Kidneys retrieved from brain-dead donors have impaired allograft function after transplantation compared to kidneys from living donors. Donor brain death (BD) triggers inflammatory responses, including both systemic and local complement activation. The mechanism by which systemic activated complement contributes to allograft injury remains to be elucidated. The aim of this study was to investigate systemic C5a release after BD in human donors and direct effects of C5a on human renal tissue. C5a levels were measured in plasma from living and brain-dead donors. Renal C5aR gene and protein expression in living and braindead donors was investigated in renal pretransplantation biopsies. The direct effect of C5a on human renal tissue was investigated by stimulating human kidney slices with C5a using a newly developed precisioncut method. Elevated C5a levels were found in plasma from brain-dead donors in concert with induced C5aR expression in donor kidney biopsies. Exposure of precision-cut human kidney slices to C5a induced gene expression of pro-inflammatory cytokines IL-1 beta, IL-6 and IL-8. In conclusion, these findings suggest that systemic generation of C5a mediates renal inflammation in brain-dead donor grafts via tubular C5a-C5aR interaction. This study also introduces a novel in vitro technique to analyze renal cells in their biological environment.

The complement system, and specifically C5a, is involved in renal ischemia-reperfusion (IR) injury. The 2 receptors for complement anaphylatoxin C5a (C5aR1 and C5aR2) are expressed on leukocytes as well as on renal epithelium. Extensive evidence shows that C5aR1 inhibition protects kidneys from IR injury; however, the role of C5aR2 in IR injury is less clear as initial studies proposed the hypothesis that C5aR2 functions as a decoy receptor. By Using wild-type, C5aR1, and C5aR2 mice in a model of renal IR injury, we found that a deficiency of either of these receptors protected mice from renal IR injury. Surprisingly, C5aR2 mice were most protected and had lower creatinine levels and reduced acute tubular necrosis. Next, an migration study demonstrated that leukocyte chemotaxis was unaffected in C5aR2 mice, whereas neutrophil activation was reduced by C5aR2 deficiency. To further investigate the contribution of renal cell-expressed C5aR2 leukocyte-expressed C5aR2 to renal IR injury, bone marrow chimeras were created. Our data show that both renal cell-expressed C5aR2 and leukocyte-expressed C5aR2 mediate IR-induced renal dysfunction. These studies reveal the importance of C5aR2 in renal IR injury. They further show that C5aR2 is a functional receptor, rather than a decoy receptor, and may provide a new target for intervention.-Poppelaars, F., van Werkhoven, M. B., Kotimaa, J., Veldhuis, Z. J., Ausema, A., Broeren, S. G. M., Damman, J., Hempel, J. C., Leuvenink, H. G. D., Daha, M. R., van Son, W. J., van Kooten, C., van Os, R. P., Hillebrands, J.-L., Seelen, M. A. Critical role for complement receptor C5aR2 in the pathogenesis of renal ischemia-reperfusion injury.

SET domain-containing 2 (SETD2) is responsible for the trimethylation of histone H3 lysine36 (H3K36me3) and is one of the genes most frequently mutated in clear cell renal cell carcinoma (ccRCC). It is located at 3p21, one copy of which is lost in the majority of ccRCC tumors, suggesting that SETD2 might function as a tumor suppressor gene. However, the manner in which loss of SETD2 contributes to ccRCC development has not been studied in renal primary tubular epithelial cells (PTECs). Therefore, we studied the consequences of SETD2 knockdown through lentiviral shRNA in human PTECs. Consistent with its known function, SETD2 knockdown (SETD-KD) led to loss of H3K36me3 in PTECs. In contrast to SETD2 wild-type PTECs, which have a limited proliferation capacity; the SETD2-KD PTECs continued to proliferate. The expression profiles of SETD2-KD PTECs showed a large overlap with the expression profile of early-passage, proliferating PTECs, whereas nonproliferating PTECs showed a significantly different expression profile. Gene set enrichment analysis revealed a significant enrichment of E2F targets in SETD2-KD and proliferating PTECs as compared with nonproliferating PTECs and in proliferating PTEC compared with SETD2-KD. The SETD2-KD PTECs maintained low expression of CDKN2A and high expression of E2F1, whereas their levels changed with continuing passages in untreated PTECs. In contrast to the nonproliferating PTECs, SETD2-KD PTECs showed no β-galactosidase staining, confirming the protection against senescence. Our results indicate that SETD2 inactivation enables PTECs to bypass the senescence barrier, facilitating a malignant transformation toward ccRCC.