56±22.32) and C57 group (132.13±14.26) (P<0.001). (3) In three groups, the number of podocytes was decreased after LPS injection and the decrease in the KO group was the slightest. (4) Compared to WT and C57 group, podocyte foot process effacement was less obvious in KO group after LPS injection in transmission electron microscopy. (5) Immunofluorescence results showed that after LPS injection, integrin-β1 was decreased in three groups, but most significantly in KO group. Integrin-β3 was increased in three groups, but less obvious in KO group. (6) Westernblot results showed consistency with immunofluorescence. The expression of uPAR protein was increased in three groups, with the increase in KO group being the slightest. Conclusions Podocyte-specific knockout of RANK reduces LPS-induced proteinuria, suggesting that RANK might be involved in the development of proteinuria in podocyt injury. Its mechanism is probably related with integrin-β1, integrin-β3 and uPAR. Key words: Podocyte; Mice, knockout; Lipopolysaccharides; Receptor activator of nuclear factor-kappa B