Ischemia-reperfusion injury is the major cause of delayed graft function in transplanted kidneys, an early event significantly affecting long-term graft function and survival. Several studies in rodents suggest that the alternative pathway of the complement system plays a pivotal role in renal ischemia-reperfusion injury. However, limited information is currently available from humans and larger animals. Here we demonstrated that 30 minutes of ischemia resulted in the induction of C4d/C1q, C4d/MLB, and MBL/MASP-2 deposits in a swine model of ischemia-reperfusion injury. The infusion of C1-inhibitor led to a significant reduction in peritubular capillary and glomerular C4d and C5b-9 deposition. Moreover, complement-inhibiting treatment significantly reduced the numbers of infiltrating CD163 ؉ , SWC3a ؉ , CD4a ؉ , and CD8a

Our data demonstrate a critical role for complement in the acute induction of EndMT via the Akt pathway. Therapeutic inhibition of these systems may be essential to prevent vascular damage and tissue fibrosis in transplanted kidney.

Sepsis remains a serious cause of morbidity and mortality in critically ill patients, with limited therapeutic options available. Of the several disorders connected with sepsis, acute kidney injury (AKI) is one of the major complications. The pathophysiology of sepsis-induced AKI is characterized by severe inflammation in renal parenchyma with endothelial dysfunction, intra-glomerular thrombosis and tubular injury. Endothelial dysfunction is regulated by several mechanisms implicated in cellular de-differentiation, such as endothelial-to-mesenchymal transition (EndMT). Gram-negative bacteria and their cell wall component lipopolysaccharides (LPSs) are frequently involved in the pathogenesis of AKI. The host recognition of LPS requires a specific receptor, which belongs to the Toll-like receptor (TLR) family of proteins, called TLR4, and two carrier proteins, namely the LPS-binding protein (LBP) and cluster of differentiation 14 (CD14). In particular, LBP is released as a consequence of Gram-negative infection and maximizes the activation of TLR4 signalling. Recent findings regarding the emerging role of LBP in mediating sepsis-induced AKI, and the possible beneficial effects resulting from the removal of this endogenous adaptor protein, will be discussed in this review.

Pentraxin-3 (PTX3) belongs to the pentraxine family, innate immune regulators involved in angiogenesis, proliferation and immune escape in cancer. Here, we evaluated PTX3 tissue expression and serum levels as biomarkers of clear cell renal cell carcinoma (ccRCC) and analyzed the possible role of complement system activation on tumor site. A 10-year retrospective cohort study including patients undergoing nephrectomy for ccRCC was also performed. PTX3 expression was elevated in both neoplastic renal cell lines and tissues, while it was absent in both normal renal proximal tubular cells (HK2) and normal renal tissues. Analysis of complement system activation on tumor tissues showed the co-expression of PTX3 with C1q, C3aR, C5R1 and CD59, but not with C5b-9 terminal complex. RCC patients showed higher serum PTX3 levels as compared to non-neoplastic patients (p<0.0001). Higher PTX3 serum levels were observed in patients with higher Fuhrman grade (p<0.01), lymph node (p<0.0001), and visceral metastases (p<0.001). Patients with higher PTX3 levels also showed significantly lower survival rates (p=0.002). Our results suggest that expression of PTX3 can affect the immunoflogosis in the ccRCC microenvironment, by activating the classical pathway of CS (C1q) and releasing pro-angiogenic factors (C3a, C5a). The up-regulation of CD59 also inhibits the complementmediated cellular lysis.

Epigenetic mechanisms, such as DNA methylation, affect tubular maladaptive response after Acute Kidney Injury (AKI) and accelerate renal aging. Upon ischemia/reperfusion (I/R) injury, Complement activation leads to C5a release that mediates damage; however, little is known about the effect of C5a-C5a Receptor (C5aR) interaction in Renal Tubular Epithelial Cells (RTEC). Through a whole-genome DNA methylation analysis in cultured RTEC, we found that C5a induced aberrant methylation, particularly in regions involved in cell cycle control, DNA damage and Wnt signaling. The most represented genes were BCL9 , CYP1B1 and CDK6 . C5a stimulation of RTEC led to up-regulation of SA-β Gal and cell cycle arrest markers such as p53 and p21. C5a increased also IL-6 , MCP-1 and CTGF gene expression, consistent with SASP development. In accordance, in a swine model of renal I/R injury, we found the increased expression of Wnt4 and βcatenin correlating with SA-β Gal, p21, p16 and IL-6 positivity. Administration of Complement Inhibitor (C1-Inh), antagonized SASP by reducing SA-β Gal, p21, p16, IL-6 and abrogating Wnt4/βcatenin activation. Thus, C5a affects the DNA methylation of genes involved in tubular senescence. Targeting epigenetic programs and Complement may offer novels strategies to protect tubular cells from accelerated aging and to counteract progression to Chronic Kidney Disease