Antibody-induced allograft injury in renal transplantation

Donor reactive antibodies (DRA) may critically contribute to renal allograft rejeetion. Antibody mediated rejection (AMR) is frequently resistant to conventional (anti-cellular) immunosuppressive therapy. Acute AMR is characterized by rapid deterioration of renal function and an accelerated course towards terminal renal failure. Knowledge about the molecular basis of severe organ dysfunction commonly observed in acute AMR is still fragmentary. Endothelial cells (EC), forming the Interface between a grafted organ and the recipient`s immune System, are prominently involved in allograft rejection. Various molecular mechanisms have been described in several models of antibody mediated EC injury. The significance of these molecular pathways for AMR has not yet been determined to a clinically relevant degree. In this study we therefore plan to identify early DRA/complement mediated cellular events in cases of serologically, morphologically and clinically confirmed AMR. We will establish an "autologous" in-vitro test system using microvascular EC of donor origin to be incubated with antibodies of the corresponding recipient. The molecular response of EC to antibody binding (and complement activation) will be analyzed by genome wide transeriptional gene expression profiling employing GeneChips (Affymetrix). Array derived expression data will first be confirmed by real time PCR. Among other strategies for subsequent validation of gene expression at the protein level, our study will provide the unique opportunity to verify expression of candidate molecules in allograft biopsies of the very same patient from whom array data derived. Our group has extensive experience in isolation, cultivation and flow cytometric analysis of donor EC. All technical procedures related to EC culture, EC treatment with recipient antibodies (and complement), RNA Isolation, hybridization to and Scanning of GeneChips and subsequent bioinformatical analysis of expression data are already established at our institution. In summary, with our approach of creating an "autologous" in-vitro test System, we hope to provide biologically and clinically meaningful insights into the molecular mechanisms of AMR in renal transplantation that eventually might pave the way for the development of novel strategies in diagnosis and treatment of AMR.