Renal endothelial cells (ECs) play crucial roles in vasorelaxation, ultrafiltration, and selective transport of electrolytes and water, but also in leakage of the glomerular filtration barrier and inflammatory processes like complement activation and leukocyte recruitment. In addition, they are target cells for both cellular and antibody mediated rejection in the transplanted kidney. In order to study the molecular and cellular processes underlying EC behavior in renal disease, well characterized primary renal ECs are indispensible. In this report we describe a straightforward procedure to isolate ECs from the perfusion fluid of human donor kidneys by a combination of negative selection of monocytes/macrophages, positive selection by CD31 Dynabeads and propagation in endothelial specific culture medium. Thus, we isolated and propagated renal ECs from 102 donor kidneys, representative of all blood groups and major HLA class I and II antigens. Obtained ECs were positive for CD31 and von Willebrand Factor, expressed other endothelial markers such as CD34, VEGFR-2, TIE2, and PV-1 to a variable extent, and were negative for monocyte marker CD14 and lymphatic endothelial marker podoplanin. HLA class II was either constitutively expressed or could be induced by IFN-γ. Furthermore, we showed the diagnostic value of this renal endothelial biobank in renal endothelial specific cross matching tests for HLA antibodies.

BackgroundThe role of the complement system in antibody-mediated rejection (ABMR) is insufficiently understood. We aimed to investigate the role of local and systemic complement activation in active (aABMR). We quantified complement activation markers, C3, C3d, and C5b-9 in plasma of aABMR, and acute T-cell mediated rejection (aTCMR), and non-rejection kidney transplant recipients. Intra-renal complement markers were analyzed as C4d, C3d, C5b-9, and CD59 deposition. We examined in vitro complement activation and CD59 expression on renal endothelial cells upon incubation with human leukocyte antigen antibodies.MethodsWe included 50 kidney transplant recipients, who we histopathologically classified as aABMR (n=17), aTCMR (n=18), and non-rejection patients (n=15).ResultsComplement activation in plasma did not differ across groups. C3d and C4d deposition were discriminative for aABMR diagnosis. Particularly, C3d deposition was stronger in glomerular (P<0,01), and peritubular capillaries (P<0,05) comparing aABMR to aTCMR rejection and non-rejection biopsies. In contrast to C3d, C5b-9 was only mildly expressed across all groups. For C5b-9, no significant difference between aABMR and non-rejection biopsies regarding peritubular and glomerular C5b-9 deposition was evident. We replicated these findings in vitro using renal endothelial cells and found complement pathway activation with C4d and C3d, but without terminal C5b-9 deposition. Complement regulator CD59 was variably present in biopsies and constitutively expressed on renal endothelial cells in vitro.ConclusionOur results indicate that terminal complement might only play a minor role in late aABMR, possibly indicating the need to re-evaluate the applicability of terminal complement inhibitors as treatment for aABMR.