The Role For Toll-like Receptor 9 (TLR9) In Podocyte Injury

Objective:Toll-like Receptors (TLRs) and their signaling are the essential mechanism by which the organisms protect themselves from pathogens through both innate and acquired immunity. It has been reported that TLR4 is expressed in podocyte, and involved in podocyte injury. Recently TLR9 has also been shown to be de novo expressed in the podocytes of the patients of several glomerular diseases, but its role in podocyte injury is not known. This study aims to determine whether TLR9 mediates podocyte injury by using PAN-induced podocyte injury model.Methods:1) PAN was used to induce injury in cultured podocytes, and the expression or activation of TLR9 and its signaling components, IRAK1, TRAF6, NF-κB p65, p38 MAPK and IL-12, were examined by qRT-PCR or Western blotting; in addition, NF-κB p65 nuclear translocation was examined by immunofluorescence staining, and podocyte apoptosis was assayed by Annexin V staining followed by flow cytometry. Besides, PAN-induced podocyte injury rat model was also generated and TLR9 expression in the podocytes/glomeruli was examined by immunohistochemistry and qRT-PCR.2) To explore the role of TLR9 signaling in podocyte injury, podocytes were transfected with TLR9 siRNA expresssing plasmid or control plasmid, followed by PAN treatment, and p65 or p38 phosphorylation was then examined and compared by Western blotting, IL12 mRNA levels by qRT-PCR, and cell apoptosis by Annexin V-flow cytometry.Results:1) PAN increased the mRNA levels of TLR9, IRAK1, TRAF6 and IL12, the phosphorylation of p65 and p38, p65 nuclear translocation and apoptosis in the podocytes. Both TLR9 mRNA and protein were upregulated in the podocytes/glomeruli of rats treated with PAN.2) Compared with control plasmid, the transfection with TLR9 siRNA plasmid decreased TLR9 mRNA and protein, p65 and p38 phosphorylation, apoptosis, and IL12 mRNA in the podocytes treated with PAN.Conclusions:TLR9 signaling is involved in the podocyte injury induced by PAN. This finding may have provided a novel mechanism underlying podocyte apoptosis in certain glomerular diseases in which TLR9 is de novo expressed.