Danilo Schmidt

Anti-human leukocyte antigen and donor-specific antibodies detected by luminex posttransplant serve as biomarkers for chronic rejection of renal allografts.

Background. Although the incidence of early acute rejection could have been diminished in the past, the long-term renal allograft survival could not benefit from the introduction of more effective immunosuppressive regimens mainly aiming at cellular rejection mechanisms. The cause of chronic rejection is still discussed controversially. Here, we demonstrate to what extent human leukocyte antigen (HLA) antibodies (HLAab) posttransplant contribute to late graft outcome. Methods. A total of 1014 deceased kidney transplant recipients transplanted at the Charité hospital were monitored in a cross-sectional manner for the development of HLAab using Luminex Single Antigen beads. Patients with stable kidney function at a median of 5-years posttransplant were tested once for HLAab and monitored for 5.5 years after testing. Results. Thirty percent of recipients showed HLAab. Donor-specific antibodies (DSA) were found in 31% of antibody positive patients. The presence of DSA was associated with a significantly lower graft survival of 49% vs. 83% in the HLAab negative group (P≤0.0001). Non-DSAs also had an adverse effect on graft survival (70% vs. 83%; P=0.0001). In a prospective analysis of 195 patients with repeatedly no detectable HLAab, the survival probability was 94% as opposed to 79% survival among patients who developed HLAab de novo after the first testing (P=0.05). Conclusions. We confirmed that HLAab produced even late after transplantation are detrimental to graft outcome. DSA were proven to have a strong adverse impact on graft survival. The results indicate that a posttransplant HLAab monitoring routine could be appropriate to improve long-term results.

Donor‐Specific HLA Antibodies in a Cohort Comparing Everolimus With Cyclosporine After Kidney Transplantation

Donor‐specific HLA antibodies (DSA) have a negative impact on kidney graft survival. Therefore, we analyzed the occurrence of DSA and antibody‐mediated rejection (AMR) in patients from two prospective randomized trials in our center. At 3–4.5 months posttransplant 127 patients were randomized to continue cyclosporine or converted to everolimus therapy. The presence of DSA was prospectively assessed using Luminex assays. AMR was defined according to the Banff 2009 classification. Antibody screening was available in 126 patients with a median follow‐up of 1059 days. Seven out of 65 (10.8%) patients on cyclosporine developed DSA after a median of 991 days. In comparison, 14/61 patients (23.0%) randomized to everolimus developed DSA after 551 days (log‐rank: p = 0.048). Eight patients on everolimus compared to two patients on cyclosporine developed AMR (log‐rank: p = 0.036). Four of 10 patients with AMR—all in the everolimus group—lost their graft. A multivariate regression model revealed everolimus, >3 mismatches and living donor as significant risk factors for DSA. Acute rejection within the first year, >3 mismatches, everolimus and living donor were independent risk factors for AMR. This single center analysis demonstrates for the first time that everolimus‐based immunosuppression is associated with an increased risk for the development of DSA and AMR.

The severity of acute cellular rejection defined by Banff classification is associated with kidney allograft outcomes.

Background It is unclear if the severity or the timing of acute cellular rejection (ACR) defined by Banff classification 2009 is associated with graft survival. Methods Borderline changes, TCMR I (interstitial rejection), and TCMR II/III (vascular rejection) were defined as low, moderate, and high ACR severity, respectively. Approximately 270 patients who had at least one episode of ACR were enrolled, 270 biopsies were chosen which showed the highest ACR severity of each patient and were negative for donor-specific antibodies (DSA), C4d, and microcirculation changes (MC). Six months were used as the cutoff to define early and late ACR; 370 patients without biopsy posttransplantation were recruited in the control group. Results Up to 8-year posttransplantation, death-censored graft survival (DCGS) rates of control, borderline, TCMR I, and TCMR II/III groups were 97.6%, 93.3%, 79.6%, and 73.6% (log rank test, P<0.001); the control group had significantly higher DCGS rate than the three ACR groups (each pairwise comparison yields P<0.05). The DCGS rate of late ACR was significantly lower compared with early ACR (63.6% vs. 87.4%, P<0.001). Intimal arteritis (Banff v-lesion) was an independent histologic risk factor correlated with long-term graft loss regardless of the timing of ACR. The v-lesions with minimal or high-grade tubulitis displayed similar graft survival (72.7% vs. 72.9%, P=0.96). Conclusion All types of ACR affect long-term graft survival. Vascular or late ACR predict poorer graft survival; the extent of tubulointerstitial inflammation (TI) is of no prognostic significance for vascular rejection.

The ‘blood group O problem’ in kidney transplantation—time to change?

BACKGROUND Patients with blood group O have disadvantages in the allocation of deceased donor organs in the Eurotransplant Kidney Allocation System and fewer ABO-compatible living donors. In order to investigate the consequences of this dilemma, we analysed the outcome of patients with blood group O in our transplantation programme. METHODS A single-centre analysis of 1186 waitlisted patients for first deceased donor kidney transplantations between 1996 and 2008 was performed, and the mechanisms of blood group-dependent differences for graft and recipient outcome were assessed. RESULTS Median follow-up time until death or end of observation for all waitlisted patients was 66 months (range, 0-158 months) and for 589 recipients of a kidney graft was 61 months (range, 0-158 months). Patients with blood group O had significantly longer waiting times for deceased donor kidney grafts, compared to non-group O recipients (median waiting time, 85 vs 59 months). As a consequence, blood group O patients had an increased risk for death without transplantation (13.1% for O patients vs 9.6% for non-O patients; P < 0.05). Despite a good human leukocyte antigen match, graft outcome tended to be worse in O recipients; 14.1% (95% CI, 8.2-19.9%) of all O kidneys from deceased donors were transplanted into non-O recipients, leading to the accumulation of O recipients on the waiting list. CONCLUSIONS The export of blood group O donor kidneys to other blood groups leads to longer waiting times, to a higher death rate and to accumulation of blood group O patients on the waiting list, which will further aggravate the problem in the future. Our results should prompt further research on the issues associated with blood group O. Current allocation systems and living donor kidney exchange programmes should be re-evaluated to address this problem.

Recurrent Primary Focal Segmental Glomerulosclerosis Managed With Intensified Plasma Exchange and Concomitant Monitoring of Soluble Urokinase-Type Plasminogen Activator Receptor-Mediated Podocyte β3-integrin Activation.

Background Primary focal segmental glomerulosclerosis (FSGS) often causes nephrotic proteinuria and frequently results in end-stage renal disease and recurrence after kidney transplantation. Recent studies describe soluble urokinase-type plasminogen activator receptor (suPAR) as a circulating factor implicated in FSGS. Methods This single-center study included 12 adult patients with histologically proven primary FSGS (n = 2) or recurrent FSGS after transplantation (n = 10). The effect of plasma exchange (PE) on clinical outcome, suPAR levels, and in vitro podocyte &bgr;3-integrin activation was investigated over a median of 11 (6-18) sessions of PE. Results The course of treatment was monitored in a total of 70 sessions of PE, which partly eliminated suPAR, with a mean reduction of 37 ± 12% of serum concentration per session. However, a substantial rebound was observed between sessions, with suPAR levels reaching 99 ± 22% of the pretreatment levels after a median of 4 days. Podocyte &bgr;3-integrin activation dropped significantly after PE but rebounded within 4 days concomitant with a rising suPAR level. In 11 of 12 patients, multimodal treatment (including extensive PE) reduced proteinuria significantly (from 5.3 [2.0-7.8] to 1.0 [0.4-1.6] g/d), indicating clinical efficacy of the therapy. One patient suffered allograft loss due to FSGS recurrence. A persisting response was independent of a lasting reduction in the level of total suPAR because there was no sustained significant change in suPAR levels before and after the course of intensified treatment (3814 ± 908 to 3595 ± 521 pg/mL; P = 0.496). Conclusions We conclude that multimodal therapy including extensive PE was associated with stabilization of recurrent FSGS and a temporary lowering of plasma suPAR as well as podocyte &bgr;3-integrin activation. Whether a sustained lowering of total suPAR results in further improved outcomes requires additional study.

Acute cellular rejection with isolated v-lesions is not associated with more favorable outcomes than vascular rejection with more tubulointerstitial inflammations

The impact of isolated v‐lesions on clinical outcome in biopsies with acute cellular rejection (ACR) is unclear.

Identification of T Cell-Mediated Vascular Rejection After Kidney Transplantation by the Combined Measurement of 5 Specific MicroRNAs in Blood.

Background MicroRNAs (miRNAs, miR) hold important roles in the posttranscriptional regulation of gene expression. Their function has been correlated with kidney disease, and they might represent a new class of biomarkers for frequent evaluation of renal graft status. We analyzed their potential in identifying severe T cell–mediated vascular rejection (TCMVR) (Banff 4-II/III) in kidney transplanted patients. Methods Microarray experiments and semiquantitative real-time reverse transcription polymerase chain reaction were performed with total RNA isolated from blood cells of kidney graft recipients. Initial microarray analysis revealed 23 differentially expressed miRNAs distinguishing patients with TCMVR from patients with stable grafts. From these, we validated and further determined the expression of 6 differentially expressed miRNAs and 2 control miRNAs in 161 samples from patients with T cell–mediated rejection (Banff 3-Borderline, Banff 4-I/II/III), Banff-2 antibody-mediated rejection, Banff-5 interstitial fibrosis/tubular atrophy, in samples from stable patients and in samples from patients with urinary tract infection using real-time reverse transcription polymerase chain reaction. Results Expression levels of all 6 candidate miRNAs were significantly downregulated in blood of TCMVR patients compared to the other groups and displayed high sensitivities and specificities for diagnosing TCMVR. The combination of 5 miRNAs, identified by an unbiased multivariate logistic regression followed by cross-validation, enhanced the sensitivity and specificity for the diagnosis of TCMVR after renal transplantation. Conclusions The combined measurement of miRNA-15B, miRNA-16, miRNA-103A, miRNA-106A, and miRNA-107 may help to better identify TCMVR after renal transplantation in a precise and clinically applicable way.

Experience with belatacept rescue therapy in kidney transplant recipients.

In kidney transplant recipients with chronic graft dysfunction, long-term immunosuppression with calcineurin inhibitors (CNIs) or mTOR inhibitors (mTORi) can be challenging due to adverse effects, such as nephrotoxicity and proteinuria. Seventy-nine kidney transplant recipients treated with CNI-based or mTORi-based maintenance immunosuppression who had CNI-induced nephrotoxicity or severe adverse events were switched to belatacept. Mean time from transplantation to belatacept conversion was 69.0 months. Mean estimated glomerular filtration rate (eGFR) ± standard deviation at baseline was 26.1 ± 15.0 ml/min/1.73 m2 , increasing to 34.0 ± 15.2 ml/min/1.73 m2 at 12 months postconversion (P < 0.0005). Renal function improvements were also seen in patients with low eGFR (<25 ml/min/1.73 m2 ) or high proteinuria (>500 mg/l) at conversion. The Kaplan-Meier estimates for patient and graft survival at 12 months were 95.0% and 85.6%, respectively. The discontinuation rate due to adverse events was 7.9%. One case of post-transplant lymphoproliferative disorder occurred at 17 months postconversion. For comparison, a historical control group of 41 patients converted to mTORi-based immunosuppression because of biopsy-confirmed CNI-induced toxicity was examined; eGFR increased from 27.6 ± 7.2 ml/min/1.73 m2 at baseline to 31.1 ± 11.9 ml/min/1.73 m2 at 12 months (P = 0.018). Belatacept-based immunosuppression may be an alternative regimen for kidney transplant recipients with CNI- or mTORi-induced toxicity.

Pretransplant virtual PRA and long-term outcomes of kidney transplant recipients

Virtual panel‐reactive antibodies (vPRA) have been implemented to gauge sensitization worldwide. It is unclear how it associates with long‐term outcomes, and its correlation with peak (pPRA) or actual (aPRA) has not been studied. We retrospectively reviewed data from 18‐ to 65‐year‐old kidney‐only transplant patients during 1.1.1996–31.7.2011 in our center. PRAs were calculated based on solid‐phase techniques. Of the 726 qualified cases, regardless of the PRA type, sensitized patients (PRA > 5%) had more females and previous transplant. Highly sensitized (HS, PRA > 50%) had longer waiting time, lower transplant rate, less living donor, more delayed graft function, and acute rejection. The conformity between vPRA and pPRA in HS was 75%, 57% between pPRA and aPRA. Forty‐three percent (61/142) patients whose pPRA was >5% had no detectable aPRA and maintained similar outcomes as sensitized patients. Multivariate analysis showed consistently lower death‐censored graft survival in HS defined by vPRA [HR 2.086 (95% CI 1.078–4.037), P < 0.05] and pPRA [HR 2.139 (95% CI 1.024–4.487), P < 0.05]. Both vPRA and pPRA provided reliable way estimating sensitization and predicting long‐term graft survival, while aPRA might underestimate true sensitization. vPRA might be the most objective parameter to gauge sensitization.

Impaired thymic function and CD4 + T lymphopenia, but not mannose-binding lectin deficiency, are risk factors for Pneumocystis jirovecii pneumonia in kidney transplant recipients

Pneumocystis jirovecii pneumonia (PCP) incidence is increasing in kidney transplant recipients (KTR), but risk factors remain poorly defined. CD4+ T lymphopenia and mannose-binding lectin (MBL) deficiency are common immunodeficiencies in KTR. Here, we investigated whether CD4+ T lymphopenia and/or MBL deficiency would be risk factors for PCP in KTR. Furthermore, the role of thymic function in CD4+ T lymphopenia and outcome was studied by assessing CD45RA+CD31+CD4+ T cell numbers (RTE, recent thymus emigrants). In 321 de novo KTR serial determinations of peripheral T lymphocyte subsets (n=281, mean 4.2 times between days 0-365) and/or MBL levels (n=112, mean 1.8 times between days 30-180) were performed. 22/321 patients developed a PCP episode on average at day 199 (107-398) post-Tx. Age correlated inversely with RTE, CD4+ and CD8+ T-cell counts until day 180 post-Tx. RTE correlated with CD4+ T-cell counts at all time-points pre- and post-Tx. PCP patients had more CMV infections (p=0.045) within the first 3 months compared to controls. Importantly, PCP patients were older (p=0.008), and had lower RTE (p=0.046) pretransplant, and lower CD4+ T-cell counts pretransplant (p=0.017), at day 60 (p=0.032) and for the average of all post-Tx values (p=0.027) compared to controls. Though treatment with T-cell depleting antibodies was associated with consecutive CD4+ T lymphopenia in the whole cohort, the number of patients who received T-cell depleting antibodies was comparable between PCP and control patients (p=0.754). A multivariate stepwise logistic regression model identified only pretransplant CD4+ T-cell counts (OR 0.011, p=0.010) and acute rejection (OR 4.66, p=0.023) as predictors of PCP. In contrast, MBL levels and incidence of MBL deficiency (<500 ng/ml) at days 30, 90 and 180 post-Tx were not different between PCP patients and controls. In conclusion, PCP risk was associated with higher age and related to both thymic functional impairment and long-lasting CD4+ T-lymphopenia that started already before transplantation. Despite frequent occurrences in KTR, low levels of serum MBL were not associated with increased risk for PCP. CD4+ T-cell counts and function should be addressed in prospective studies for more individualized approaches to PCP prophylaxis.