Abstract
In renal transplantation, complement is involved in ischemia reperfusion injury, graft rejection and dysfunction. However, it is still unclear how induction of complement and its activation are initiated. Using allograft biopsies of a well-characterized cohort of 28 renal transplant patients with no rejection (Ctrl), delayed graft function (DGF), acute T-cell-mediated (TCMR) or antibody-mediated rejection (ABMR) we analyzed differences in complement reaction. For that mRNA was isolated from FFPE sections, quantified with a multiplex gene expression panel and correlated with transplant conditions and follow-up of patients. Additionally, inflammatory cells were quantified by multiplex immunohistochemistry. In allograft biopsies with TCMR and ABMR gene expression of C1QB was 2-4 fold elevated compared to Ctrl. In TCMR biopsies, mRNA counts of several complement-related genes including C1S, C3, CFB and complement regulators CFH, CR1 and SERPING1 were significantly increased compared to Ctrl. Interestingly, expression levels of about 75% of the analyzed complement related genes correlated with cold ischemia time (CIT) and markers of inflammation. In conclusion, this study suggest an important role of complement in transplant pathology which seems to be at least in part triggered by CIT. Multiplex mRNA analysis might be a useful method to refine diagnosis and explore new pathways involved in rejection.
Highlights
Abbreviations antibody-mediated rejection (ABMR) Antibody-mediated rejection body mass index (BMI) Body mass index CAD Coronary artery disease cold ischemia time (CIT) Cold ischemia time control group without rejection (Ctrl) Control delayed graft function (DGF) Delay graft function follicular dendritic cells (FDCs) Follicular dendritic cells formalin-fixed paraffin-embedded (FFPE) Formalin-fixed paraffin-embedded GFR Glomerular filtration ratio HLA Human leucocyte antigen IRI Ischemia reperfusion injury membrane attack complex (MAC) Membrane attack complex PC Principal component T-cell-mediated rejection (TCMR) T-cell mediated rejection WIT Warm ischemia time
The terminal complement pathway is activated by cleavage of C5 through the C5 convertase, resulting in formation of the membrane attack complex (MAC) C5b-9, which forms pores in the target m embrane[8]
Expression of several complement initiator molecules, proteases and factors was upregulated in biopsies of renal transplants with TCMR and ABMR
Summary
Abbreviations ABMR Antibody-mediated rejection BMI Body mass index CAD Coronary artery disease CIT Cold ischemia time Ctrl Control DGF Delay graft function FDC Follicular dendritic cells FFPE Formalin-fixed paraffin-embedded GFR Glomerular filtration ratio HLA Human leucocyte antigen IRI Ischemia reperfusion injury MAC Membrane attack complex PC Principal component TCMR T-cell mediated rejection WIT Warm ischemia time. Previous mRNA expression studies in kidney transplants were performed using microarrays (cryo or RNAlater-fixed material) but required a 2nd biopsy taken for this purpose only These studies have already shown that the innate immune system plays a significant role in graft rejection, but have never focused directly on the complement system[11,12]. In our study transcript analysis was conducted with the NanoString nCounter® FLEX Analysis System using the Human Organ Transplant Panel, which was created through a collaboration between NanoString and the Banff Foundation for Allograft Pathology[13] This high through-put gene expression platform has the ability of analyzing up to 800 genes per sample and works on formalin-fixed paraffin-embedded (FFPE) tissue[14,15]. We correlated our results with relevant clinical data like cold and warm ischemia time and recipient and donor data like age, body mass index (BMI), serum creatinine, renal inflammation and living or deceased donor state
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