Extracellular vesicle‐bound DNA (evDNA) is an understudied extracellular vesicle (EV) cargo, particularly in cancer‐unrelated research. Although evDNA has been detected in urine, little is known about its characteristics, localization, and biomarker potential for kidney pathologies. To address this, we enriched EVs from urine of well‐characterized kidney transplant recipients undergoing allograft biopsy, characterized their evDNA and its association to allograft injury. The SEC‐based method enriched pure EVs from urine of kidney transplant recipients, regardless of the allograft injury. Urinary evDNA represented up to 29.2 ± 8% (mean ± SD) of cell‐free DNA (cfDNA) and correlated with cfDNA in several characteristics but was less fragmented (P < 0.001). Importantly, using DNase treatment and immunogold labelling TEM, we demonstrated that evDNA was bound to the surface of urinary EVs. Normalised evDNA yield (P = 0.042) and evDNA copy number (P = 0.027) significantly differed between patients with normal histology, rejection injury and non‐rejection injury, the later groups having significantly larger uEVs (mean diameter, P = 0.045) and more DNA bound per uEV. ddDNA is detectable in uEV samples of kidney allograft recipients, but its quantity is highly variable. In a proof‐of‐principle study, several evDNA characteristics correlated with clinical and histological parameters (P = 0.040), supporting that the potential of evDNA as a biomarker for kidney allograft injury should be further investigated.

Most kidney transplant patients who undergo biopsies are classified as having no rejection based on consensus thresholds. However, we hypothesized that because these patients have normal adaptive immune systems, T cell-mediated rejection (TCMR) and antibody-mediated rejection (ABMR) may exist as subthreshold activity in some transplants currently classified as no rejection. To examine this question, we studied genome-wide microarray results from 5086 kidney transplant biopsies (4170 patients). An updated archetypal analysis designated 56% of biopsies as no rejection. Subthreshold molecular TCMR and/or ABMR activity molecular activity was detectable as elevated classifier scores in many biopsies classified as no rejection, with ABMR activity in many TCMR biopsies and TCMR activity in many ABMR biopsies. In biopsies classified as no rejection histologically and molecularly, molecular TCMR classifier scores correlated with increases in histologic TCMR features and molecular injury, lower eGFR, and higher risk of graft loss, and molecular ABMR activity correlated with increased glomerulitis and donor-specific antibody. No rejection biopsies with high subthreshold TCMR or ABMR activity had a higher probability of having TCMR or ABMR respectively diagnosed in a future biopsy. We conclude that many kidney transplant recipients have unrecognized subthreshold TCMR or ABMR activity, with significant implications for future problems.

Proteinuria after kidney transplantation is accompanied by an increased risk of graft failure. In this single‐center, placebo‐controlled, double‐blind trial we studied whether vitamin D receptor activator paricalcitol might reduce proteinuria. Patients with urinary protein‐to‐creatinine ratio (UPCR) ≥20 mg/mmol despite optimization of the RAAS blockade were randomly assigned to receive 24 weeks’ treatment with 2 μg/day paricalcitol or placebo. Primary endpoint was change in UPCR, and main secondary endpoints were change in urinary albumin‐to‐creatinine ratio (UACR) and 24‐hour proteinuria. Analysis was by intention to treat. One hundred and sixty‐eight patients undergo randomization, and 83 were allocated to paricalcitol, and 85 to placebo. Compared with baseline, UPCR declined in the paricalcitol group (‐39%, 95% CI ‐45% to ‐31%) but not in the placebo group (21%, 95% CI 9% to 35%), with a between group difference of ‐49% (95% CI ‐57% to ‐41%; P<0.001). UACR and 24‐hour proteinuria decreased only on paricalcitol therapy and significantly differed between groups at end‐of‐treatment (P<0.001). Paricalcitol was well tolerated but incidence of mild hypercalcemia was higher than in placebo. In conclusion, addition of 2 μg/day paricalcitol lowers residual proteinuria in kidney transplant recipients. Long‐term studies are needed to determine if the reduction in proteinuria improves transplant outcomes.

Background.: At Eurotransplant (ET), kidneys are transferred to “rescue allocation” (RA), whenever the standard allocation (SA) algorithms Eurotransplant Kidney Allocation System (ETKAS) and Eurotransplant Senior Program (ESP) fail. We analyzed the outcome of RA. Methods.: Retrospective patient clinical and demographic characteristics association analyses were performed with graft outcomes for 2422 recipients of a deceased donor renal transplantation (DDRT) after RA versus 25 481 after SA from 71 centers across all ET countries from 2006 to 2018. Results.: Numbers of DDRTs after RA increased over the time, especially in Germany. RA played a minor role in ESP versus ETKAS (2.7% versus 10.4%). RA recipients and donors were older compared with SA recipients and donors, cold ischemia times were longer, waiting times were shorter, and the incidence of primary nonfunction was comparable. Among ETKAS recipients, HLA matching was more favorable in SA (mean 3.7 versus 2.5). In multivariate modeling, the incidence of graft loss in ETKAS recipients was reduced in RA compared with SA (subdistribution hazard ratio, 0.80; 95% confidence interval [0.70-0.91], P < 0.001), whereas other outcomes (mortality, death with functioning graft (DwFG)) were not significantly different. None of the 3 outcomes were significantly different when comparing RA with SA within the ESP program. Conclusions.: Facing increased waiting times and mortality on dialysis due to donor shortage, this study reveals encouragingly positive DDRT outcomes following RA. This supports the extension of RA to more patients and as an alternative tool to enable transplantation in patients in countries with prohibitively long waiting times or at risk of deterioration.