Aravind Cherukuri

The clinical significance of early proteinuria after renal transplantation.

Background. Late-onset proteinuria after renal transplantation has been universally associated with poor allograft outcomes. However, the significance of early low-grade posttransplant proteinuria remains uncertain. Methods. We analyzed the effect of proteinuria 3 months posttransplantation on death-censored graft loss, death with a functioning graft, vascular events within the grafts life, and estimated glomerular filtration rate at 5 years. Four hundred seventy-seven renal transplants from a single center (1988–2003) with a mean follow-up of 122 months were divided into four groups based on the median protein creatinine ratio (PCR) during the 3rd posttransplant month (PCR<0.15 [group 1, n=85]; PCR 0.15–0.5 [group 2, n=245]; PCR 0.5–1.00 [group 3, n=96]; PCR>1.00 [group 4, n=51]). Cox proportional hazards analysis was performed to study the impact of proteinuria on the various outcomes. Results. Multivariate analysis revealed that even low-level proteinuria at 3 months predicted death-censored graft failure (group 1 [reference]—hazard ratio [HR]=1, group 2—HR=7.1, group 3—HR = 10.5, group 4—HR 16.0; P=0.001). The impact on death and the occurrence of vascular events was only significant for group 4 (HR: 2.6; P=0.01 for death and HR: 2.2; P=0.04 for vascular events). Estimated glomerular filtration rate at 5 years was group 1, 48.5 mL/min; group 2, 41.2 mL/min; group 3, 31.1 mL/min; and group 4, 24.5 mL/min (P<0.001). Continued observation of group 2 to 1 year revealed adverse outcomes with increasing proteinuria. Conclusions. Low-grade proteinuria at 3 months is associated with adverse clinical outcomes and identifies high-risk group of patients who may benefit from further intervention.

Early BK Polyomavirus (BKV) Reactivation in Donor Kidney Is a Risk Factor for Development of BKV-Associated Nephropathy

The pathogenesis of BK polyomavirus (BKV) infection and associated nephropathy in renal transplant recipients is not clearly understood. To gain insight, urine and plasma samples were collected from 112 renal transplant recipients before and after transplantation and tested for the presence of BKV by polymerase chain reaction. Detection of BKV infection very early (ie, 5 days) after transplantation was identified as a risk factor for subsequent BKV viremia and BKV-associated nephropathy. Phylogenetic analysis of VP1 sequences with corresponding ethnicity data suggests that reactivation was of donor origin. Thus, early testing of urine samples from renal transplant recipients may identify those at risk for BKV-associated nephropathy.

Who should have pelvic vessel imaging prior to renal transplantation

Subesinghe M, Cherukuri A, Ecuyer C, Baker RJ. Who should have pelvic vessel imaging prior to renal transplantation?
Clin Transplant 2011: 25: 97–103.

Pro-inflammatory B Cells Predict Progressive Minimal Early Renal Allograft Inflammation Which Is Associated with Poor Graft Outcomes

Introduction The clinical significance of minimal tubulointerstitial inflammation (MTI: ‘t’+’i’ scores 0.5-1.5) and Banff Borderline changes (BBC: ‘t’ + ‘i’ scores 2-3.0) in early renal allograft biopsies is unclear. The rate and significance of progression of these lesions to late acute rejection (AR) also remains unknown. In this study, we assessed the clinical significance of early tubulointerstitial inflammation (< Banff 1A rejection). Methods Our center performs 2 protocol biopsies (3 & 12 months) along with for-cause biopsies. This allowed us to assess the clinical impact of early (0-4 months) MTI and BBC on graft outcomes. Furthermore, we examined the relationship between peripheral blood B cell cytokines and early MTI and BBC. Results 208/372 patients transplanted between 1/13-11/14 had either no inflammation (NI, 36%, 76/208), MTI (34%, 70/208) or BBC (30%, 62/208) on early biopsies (0-4 months). Patients with NI (17%), MTI (24%) and BBC (34%) at 0-4 months exhibited increasing rates (in parentheses) of progression to AR (≥Banff 1A) by 12mo. Further, patients with MTI or BBC (0-4 months) had increased graft loss or impending graft loss (eGFR<30ml/min and >30% decline from baseline) by 50 months when compared to those with NI (Fig. 1A). While graft outcome in the NI group was not affected by progression to late AR (p=0.85), patients with early MTI (Fig 1B) or BBC (Fig 1C) had significantly worse outcomes if they developed late AR. Thus, early allograft inflammation (either MIT or BBC) was not only associated with increased progression to late AR, but those who progressed had worse outcomes. Thus, MTI and BBC, particularly in patients who will progress, represent a clinical phenotype at risk for poor outcomes. Clinical factors, including 3 months histology, could not predict progression of MTI or BBC to late AR. Based on previous results, we asked whether peripheral blood B cell cytokines could predict progression to AR in these patients. 72 patients with either MTI (n=26) or BBC (n=46) had their B cells and cytokines analyzed at 3 months. IL-10:TNF&agr; expression ratio within T1 transitional B cells was significantly lower in both the MTI (6.3X) and BBC (4.6X) patients who progressed compared to those who were stable (p<0.0004). Finally, a low T1 B cytokine ratio strongly predicted late progression to AR (ROC AUC 0.94 p<0.0001, Sen 92%, Spec 88%) in patients with early allograft inflammation (MIT or BBC). Conclusion Patients with early minimal allograft inflammation that progress to AR at 1 year represent a high-risk cohort for graft dysfunction. This group could be identified by 2-4 mo, using the T1 B IL-10:TNF&agr; ratio – allowing early intervention.

DSA with TCMR Identifies High Risk Renal Transplant Recipients that can be Predicted by Proinflammatory Transitional B Cells

Introduction Post-transplant DSA is strongly associated with poor graft outcomes but has limited predictive value. In this prospective study, we aimed to risk stratify patients with early post-transplant DSA to allow timely identification of individuals at risk for poor clinical outcomes. Methods Patients were screened for DSA at 0, 1, 3, 6, 9 & 12 months and analyzed in relation to protocol biopsies at 3 and 12 months and any for-cause biopsies within the first year. To risk stratify DSA positive patients, we analyzed B cell subsets and cytokines of those who had PBMC available at 3 months. Results 294/ 372 of patients transplanted between 01/2013 and 11/2014 with at least one biopsy in the first post-transplant year were included in the analysis. The immunosuppressive regimen was Thymoglobulin induction followed by MPA and Tacrolimus as maintenance therapy. 67/294 (22.8%) of these patients developed DSA. DSA was detected early (< 3 months) in 76% of the patients. DSA was associated with significantly increased rates of subclinical and clinical TCMR (58%) compared to patients lacking DSA (33%, %; p<0.0001). Importantly, patients with DSA plus TCMR had significantly worse chronic allograft changes (1 year protocol biopsy) and increased graft loss or impending graft loss (eGFR < 30ml/min and > 30% decline from baseline) at 4 years compared to those with DSA or TCMR alone (Fig. 1A). Thus, DSA plus TCMR identifies high-risk patients, in whom early identification would allow pre-emptive intervention. Based on prior findings, we asked whether cytokine expression by peripheral blood B cell subsets could predict TCMR in patients with DSA. 43/67 of DSA positive patients had their B cell cytokines analyzed at 3 months by flow cytometry (after 24 h stimulation with CpG and CD40L). Of the markers analyzed, the ratio of IL-10/TNF&agr; expression by T1 transitional B cells (T1B) was significantly lower in patients with DSA plus TCMR compared to those with DSA alone (ratio: 0.94 vs. 4.9, p<0.0001). A low T1B cytokine ratio was a strong predictor of DSA plus TCMR (ROC AUC 0.94, p<0.0001; Fig. 1B). At a cut-off value of 1.26, the T1B cytokine ratio predicted DSA plus TCMR with a positive predictive value of 81% and a negative predictive value of 94%. Reanalysis of the data after removing the 5/43 patients whose DSA was detected after TCMR again showed that the T1 B cytokine ratio strongly predicted concomitant or ensuing TCMR in patients with DSA (ROC AUC 0.94, p<0.0001). Figure. No caption available. Conclusion Thus, patients with DSA plus TCMR represent a high-risk population for adverse graft outcomes, and this outcome can be predicted in DSA positive patients using the T1B cytokine ratio as a biomarker. Deutsche Forschungsgesellschaft (DFG). American Society of Transplantation (AST).

Prospective Multicenter Validation of Human Transitional B cell Cytokines as a Predictive Biomarker in Renal Transplantation

Introduction In renal transplantation, non-invasive biomarkers are needed to identify patients at risk for poor outcomes and guide pre-emptive therapy. In this prospective multicenter study, we assessed B cells and their cytokines as a predictive biomarker. Methods Based on prior data, we tested the ratio of IL-10:TNF&agr; expression in peripheral blood T1 transitional B cells (T1B) 2-4mo post-transplant, to perform as an early biomarker of clinical course. (Cytokines were examined by flow cytometry after 24hr stimulation with CD40L & CpG). Results 165 patients (transplanted in 2013-14) with serial biopsies (Bxs) (including 3&12mo protocol Bxs + for-cause Bxs) and serial blood draws (0, 1, 3, 6 & 12mos) served as the training set. Immunosuppression utilized Thymoglobulin induction followed by MPA+TAC maintenance. A low T1B IL10:TNF&agr; ratio at 3mo predicted acute rejection (AR) within the 1st yr (Fig1A). Notably, in patients with a normal Bx at 2-4mo, a low T1B cytokine ratio strongly predicted late AR (6-12mo; AUC 0.9, p<0.0001) with a lead time of > 7mo in 80% of the patients. These data were validated in an independent internal cohort (2015, n=74). Further, a low T1B cytokine ratio was associated with IFTA (12 mo) and graft loss/impending graft loss (eGFR<30ml/min & >30% fall from baseline) at 4yrs (Fig1B). The predictive value of this biomarker was not influenced by either opportunistic viral infections or by non-adherence. Figure. No caption available. Next, T1B cytokine ratio was validated in an external cohort (n=100) from the UK. Immunosuppression utilized Basiliximab induction followed by TAC+MMF maintenance. As protocol Bxs were not performed in this cohort, all AR was clinical. Again, the T1B cytokine ratio at 3mo strongly predicted AR within the 1st yr (Fig1C), and was associated with significantly greater graft loss/impending graft loss (Fig1D). B cells in high-risk patients express excessive TNF&agr; relative to IL-10. Culture of B cells from such patients with &agr;-TNF, reduced their TNF&agr; and increased their IL10 expression. Such B cells subsequently suppressed autologous ∝-CD3 stimulated T cell TNF&agr; while increasing IL10 expression. Thus, TNF blockade restored a normal B cell cytokine balance (Breg activity) in high-risk patients. Finally, &agr;-TNF inhibited in vitro differentiation of B cells to plasma cells, reduced their Ab secretion, and selectively increased their IL-10 expression. Conclusion Thus, T1B IL10:TNF&agr; ratio (2-4 mo) was tested and validated as a strong biomarker for subsequent renal allograft rejection and clinical course. Importantly, our data not only identifies patients in need of pre-emptive therapy, but provides rationale for therapeutic intervention based on TNF blockade. American Society of Transplantation. Roche Organ Transplant Reseacrh Foundation.

Non-Compliance Assessed By Variability of Blood Calcineurin Inhibitor (CNI) Levels Is Associated With Poor Long-Term Outcomes in Kidney Transplant Recipients (KTRs).: Abstract# 2944

Abstract# 2944 Non-Compliance Assessed By Variability of Blood Calcineurin Inhibitor (CNI) Levels Is Associated With Poor Long-Term Outcomes in Kidney Transplant Recipients (KTRs). A. Cherukuri,1,2 S. Balasubramanian, T. Abheygunaratne, J. Tattersall, R. Baker. 1University of Pittsburgh, Pittsburgh, PA; 2Leeds Teaching Hospitals, Leeds, United Kingdom. Non-compliance with treatment is a major problem in clinical renal transplantation. There is an emerging interest in the development of objective markers of noncompliance. Variability of CNI drug levels is one such marker. In this large single centre retrospective analysis, we examined the relationship between the overall graft outcomes and CNI variability. A total of 10 plasma CNI levels were examined between the 2nd and 3rd post-transplant year for the analysis in 1235 consecutive KTRs. Patients were divided into three tertiles based on their CNI (2y-3y) variability. Grafts lost prior to third transplant year were excluded to minimize the effect of early therapeutic modifi cations. Overall graft survival was examined over 10 years by Kaplan Meier analysis. The mean variability was signifi cantly higher for younger KTRs {age lowest tertile (20yrs), 24.7 vs. middle tertile (40yrs), 21.9, vs. highest tertile (59yrs), 22.9; P<0.05, ANOVA}. Overall, patients in the highest tertile group of CNI (2y-3y) variability had the worst overall graft survival when compared to the other tertiles. When the analysis was stratifi ed by the age of the KTRs, patients with high variability in the lowest tertile group of age (i.e. youngest age group) had the worst outcomes. CNI (2y-3y) variability was not strongly associated with increased overall graft loss in older KTRs. The utility of CNI variability was re-examined in 118 KTRs with allograft dysfunction. These patients underwent an indication biopsy for either creeping creatinine or proteinuria. Patients in the highest tertile group of CNI variability had higher microcirculation infl ammation score (glomerulitis+peritubular capillaritis) and a higher percentage of these grafts were positive for C4d deposition despite no signifi cant difference in the prevalence of DSA in the serum. There was no signifi cant difference in the mean score for scarring (ct+ci). However, patients in the lowest tertile group for CNI level variability had signifi cantly superior post-biopsy graft survival when compared to the highest two tertiles in this selective group of patients with allograft dysfunction. To conclude, CNI drug level variability is a potential marker for predicting allograft outcomes especially in the younger patient cohort where non-compliance is a major problem. This has to be validated in further prospective studies

Histological, But Not Clinical Phenotype Is Associated With Adverse Graft Survival After an Indication Biopsy in Troubled Renal Allografts.: Abstract# A246

A246 Histological, But Not Clinical Phenotype Is Associated With Adverse Graft Survival After an Indication Biopsy in Troubled Renal Allografts. A. Cherukuri,1,2 S. Balasubramanian,2 P. Prasad,2 R. Baker.2 1University of Pittsburgh, Pittsburgh; 2Leeds Teaching Hospitals, Leeds, United Kingdom. Deteriorating renal allograft function as evidenced by either a rise in serum creatinine or proteinuria remains the most important indication for a late transplant biopsy. In this study we analysed the histology of 135 ‘indication biopsies’ performed at our centre, in relation to the presenting indication. We have also collected serum samples for the detection of Donor Specifi c Antibody (DSA). Allografts were classifi ed into those with creeping creatinine and no proteinuria (CC, n=32), isolated proteinuria (IP, n=44) and proteinuria with creeping creatinine (PC, n=59). Creeping creatinine was defi ned by slope(>0.35, P<0.05) on the regression analysis of 1/creatinine over 2 years prior to the biopsy. Proteinuria was defi ned by a cut-off value of >0.5g/day. We found that histology was quite heterogeneous with patients in all the clinical groups with similar frequencies of the broad histological classes of glomerulonephritis (GN), IFTA, antibody mediated changes (AMC, i.e. glomerulitis (g) or peritubular capillaritis (ptc) or Transplant glomerulopathy (tg) or peritubular capillary C4d deposition), T cell rejection and others. Thus there is no relationship between histological phenotype and clinical indication for the biopsy. Patients who had signifi cant chronic damage (high ‘ct’, ‘ci’, ‘cv’ scores) and those with AMC and GN had worse graft survival after the biopsy in a multivariate analysis. In the 47 patients with AMC, c4d deposition in the peritubular capillaries, the degree of scarring (high ‘ct’, ‘ci’, ‘cv’ scores) and the microcirculation infl ammation (MI) score (g+ptc) ≥ 3 were signifi cantly associated with adverse graft survival at 3 year follow-up; with clinical presentation or DSA detection not having any impact on the outcome. Finally patients with MI score ≥3 and C4d positivity had the worst graft survival when compared to those with either MI score ≥3 or C4d positivity. In summary, we propose that the histology of late “for cause” biopsies is heterogeneous irrespective of the indication and key histological features rather than clinical presentation affects graft outcomes. Abstract# A247 Signifi cance of CD56+ Cell Aggregation in the Graft Following Kidney Transplantation. S. Shin,1 Y. Cho,2 Y. Park,2 Y. Kim,1 B. Choi,1 J. Jung,1 H. Cho.1 1General Surgery, Asan Medical Center, Seoul, Korea, Republic of; 2Pathology, Asan Medical Center, Seoul, Korea, Republic of. Objective In the context of kidney transplantation, little is known about the involvement of natural killer (NK) cells in the immune reaction leading to either rejection or graft failure. We investigated the signifi cance of CD56+ cell aggregation in human kidney transplant biopsies in regard of graft function and survival. Methods One hundred seventy-four clinically indicated transplant biopsies were included in this analysis. Immunohistochemical stainings for C4d and CD56 were performed and clinical correlation was assessed by serum creatinine and graft status. ResultsTen or more CD56+ cells/high power fi eld (HPF) were associated with acute cellular rejection (P=0.001), chronic active antibody-mediated rejection (P<0.001), glomerular disease (P=0.022), and interstitial fi brosis and tubular atrophy (P=0.002). There was a signifi cant positive correlation between the degree of C4d deposits and the number CD56+ cells (r=0.23, P=0.002). Compared with patients with 0 to 9 cells/HPF, those with 10 to 29 cells/HPF and those with greater than 30 cells/ HPF had worse death-censored graft survival 1 year postbiopsy with a hazard ratio of 8.2 (95% CI 1.05–64.39, P=0.045) and 13.3 (95% CI 1.65–106.55, P=0.015), respectively in multivariate Cox regression. In addition, the postbiopsy mean levels of serum creatinine in patients with 10 or more cells/HPF were signifi cantly higher than in those with 0 to 9 cells/HPF during the 1 year follow-up period which was confi rmed by a linear mixed effect model (P<0.001). Conclusions CD56+ cell aggregation in human kidney transplant biopsies can be a predictive marker for graft function and survival. A247 Signifi cance of CD56+ Cell Aggregation in the Graft Following Kidney Transplantation. S. Shin,1 Y. Cho,2 Y. Park,2 Y. Kim,1 B. Choi,1 J. Jung,1 H. Cho.1 1General Surgery, Asan Medical Center, Seoul, Korea, Republic of; 2Pathology, Asan Medical Center, Seoul, Korea, Republic of. Objective In the context of kidney transplantation, little is known about the involvement of natural killer (NK) cells in the immune reaction leading to either rejection or graft failure. We investigated the signifi cance of CD56+ cell aggregation in human kidney transplant biopsies in regard of graft function and survival. Methods One hundred seventy-four clinically indicated transplant biopsies were included in this analysis. Immunohistochemical stainings for C4d and CD56 were performed and clinical correlation was assessed by serum creatinine and graft status. ResultsTen or more CD56+ cells/high power fi eld (HPF) were associated with acute cellular rejection (P=0.001), chronic active antibody-mediated rejection (P<0.001), glomerular disease (P=0.022), and interstitial fi brosis and tubular atrophy (P=0.002). There was a signifi cant positive correlation between the degree of C4d deposits and the number CD56+ cells (r=0.23, P=0.002). Compared with patients with 0 to 9 cells/HPF, those with 10 to 29 cells/HPF and those with greater than 30 cells/ HPF had worse death-censored graft survival 1 year postbiopsy with a hazard ratio of 8.2 (95% CI 1.05–64.39, P=0.045) and 13.3 (95% CI 1.65–106.55, P=0.015), respectively in multivariate Cox regression. In addition, the postbiopsy mean levels of serum creatinine in patients with 10 or more cells/HPF were signifi cantly higher than in those with 0 to 9 cells/HPF during the 1 year follow-up period which was confi rmed by a linear mixed effect model (P<0.001). Conclusions CD56+ cell aggregation in human kidney transplant biopsies can be a predictive marker for graft function and survival. Abstract# A248 Kynurenine as Biomarker in Long-Term After Renal Transplantation. D. Abendroth,1 M. Marzinzig,1 J. Kaden,2 M. Stangl.3 1Center of Surgery, University of Ulm, Ulm, Germany; 2Renal Transplant Center, Municipal Friedrichshain Hospital, Berlin, Germany; 3Ludwig-MaximiliansUniversity, Munich, Germany. Introduction If renal function after a course of acute rejection recovers fully, there appears to be no survival disadvantage (CTS-Study). We investigated the specifi city of (subclinical) chronic infl ammatory responses (SCAR) induced either by not successful treated rejection episodes or using different CNI ́s. We used a highly sensitive parameter for infl ammation, kynurenine (Kyn), in two extended cohorts after renal transplantation in correlation to graft survival and graft function. Patients and Methods In a retro(A, n=224, up to 12 years) and prospective study (B, n=116, up to 3 years) of renal transplant recipients we evaluated the Kyn [μM] behavior for the long-term run. Both groups showed comparable basic demographic data (age, time on dialysis, PRA and waiting time). Results Kyn in normal controls (n=292) was 2,7+0,5 μM, in stable renal transplanted patients (n=311) 5,7+1,8μM. We identifi ed in both groups already end of the fi rst week posttransplant a statistically signifi cant difference (A:17,2 vs. 7,0 (week 1, median, p<.005); 8,1vs.4,8 (week 6, median, p<.001); B:12,3 vs. 6,9 (week 1, median, p<.001); 6,7 vs. 4,2 (week 6, median, p<.001)) in Kyn levels between patients with/ without acute rejection. Patients with a stable Kyn-level > 5,5 in the follow up (min. 3 weeks) showed a signifi cant lower graft survival. In A (Kyn < 5,0) 1/5/10 year graft survival was 98/88/69% compared to >5,5 and 88/57/34% (death censored). There was no difference between both CNI ́s. Rejection rate in A was allover 18%, in B 19,5 % (BPAR). Conclusion Serum kynurenine measurement showed up as a sensitive and reliable marker for infl ammatory processes. Obvious, not the rejection episode itself but a subclinical ongoing infl ammatory process is leading to reduced long-term results. Once the rejection episode is treated successfully the reestablishment of the infl ammatory homeostasis the chance for the graft survival is still given. Kynurenine monitoring is a helpful tool to identify very early (subclinical) acute rejection, the best rejection treatment and the optimization of an immunosuppressive regimen. A248 Kynurenine as Biomarker in Long-Term After Renal Transplantation. D. Abendroth,1 M. Marzinzig,1 J. Kaden,2 M. Stangl.3 1Center of Surgery, University of Ulm, Ulm, Germany; 2Renal Transplant Center, Municipal Friedrichshain Hospital, Berlin, Germany; 3Ludwig-MaximiliansUniversity, Munich, Germany. Introduction If renal function after a course of acute rejection recovers fully, there appears to be no survival disadvantage (CTS-Study). We investigated the specifi city of (subclinical) chronic infl ammatory responses (SCAR) induced either by not successful treated rejection episodes or using different CNI ́s. We used a highly sensitive parameter for infl ammation, kynurenine (Kyn), in two extended cohorts after renal transplantation in correlation to graft survival and graft function. Patients and Methods In a retro(A, n=224, up to 12 years) and prospective study (B, n=116, up to 3 years) of renal transplant recipients we evaluated the Kyn [μM] behavior for the long-term run. Both groups showed comparable basic demographic data (age, time on dialysis, PRA and waiting time). Results Kyn in normal controls (n=292) was 2,7+0,5 μM, in stable renal transplanted patients (n=311) 5,7+1,8μM. We identifi ed in both groups