Irina Scheffner

Urinary miR‐210 as a Mediator of Acute T‐Cell Mediated Rejection in Renal Allograft Recipients

MicroRNAs (miRNAs) are small ribonucleotides regulating gene expression. Circulating miRNAs are remarkably stable in the blood. We tested whether miRNAs are also detectable in urine and may serve as new predictors of outcome in renal transplant patients with acute rejection. We profiled urinary miRNAs of stable transplant patients and transplant patients with acute rejection. The miR‐10a, miR‐10b and miR‐210 were strongly deregulated in urine of the patients with acute rejection. We confirmed these data in urine of a validation cohort of 62 patients with acute rejection, 19 control transplant patients without rejection and 13 stable transplant patients with urinary tract infection by quantitative RT‐PCR. The miR‐10b and miR‐210 were downregulated and miR‐10a upregulated in patients with acute rejection compared to controls. Only miR‐210 differed between patients with acute rejection when compared to stable transplant patients with urinary tract infection or transplant patients before/after rejection. Low miR‐210 levels were associated with higher decline in GFR 1 year after transplantation. Selected miRNAs are strongly altered in urine of the patients with acute renal allograft rejection. The miR‐210 levels identify patients with acute rejection and predict long‐term kidney function. Urinary miR‐210 may thus serve as a novel biomarker of acute kidney rejection.

Acute Tubular Injury in Protocol Biopsies of Renal Grafts: Prevalence, Associated Factors and Effect on Long-Term Function

Acute tubular injury (ATI) is commonly observed in renal allografts, especially early after transplantation. This study analyzes prevalence and associated clinical conditions of ATI in serial protocol biopsies (pBx) and indication biopsies (iBx), and its impact on long‐term graft function.

Arteriolar Lesions in Renal Transplant Biopsies: Prevalence, Progression, and Clinical Significance

Arteriolar hyalinosis in kidney transplants is considered the histopathologic hallmark of chronic calcineurin inhibitor (CNI) toxicity. However, the lesion is not specific. We assessed prevalence, progression, and clinical significance of arteriolar lesions in 1239 renal transplant sequential protocol biopsy samples and 408 biopsy for cause samples in 526 patients. Associations between arteriolar lesions and presumed risk factors, concomitant histopathologic lesions, demographic factors, and graft function were evaluated. The frequency of arteriolar lesions was stable during the first 2 years after transplantation, and increased thereafter (14.8% at 6 months versus 48.6% at >2 years; P < 0.0001). We were unable to find associations with diabetes, hypertension, or CNI therapy. However, patients with early arteriolar lesions received grafts from older donors (mean ± SD age, 54.4 ± 13.4 years versus 43.1 ± 16.6 years; P < 0.0001), and had inferior graft function (estimated glomerular filtration rate 55 ± 21 mL/min versus 63 ± 24 mL/min at 6 weeks, 53 ± 19 mL/min versus 60 ± 23 mL/min at 1 year, and 49 ± 19 mL/min versus 59 ± 22 mL/min at 2 years; P < 0.05). Evaluation of late biopsy samples from patients not receiving CNI therapy revealed a high prevalence of AH without clear-cut identifiable underlying cause. Reproducibility of arteriolar lesions was at best moderate (κ ≤ 0.62). Sampling error in sequential biopsy samples was frequent. In conclusion, in samples from sequential protocol biopsies and biopsies for cause in individual patients, arteriolar lesions in renal transplants not only increase over time without being specific for CNI toxicity but are affected by sampling error and limited reproducibility.

Impact of CMV infection on acute rejection and long-term renal allograft function: a systematic analysis in patients with protocol biopsies and indicated biopsies

BACKGROUND Higher rates of acute rejection (AR) and reduced graft survival have been reported in patients with cytomegalovirus (CMV) infection, but an association between these factors remains controversial. METHODS In this study, serial protocol biopsies (PBs) and clinically indicated biopsies (IBs) from a large cohort of renal allograft recipients (n ¼ 594) were analyzed to examine the relation between CMV and AR. RESULTS Patients with CMV were more likely to receive IB (85 of the 153 patients; 56%) compared to patients without CMV (138 of 441 patients; 32%; P = 0.003). However, this did not translate into a greater number of patients with episodes of acute cellular rejection on histopathology in IBs. Analysis of PBs revealed a significantly higher number of episodes of rejection per patient with CMV infection (P = 0.04), but only in a subgroup of patients with triple immunosuppression. Long-term graft function post-transplantation was analyzed in four different subgroups according to CMV infection and/or AR. Differences in renal function were apparent within the first 6 weeks after transplantation and persisted during follow-up, with the best renal function in patients without AR or CMV, whereas patients with both AR and CMV had the worst (P < 0.012 at 1 year; P < 0.001 at 2 years). On average, the latter group had significantly older donors and more often delayed graft function. CONCLUSIONS Our data suggests that the link between CMV and AR is far less significant than previously thought. Outcome in patients with CMV may be more determined by coexisting conditions like high donor age and delayed graft function.

Beyond C4d: the ultrastructural appearances of endothelium in ABO-incompatible renal allografts

BACKGROUND ABO incompatibility is no longer a barrier in kidney transplantation. C4d is frequently positive in ABO-incompatible (iABO) biopsies without further signs of microcirculation injury. This phenomenon is assumed to represent graft accommodation. However, ultrastructural examination of glomerular and peritubular capillary endothelium might reveal subtle endothelial damage. METHODS We studied the ultrastructural appearance of the endothelium in 67 biopsies from 21 patients with iABO allografts and compared it with 20 patients (29 biopsies) with ABO-compatible (cABO) grafts with C4d positivity and 25 ABO-compatible control patients (25 biopsies) without serological or histological evidence of humoral rejection (C4d negative). Ten ultrastructural parameters indicative of chronic and acute glomerular and peritubular capillary damage in transmission electron microscopy (TEM) were semi-quantitatively graded and expressed in a sum score. Clinico-pathological data were compared as well as graft function at the time of biopsy and follow-up. RESULTS Ultrastructural parameters did not significantly differ between iABO and controls. In contrast, C4d-positive cABO had the highest TEM sum score (P = 0.001 versus iABO, P = 0.002 versus controls). The sum score did not differ between C4d-positive and C4d-negative iABO but did differ between patients with and without anti-HLA donor-specific antibodies (DSA). Graft function in iABO at the time of biopsy and at follow-up was similar to controls. CONCLUSIONS Our ultrastructural observations support the concept of endothelial accommodation in iABO renal transplants. C4d positivity in the ABO-incompatible situation does not indicate injurious activation of the complement cascade and does not seem to impact on the graft function, in contrast to C4d deposition in cABO with antibody-mediated rejection.

Urinary NGAL Ratio Is Not a Sensitive Biomarker for Monitoring Acute Tubular Injury in Kidney Transplant Patients: NGAL and ATI in Renal Transplant Patients.

Urinary neutrophil gelatinase-associated lipocalin (uNGAL) is known to predict the prolonged delayed graft function after kidney transplantation. We examined the relation of uNGAL with histological findings of acute tubular injury (ATI). Analyses were made in biopsies taken at 6 weeks, 3 months, and 6 months after kidney transplantation. uNGAL was measured in the spot urines, normalized to urinary creatinine excretion, and correlated to biopsy findings and clinical, laboratory, and demographic variables. Controls included healthy individuals, individuals after kidney donation and ICU patients with acute kidney failure. Renal transplant recipients without ATI did not display elevated uNGAL levels compared to the healthy controls. Transplant patients with ATI had a higher uNGAL excretion at 6 weeks than patients without ATI (27,435 versus 13,605 ng/g; P = 0.031). This increase in uNGAL was minor compared to ICU patients with acute renal failure (2.05 × 106 ng/g). Patients with repeated findings of ATI or severe ATI did not have higher urinary NGAL levels compared to those with only one ATI finding or moderate ATI. Female recipient gender and urinary tract infection were identified as potential confounders. uNGAL has a relation with histological signs of acute tubular injury. The usability of this biomarker in renal allograft recipients is limited because of the low sensitivity.

Long-term impact of CMV infection on allografts and on patient survival in renal transplant patients with protocol biopsies

Cytomegalovirus (CMV) infection is a frequent complication of early posttransplantation. This study examines its impact on chronic allograft changes, long-term graft loss, and patient survival. We studied 594 patients who had protocol biopsies at 6 wk, and 3 and 6 mo posttransplantation. Chronic allograft changes were evaluated according to the updated Banff classification [interstitial fibrosis/tubular atrophy (IF/TA), vascular and glomerular lesions]. Follow-up data were available for up to 10 yr. CMV infection was diagnosed in 153 of 594 patients (26%) in the first year after transplantation, mostly within the first 3 mo. Graft survival was reduced in patients with CMV (P = 0.03) as well as the combined allograft/patient survival (P = 0.008). Prevalence of IF/TA at 6 wk after transplantation was already threefold higher in patients who experienced CMV infection later on compared with patients without CMV (P = 0.005). In multivariate analyses, CMV viremia or disease was not a significant factor for graft loss or death. In conclusion, patients with CMV infection posttransplantation show more chronic allograft changes early on, even before CMV infection, and development of IF/TA is not more prevalent in patients with CMV. Our data do not support a significant role of CMV in patient and graft outcomes.

High impact of rejection therapy on the incidence of post-transplant diabetes mellitus after kidney transplantation.

Although major risk factors for post‐transplant diabetes (PTDM) after kidney transplantation have been identified, a systematic study on the impact of rejection and rejection therapy is missing so far.

Fetuin, Matrix-Gla Protein and Osteopontin in Calcification of Renal Allografts

Background Calcification of renal allografts is common in the first year after transplantation and is related to hyperparathyroidism. It is associated with an impaired long-term function of the graft (Am J Transplant 5∶1934-41, 2005). Aim of this study is to examine the role of the anti-calcifying/calcifying factors in the pathophysiology of renal allograft calcification. Methods We analyzed protocol allograft biopsies, blood and urine samples of 31 patients with and 27 patients without allograft calcification taken at 6 weeks, 3 and 6 months after transplantation. Patient demographical data, cold ischemia time, initial graft function and donor characteristics were comparable between the two groups. Biopsies were stained for osteopontin, fetuin, and matrix-gla-protein. Serum and urine electrolytes, matrix-gla-protein, fetuin, Vitamin D and intact parathyroid hormone in serum and osteopontin (OPN) in urine were examined. Results Serum levels of fetuin and matrix-Gla protein as well as urinary levels of OPN showed specific time dependent changes (6 weeks vs. 3 months vs. 6 months; all p<0.0001). In patients with calcifications, urinary levels of OPN were decreased by 55% at 6 weeks and increased thereafter, showing 54% higher levels at 6 months compared to patients without calcification (6 weeks: p<0.01, 6 months: p<0.05). Local protein expression of fetuin-A, matrix-Gla protein and OPN in the graft was specifically increased around calcified plaques, without differences in the mRNA tissue expression. Conclusion Anticalcifying factors show significant changes in the early phase after renal transplantation, which may be important for the prevention of allograft calcification. The differences in OPN indicate an involvement of this factor in the regulation of calcification.

Distinct morphological features of acute tubular injury in renal allografts correlate with clinical outcome

Acute tubular injury (ATI) is common in renal allografts and is related to inferior long-term allograft function. However, it is unknown which of the morphological features of ATI can predict outcome and how they should be graded. Here, we examine features of ATI systematically in protocol biopsies and biopsies for cause to define the most predictive features. Analyses included 521 protocol biopsies taken at 6 wk, 3 mo, and 6 mo after transplantation and 141 biopsies for cause from 204 patients. Features of ATI included brush border loss, tubular epithelial lucency, flattening, pyknosis, nuclei loss, and luminal debris, each graded semiquantitatively. Additional immunohistochemical stainings were performed for markers of cell injury (neutrophil gelatinase-associated lipocalin), cell death [cleaved caspase-3, fatty acid-coenzyme A ligase 4 (FACL4)], and proliferation (Ki-67). Interobserver reliability was good for pyknosis, flattening, and brush border loss and poor for lucency, nuclei loss, and luminal debris. In protocol biopsies between 6 wk and 6 mo, the degree of ATI remained virtually unchanged. Biopsies for cause had generally higher injury scores. Deceased donor source, delayed graft function, ganciclovir/valganciclovir treatment, and urinary tract infection correlated with ATI. The degree of pyknosis, flattening, and brush border loss correlated best with impaired allograft function. FACL4 expression was observed in areas of ATI. Only patients with Ki-67 expression showed stable or improved allograft function in the longitudinal assessment. Reliable assessment of ATI is possible by semiquantitative grading of tubular epithelial cell brush border loss, flattening, and pyknosis. Examination of Ki-67 expression can help determine the potential for recovery from this damage.