Identification of c-Jun phosphorylation as a crucial mediator of complement activation in renal ischemia-reperfusion injury revealed by phosphoproteomics and functional validation.

Background: Ischemia reperfusion injury (IRI) is an unavoidable condition that primarily affects graft function in renal transplantation. Blockage of complement activation by complement receptor immunoglobulin/ factor H (CRIg/FH), a novel complement inhibitor, shows great potency to ameliorate renal IRI. Sublytic membrane attack complex (MAC) disrupts cellular functions via the activation of different protein kinases and phosphorylation of critical signal transduction factors. We aimed to investigate whether complement activation triggered shift in phosphorylation status in IRI.

Methods And Results: We performed a LC-MS/MS-based quantitative phosphoproteomic analysis of CRIg/FH-IRI, PBS-IRI and Sham mice, depicting a thorough protein phosphorylation profile. C3d and MAC staining were conducted to study the complement activation status. In vitro model mimicking complement mediated IRI tubular injury was achieved by applying normal human serum (NHS) to TCMK cells. By hierarchical clustering, we observed that CRIg/FH treatment reversed the hyperphosphorylation status triggered by IRI. Differentially expressed phosphoproteins (DEPs) were associated with focal adhesion, integrin activation, actin cytoskeleton organization and cell junction. We identified c-Jun as the most differentially phosphorylated transcriptional factor regulated by complement activation, the S63 phosphorylation of which was verified both in vitro and in vivo and screened for its downstream targets. JNK inhibitor reduced the phosphorylation of c-Jun and attenuated accumulation of the C3d on the tubular epithelial cells.

Conclusion: We proposed a crucial role of c-Jun phosphorylation in complement activation induced by renal IRI by combining phosphoproteomic approaches and protein validation, which hopefully could provide novel insights into the pathological mechanisms of IRI.