Abstract:

ABSTRACT  Objective: We have found that Toll-like receptor 9 (TLR9) was upregulated in the podocytes treated with puromycin aminonucleoside (PAN). This study aims to determine whether TLR9 mediates PAN-induced podocyte injury Methodology: 1)  PAN was used to induce injury in cultured podocytes, and the expression or activation of TLR9 and the components of the pathway was examined by qRT-PCR or Western blotting. Podocyte apoptosis was assayed by Annexin V staining followed by flow cytometry. PAN-induced podocyte injury model of rat was generated and TLR9 expression in the glomeruli was examined by immunohistochemistry and qRT-PCR. 2) To explore the role of TLR9 signaling in podocyte injury, we transfected podocytes with TLR9 siRNA expresssing plasmid and control plasmid, respectively, followed by PAN treatment. We then compared the p65 and p38 phosphorylation by Western blotting, and IL12 mRNA levels by qRT-PCR, and cell apoptosis by Annexin V-flow cytometry between the cells transfected with TLR9 or control siRNA plasmid.  Results: 1) The mRNA levels of TLR9, IRAK1, TRAF6 and IL12, as well as the phosphorylation of p65 and p38, and apoptosis in the podocytes was increased by PAN. Both TLR9 mRNA and protein were upregulated in the podocytes of rats treated with PAN. 2) Compared with control plasmid, the transfection with TLR9 siRNA plasmid decreased TLR9 mRNA and protein levels, p65 and p38 phosphorylation, as well as podocyte apoptosis; in addition, mRNA levels of IL12 was also downregulated. Conclusion: TLR9 signaling was involved in the podocyte injury induced by PAN. This finding may provide a novel mechanism underlying podocyte apoptosis in glomerular diseases.

Key words:  TLR9, podocytes, apoptosis, puromycin aminonucleoside, p38 MAPK