Complement induced aberrant epigenetic modifications in renal tubular epithelial cells and accelerates senescence after kidney transplantation by Wnt pathway

Epigenetic mechanisms as DNA methylation can affect allograft outcome after kidney transplantation, accelerating renal aging. Complement system is the major player of ischemia reperfusion (I/R) injury and Renal Proximal Tubular Cells (RPTEC) are known to express C5a receptor (C5aR). However, little is known about the downstream effect of C5a-C5aR interaction. We performed a whole-genome DNA methylation analysis on C5a stimulated RPTEC and found several regions regulating genes involved in cell cycle control, DNA damage checkpoints and WNT signaling. These most represented genes were BCL9, CYP1B1 and CDK6. We showed that C5a induced-aberrant gene methylation significantly influences the expression of these genes. Moreover, the stimulation of RTEC by C5a led to senescence by up-regulating SA-β Gal positivity and increasing p53 gene and p21 protein level. IL-6, MCP-1, CTGF gene levels increase indicated the tubular SASP (Senescence associated Secretory Phenotype) acquirement. In accordance, in a swine model of I/R injury, we found that the increase expression of WNT4/βcatenin expression correlated with the augment in SA-β Gal, p21, p16 and IL-6 positivity after 24h from reperfusion. The treatment with C1-INH, a complement inhibitor, efficiently antagonized SASP restoring SA-β Gal, p21, p16, IL-6 expression at basal level and abrogating the WNT4/βcatenin activation. Thus, C5a affects the DNA methylation of genes involved in tubular senescence leading to a persistent, low-grade, inflammatory state called inflammaging. Targeting epigenetic programs may offer novels strategies to protect tubular cells from aging and promote kidney repair and recovery. Bisulphite converted DNA from Renal cells were hybridized to the Illumina Infinium HumanMethylation450 BeadChip