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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. 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 endothelium-specific culture medium. Thus, we isolated and propagated renal ECs from 102 donor kidneys, representative of all blood groups and major human leukocyte antigen (HLA) class I and II antigens. The obtained ECs were positive for CD31 and von Willebrand factor, expressed other endothelial markers such as CD34, VEGF receptor-2, TIE2, and plasmalemmal vesicle associated protein-1 to a variable extent, and were negative for the monocyte marker CD14 and lymphatic endothelial marker podoplanin. HLA class II was either constitutively expressed or could be induced by interferon-y. Furthermore, as a proof of principle, we showed the diagnostic value of this renal endothelial biobank in renal endothelium-specific cross-matching tests for HLA antibodies.NEW & NOTEWORTHY We describe a new and widely accessible approach to obtain human primary renal endothelial cells in a \standardized fashion, by isolating from the perfusate of machine-perfused donor kidneys. Characterization of the cells showed a mixed population originating from different compartments of the kidney. As a proof of principle, we demonstrated a possible diagnostic application in an endothelium-specific cross-match. Next to transplantation, we foresee further applications in the field renal endothelial research.

An acute rise of plasma Na+ concentration associates with syndecan-1 shedding during hemodialysis. Am J Physiol Renal Physiol 319: F171-F177, 2020. First published June 15, 2020; doi:10.1152/ajprenal.00005.2020.-Endothelial dysfunction (ED) contributes to the high incidence of cardiovascular events in patients undergoing hemodialysis. Syndecan-1 in the endothelial glycocalyx can be shed into the circulation, serving as a biomarker for ED. As Na+ is a trigger for glycocalyx shedding, we now tested whether hemodialysis, with higher dialysate Na+ concentrations, is associated with more syndecan-1 shedding compared with standard hemodialysis (SHD). In this crossover study in 29 patients, plasma syndecan-1 was repeatedly measured during SHD and during Hemocontrol hemodialysis (HHD), which is characterized by initially higher dialysate and plasma Na+ levels. Courses of syndecan-1 were compared with linear mixed models. Syndecan-1 shedding was assessed by area under the curve analysis. Plasma Na+ increased early after the start of SHD and HHD, with higher values during HHD (30 min: 142.3 vs. 139.9 mM, P < 0.001). Syndecan-1 increased significantly during both conditions, but the percent change was higher (42.9% vs. 19.5%) and occurred earlier (120 vs. 180 min) during HHD. Syndecan-1 levels were significantly higher at 120 min during HHD compared with SHD (P < 0.05). Overall, syndecan-1 shedding was higher during HHD compared with SHD (means: 40.4 vs. 19.0 arbitrary units, P = 0.06). Lower predialysis plasma Na+ and osmolality were associated with greater intradialytic increases in syndecan-1 levels (both groups, P = 0.001). The rise in plasma syndecan-1 levels was more pronounced and occurred earlier during hemodialysis with higher plasma Na+ levels. Although we cannot prove that the rise in plasma syndecan-1 originates from the endothelial glycocalyx, our findings are compatible with Na+-driven endothelial glycocalyxderived syndecan-1 shedding.

Syndecan-1 is a transmembrane heparan sulfate proteoglycan involved in regenerative growth and cellular adhesion. We hypothesized that the induction of tubular syndecan-1 is a repair response to incipient renal damage in apparently stable, uncomplicated renal transplant recipients. We quantified tubular syndecan-1 in unselected renal protocol biopsies taken 1 yr after transplantation. Spearman rank correlation analysis revealed an inverse correlation between tubular syndecan-1 expression and creatinine clearance at the time of biopsy ( r = −0.483, P < 0.03). In a larger panel of protocol and indication biopsies from renal transplant recipients, tubular syndecan-1 correlated with tubular proliferation marker Ki67 ( r = 0.518, P < 0.0001). In a rat renal transplantation model, 2 mo after transplantation, mRNA expression of syndecan-1 and its major sheddase, A disintegrin and metalloproteinase-17, were upregulated (both P < 0.03). Since shed syndecan-1 might end up in the circulation, in a stable cross-sectional human renal transplant population ( n = 510), we measured plasma syndecan-1. By multivariate regression analysis, we showed robust independent associations of plasma syndecan-1 with renal (plasma creatinine and plasma urea) and endothelial function parameters (plasma VEGF-A, all P < 0.01). By various approaches, we were not able to localize syndecan-1 in vessel wall or endothelial cells, which makes shedding of syndecan-1 from the endothelial glycocalyx unlikely. Our data suggest that early damage in transplanted kidneys induces repair mechanisms within the graft, namely, tubular syndecan-1 expression for tubular regeneration and VEGF production for endothelial repair. Elevated plasma syndecan-1 levels in renal transplantation patients might be interpreted as repair/survival factor related to loss of tubular and endothelial function in transplanted kidneys.