Targeting C2 reduces ischemia-reperfusion injury-induced complement activation in preclinical human models
Kidney transplantation (KTx) is a main treatment option of end stage renal disease. KTx outcome is hampered by various factors including ischemia-reperfusion (IR) injury (IRI). Animal models suggest a role for natural IgM recognizing neoepitopes exposed on ischemic cells as a main trigger for IRI-induced complement activation. However, it is unclear if experimental data from these animal models can be extrapolated to human IRI. We used in vitro human models for kidney IRI to evaluate complement activation. First, we compared IgM binding and complement fixation on different endothelial cell (EC) sources in a 2D culture model, using primary kidney-derived ECs, primary lung-derived ECs and human umbilical vein ECs (HUVECs). These cells were exposed to hypoxia followed by reoxygenation in presence of complement-active human serum, or serum subjected to targeted complement inhibition. Next, we validated our findings in a 3D microfluidic organ-on-a-chip model for human kidney IRI using both HUVECs and renal proximal tubule epithelial cells (RPTECs). In the 2D IRI model, we observed increased binding of IgM and C3 fixation on different EC sources after ischemia and subsequent reoxygenation in presence of human serum. This was not detected when cells were exposed to normoxic culture conditions. These results were confirmed in the 3D culture model, where hypoxia followed by reperfusion with complement-active human serum also led to IgM binding and C3 fixation, particularly to HUVECs. Expression of ICAM-1, a key adhesion molecule linked to renal IRI pathophysiology, was increased on RPTECs after IR-induced complement activation on HUVECs. In both models, complement inhibition at the level of C2 inhibited the abovementioned effects of IR-induced complement activation. These results suggest classical and lectin complement pathway involvement in IR-induced damage and identify C2 as a target for therapeutic strategies.