Macrophage-Induced Pro-Fibrotic Gene Expression in Tubular Cells after Ischemia/Reperfusion Is Paralleled but Not Directly Mediated by C5a/C5aR1 Signaling

Abstract

Ischemia/reperfusion (I/R) is inevitable during kidney transplantation and causes acute kidney injury (AKI), which affects immediate outcome and leads to chronic changes such as fibrotic remodeling of the graft. We investigated pro-fibrotic signaling after I/R, focusing on the complement component and receptor C5a/C5aR1 and macrophage/tubule crosstalk. Male Dark Agouti rats were subjected to I/R and their kidneys were harvested 10 min, 6 h, 24 h, 3 days, 5 days and 8 weeks after reperfusion. The development of renal fibrosis was assessed by the detection of Vimentin (VIM), α-smooth muscle actin (α-SMA) and collagen by immunohistochemistry and Sirius Red staining, respectively. The characterization of C5a/C5aR1 activity and C5aR1+ cells was performed by multiplex mRNA analysis, ELISA, immunofluorescence flow cytometry and in situ hybridization in animal models and cell culture analyses. In the cell culture experiments, we focused on macrophage/tubule cell crosstalk in co-culture experiments and mimicked in vivo conditions by hypoxia/reoxygenation and supplementation with C5a. Already 6–24 h after the induction of I/R in the rat model, C5a concentration in the plasma was significantly increased compared to the control. The matrix components VIM and α-SMA peaked on day 5 and declined after 8 weeks, when an increase in collagen was detected using Sirius Red. In contrast to early I/R-induced C5a activation, renal C5ar1 expression was maximal at day 5 and C5 expression increased until week 8, indicating that the renal upregulation of expression is not required for early complement activation. C5aR1 mRNA was detected in neutrophils and macrophages, but not in proximal tubular cells in the injured kidneys. The macrophage/tubular cell co-culture experiments showed that macrophages were mainly responsible for the increased expression of fibrosis-associated genes in tubule cells (ACTA2, VIM, SNAI1, TGFB1 and FGF-2), and hypoxia/reoxygenation had a partially enhancing effect. A direct pro-fibrotic effect of C5a was not observed. Increased TGF-ß levels were dependent on the differentiation of macrophages to the M2 subtype. In conclusion, the early activation of mesenchymal markers in tubular epithelial cells leads to long-term fibrotic remodeling characterized by VIM expression and driven by TGF-ß-dependent macrophage/tubular crosstalk. The chemoattractive properties of complement C5a may contribute to the recruitment of pro-fibrotic macrophages.