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A major complication of primary focal segmental glomerulosclerosis (FSGS) is its recurrence after kidney transplantation that happens in 30 to 40% of the patients. The diagnosis of these relapses is not always easy as the histological lesions are not highly specific and appear after the proteinuria increase. Currently, there are no accurate biomarkers to detect FSGS recurrence. Our group identified a modified form of Apolipoprotein A-I (ApoA-I), named ApoA-Ib, specifically present in the urine of recurrent FSGS patients after kidney transplantation. Aberrant forms of ApoA-I have also been described in the urine of native primary FSGS patients, this feature has been associated with prominent staining of ApoA-I at the apical membrane of the tubular cells. In this study, we aim to analyze the ApoA-I distribution in kidney allograft biopsies of recurrent FSGS patients. We detected ApoA-I by immunohistochemistry in kidney allograft biopsies of patients with FSGS relapse after kidney transplantation and in kidney allograft biopsies of patients with a disease different from FSGS in the native kidney (non-FSGS). In recurrent FSGS patients, ApoA-I was prominently localized at the brush border of the tubular cells, while in the non-FSGS patients, ApoA-I was found along the cytoplasm of the tubular cells. The localization of ApoA-I at the brush border of the tubular cells is a specific feature of primary FSGS in relapse. This suggests that ApoA-I staining in kidney biopsies, coupled with ApoA-Ib measurement in urine, could be used as a diagnostic tool of primary FSGS relapse after kidney transplantation due to its highly specific tubular distribution.

The combination of tacrolimus (TAC) and mycophenolate is the most widely employed maintenance immunosuppression in renal transplants. Different surrogates of tacrolimus exposure or metabolism such as tacrolimus trough levels (TAC-C0), coefficient of variation of tacrolimus (CV-TAC-C0), time in therapeutic range (TTR), and tacrolimus concentration dose ratio (C/D) have been associated with graft outcomes. We explore in a cohort of low immunological risk renal transplants (n = 85) treated with TAC, mycophenolate mofetil (MMF), and steroids and then monitored by paired surveillance biopsies the association between histological lesions and TAC-C0 at the time of biopsy as well as CV-TAC-C0, TTR, and C/D during follow up. Interstitial inflammation (i-Banff score &ge, 1) in the first surveillance biopsy was associated with TAC-C0 (odds ratio (OR): 0.69, 95% confidence interval (CI): 0.50&ndash, 0.96, p = 0.027). In the second surveillance biopsy, inflammation was associated with time below the therapeutic range (OR: 1.05 and 95% CI: 1.01&ndash, 1.10, p = 0.023). Interstitial inflammation in scarred areas (i-IFTA score &ge, 1) was not associated with surrogates of TAC exposure/metabolism. Progression of interstitial fibrosis/tubular atrophy (IF/TA) was observed in 35 cases (41.2%). Multivariate regression logistic analysis showed that mean C/D (OR: 0.48, 95% CI: 0.25&ndash, 0.92, p = 0.026) and IF/TA in the first biopsy (OR: 0.43, 95% CI: 0.24&ndash, 0.77, p = 0.005) were associated with IF/TA progression between biopsies. A low C/D ratio is associated with IF/TA progression, suggesting that TAC nephrotoxicity may contribute to fibrosis progression in well immunosuppressed patients. Our data support that TAC exposure is associated with inflammation in healthy kidney areas but not in scarred tissue.

Coronavirus SARS-CoV-2; COVID-19; 2019-nCoV; Síndrome de dificultat respiratòria aguda; Trasplantament renal Coronavirus SARS-CoV-2; COVID-19; 2019-nCoV; Síndrome de dificultad respiratoria aguda; Trasplante renal Coronavirus SARS-CoV-2; COVID-19; 2019-nCoV; Acute respiratory distress syndrome; Renal transplantation Coronavirus disease 2019 (COVD-19) emerged as a pandemic in December 2019. Infection has spread quickly and renal transplant recipients receiving chronic immunosuppression have been considered a population at high risk of infection, complications and infection-related death. During this year a large amount of information from nationwide registries, multicentre and single-centre studies have been reported. The number of renal transplant patients diagnosed with COVID-19 was higher than in the general population, but the lower threshold for testing may have contributed to its better identification. Major complications such as acute kidney injury and acute respiratory distress syndrome were very frequent in renal transplant patients, with a high comorbidity burden, but further studies are needed to support that organ transplant recipients receiving chronic immunosuppression are more prone to develop these complications than the general population. Kidney transplant recipients experience a high mortality rate compared with the general population, especially during the very early post-transplant period. Despite the fact that some studies report more favourable outcomes in patients with a kidney transplant than in patients on the kidney waiting list, the higher mortality described in the very early post-transplant period would advise against performing a kidney transplant in areas where the spread of infection is high, especially in recipients >60 years of age. Management of transplant recipients has been challenging for clinicians and strategies such as less use of lymphocyte-depleting agents for new transplants or anti-metabolite withdrawal and calcineurin inhibitor reduction for transplant patients with COVID-19 are not based on high-quality evidence. The authors received grants from Red de Investigación Renal (REDinREN RD16/0009/0030) and Fondo de Investigación Sanitaria del Instituto de Salud Carlos III (PI 18/01704, PI 18/01382). B.C. is supported by a Vall d’Hebron Institute of Research grant. This article is part of a supplement supported by Fresenius Medical Care without any influence on its content.

Kidney transplant recipients might be at higher risk for severe coronavirus disease 2019 (COVID‐19). However, risk factors for relevant outcomes remain uncertain in this population. This is a multicentric kidney transplant cohort including 104 hospitalized patients between Mar 4 and Apr 17, 2020. Risk factors for death and acute respiratory distress syndrome (ARDS) were investigated, and clinical and laboratory data was analyzed. The mean age was 60 years. Forty‐seven patients (54.8%) developed ARDS. Obesity was associated to ARDS development (OR 2.63; p=0.04). Significant age differences were not found among patients developing and not developing ARDS (61.3yr vs 57.8yr, p=0.16). Seventy‐six (73%) patients were discharged while 28 (27%) died. Death was more common among the elderly (55yr and 70.8yr, p

Background and Aims Fast tacrolimus metabolizers (expressed as the blood concentration/ dose ratio; C / D; ng / mL * mg) showed poorer renal function at 2 years, a higher incidence of nephrotoxicity and BK polyomavirus infection. Greater variability of tacrolimus trough levels (CV) from six to twelve months is associated with the appearance of HLA antibodies, interstitial fibrosis / tubular atrophy (IFTA) progression and allograft loss. We evaluate the relationship between C / D, CV and IFTA progression. Method We evaluated a cohort of 87 low immunological risk renal transplants treated with prolonged-release tacrolimus, mycophenolate mofetil and steroids. We analyzed paired protocol biopsies at 4 and 18 months. Biopsies were evaluated according to the Banff classification and the progression of IFTA was defined as the difference of ci + ct score> 0 between 18 and 4 months. The C / D ratio was calculated as the average of the value recorded at 3, 6 and 12 months of follow-up. The tacrolimus CV between 6 and 12 months was calculated using all the available determinations. Results IFTA progression was observed in 36 cases (41%). In the univariate analysis, it was found that the progression of IFTA was associated with the ci + ct score at 4 months (0.92 ± 0.94 for progressors vs. 1.89 ± 1.26 not progressors, p = 0.0003), and with the average of the C / D ratio (1.70 ± 0.73 for progressors vs. 2.28 ± 1.25 not progressors; p = 0.0144, table 1). An independent association between the C/D ratio and the progression of IFTA was observed in the multivariate analysis (OR: 0.42; 95% CI: 0.22-0.82, p = 0.027). Conclusion The results of our work suggest that fast tacrolimus metabolizers (lower C / D ratio) are more susceptible to the nephrotoxic effect of tacrolimus.

The authors conducted a prospective trial to assess the feasibility of real time central molecular assessment of kidney transplant biopsy samples from 10 North American or European centers. Biopsy samples taken 1 day to 34 years posttransplantation were stabilized in RNAlater, sent via courier overnight at ambient temperature to the central laboratory, and processed (29 h workflow) using microarrays to assess T cell- and antibody-mediated rejection (TCMR and ABMR, respectively). Of 538 biopsy samples submitted, 519 (96%) were sufficient for microarray analysis (average length, 3 mm). Automated reports were generated without knowledge of histology and HLA antibody, with diagnoses assigned based on Molecular Microscope Diagnostic System (MMDx) classifier algorithms and signed out by one observer. Agreement between MMDx and histology (balanced accuracy) was 77% for TCMR, 77% for ABMR, and 76% for no rejection. A classification tree derived to provide automated sign-outs predicted the observer sign-outs with >90% accuracy. In 451 biopsy samples where feedback was obtained, clinicians indicated that MMDx more frequently agreed with clinical judgment (87%) than did histology (80%) (p = 0.0042). In 81% of feedback forms, clinicians reported that MMDx increased confidence in management compared with conventional assessment alone. The authors conclude that real time central molecular assessment is feasible and offers a useful new dimension in biopsy interpretation. ClinicalTrials.gov NCT#01299168.

The clinical significance of renal transplant biopsies displaying borderline changes suspicious for T-cell mediated rejection (TCMR) or interstitial fibrosis and tubular atrophy (IFTA) with interstitial inflammation has not been well defined. Molecular profiling to evaluate renal transplant biopsies using microarrays has been shown to be an objective measurement that adds precision to conventional histology. We review the contribution of transcriptomic analysis in surveillance and indication biopsies with borderline changes and IFTA associated with variable degrees of inflammation. Transcriptome analysis applied to biopsies with borderline changes allows to distinguish patients with rejection from those in whom mild inflammation mainly represents a response to injury. Biopsies with IFTA and inflammation occurring in unscarred tissue display a molecular pattern similar to TCMR while biopsies with IFTA and inflammation in scarred tissue, apart from T-cell activation, also express B cell, immunoglobulin and mast cell-related genes. Additionally, patients at risk for IFTA progression can be identified by genes mainly reflecting fibroblast dysregulation and immune activation. At present, it is not well established whether the expression of rejection gene transcripts in patients with fibrosis and inflammation is the consequence of an alloimmune response, tissue damage or a combination of both.

Apolipoprotein A-Ib (ApoA-Ib) is a high molecular weight form of Apolipoprotein A-I (ApoA-I) found specifically in the urine of kidney-transplanted patients with recurrent idiopathic focal segmental glomerulosclerosis (FSGS). To determine the nature of the modification present in ApoA-Ib, we sequenced the whole APOA1 gene in ApoA-Ib positive and negative patients, and we also studied the protein primary structure using mass spectrometry. No genetic variations in the APOA1 gene were found in the ApoA-Ib positive patients that could explain the increase in its molecular mass. The mass spectrometry analysis revealed three extra amino acids at the N-Terminal end of ApoA-Ib that were not present in the standard plasmatic form of ApoA-I. These amino acids corresponded to half of the propeptide sequence of the immature form of ApoA-I (proApoA-I) indicating that ApoA-Ib is a misprocessed form of proApoA-I. The description of ApoA-Ib could be relevant not only because it can allow the automated analysis of this biomarker in the clinical practice but also because it has the potential to shed light into the molecular mechanisms that cause idiopathic FSGS, which is currently unknown.

Recurrence of idiopathic focal segmental glomerulosclerosis (FSGS) is a serious complication after kidney transplantation. FSGS relapse is suspected by a sudden increase in proteinuria but there is not an accurate noninvasive diagnostic tool to confirm this entity or to detect patients at risk. We aimed to validate the diagnostic performance of ApoA-Ib to detect FSGS relapses by measuring urinary ApoA-Ib in a retrospective cohort of 61 kidney transplanted patients (37 FSGS and 24 non-FSGS). In addition, to assess the ApoA-Ib predictive ability, ApoA-Ib was measured periodically in a prospective cohort of 13 idiopathic FSGS patients who were followed during 1 year after transplantation. ApoA-Ib had a sensitivity of 93.3% and a specificity of 90.9% to diagnose FSGS relapses, with a high negative predictive value (95.2%), confirming our previous results. In the prospective cohort, ApoA-Ib predated the recurrence in four of five episodes observed. In the nonrelapsing group (n = 9), ApoA-Ib was negative in 37 of 38 samples. ApoA-Ib has the potential to be a good diagnostic biomarker of FSGS relapses, providing a confident criterion to exclude false positives even in the presence of high proteinuria. It has also the potential to detect patients at risk of relapse, even before transplantation.

Medicopsis romeroi is a melanized coelomycetous fungus, mainly found in tropical and subtropical regions and an uncommon cause of infection in solid organ transplant (SOT) recipients. We describe two cases of SOT recipients diagnosed with phaeohyphomycosis due to M romeroi and provide a comprehensive literature review. These infections should be considered in patients native to tropical countries with a localized skin and soft tissue infection. Sequencing is needed for accurate identification of uncommon melanized fungi. Surgical treatment is recommended to cure the infection and co-adjunctive oral antifungals should be considered.

Background Data are scarce on cytomegalovirus (CMV) and BK virus (BKV) infection after antibody-mediated rejection (ABMR). Objectives We hypothesized that the immunological response in patients with ABMR or the immune modulation associated with its treatment could predispose to CMV and BKV infection. Our objective was to investigate this hypothesis. Study design We conducted a single-center, matched case-control study (1:2 ratio) to analyze CMV and BKV replication during the first year after the ABMR diagnosis in kidney transplant recipients. Adult recipients with a histopathological diagnosis of ABMR between 2007–2015 were included as cases. Controls were kidney recipients who underwent transplantation immediately before and after the index case. Results Fifty-eight patients diagnosed with ABMR (33 chronic active ABMR and 25 acute ABMR), with their matched controls (116) were included. Forty-four cases received treatment for ABMR, including plasmapheresis (41), immunoglobulins (40), and rituximab (31). Within 1 year after ABMR, cases showed CMV replication more often than controls (9/58, 15.5% vs 7/116, 6%, OR = 4.21, CI 1.10–16.16, p = 0.04). Over the study period, CMV PCR determinations were requested more frequently in cases than controls (46/58, 79.3% vs 63/116, 54.3%, OR = 4.58, CI 1.92–10.9, p = 0.001). On multivariate analysis adjusted for CMV PCR determinations, retransplantation, antithymocyte globulin treatment and methylprednisolone treatment for acute rejection, CMV replication remained more common in cases than in controls (OR = 2.41, CI 0.49–11.73, p = 0.28). There were no differences in BKV replication in either urine or blood. Conclusions ABMR may be a risk factor for CMV but not for BKV replication in kidney transplant recipients.

The prevalent renal transplant population presents an opportunity to observe the adaptive changes in the alloimmune response over time, but such studies have been limited by uncertainties in the conventional biopsy diagnosis of T cell-mediated rejection (TCMR) and antibody-mediated rejection (ABMR). To circumvent these limitations, we used microarrays and conventional methods to investigate rejection in 703 unselected biopsies taken 3 days to 35 years post-transplant from North American and European centers. Using conventional methods, we diagnosed rejection in 205 biopsy specimens (28%): 67 pure TCMR, 110 pure ABMR, and 28 mixed (89 designated borderline). Using microarrays, we diagnosed rejection in 228 biopsy specimens (32%): 76 pure TCMR, 124 pure ABMR, and 28 mixed (no borderline). Molecular assessment confirmed most conventional diagnoses (agreement was 90% for TCMR and 83% for ABMR) but revealed some errors, particularly in mixed rejection, and improved prediction of failure. ABMR was strongly associated with increased graft loss, but TCMR was not. ABMR became common in biopsy specimens obtained >1 year post-transplant and continued to appear in all subsequent intervals. TCMR was common early but progressively disappeared over time. In 108 biopsy specimens obtained 10.2–35 years post-transplant, TCMR defined by molecular and conventional features was never observed. We conclude that the main cause of kidney transplant failure is ABMR, which can present even decades after transplantation. In contrast, TCMR disappears by 10 years post-transplant, implying that a state of partial adaptive tolerance emerges over time in the kidney transplant population.

The aim was to evaluate the relationship between maintenance immunosuppression, subclinical tubulo-interstital inflammation and interstitial fibrosis/tubular atrophy (IF/TA) in surveillance biopsies performed in low immunological risk renal transplants at two transplant centers. The Barcelona cohort consisted of 109 early and 66 late biopsies in patients receiving high tacrolimus (TAC-C0 target at 1-year 6-10 ng/mL) and reduced MMF dose (500 mg bid at 1-year). The Oslo cohort consisted of 262 early and 237 late biopsies performed in patients treated with low TAC-C0 (target 3-7 ng/mL) and standard MMF dose (750 mg bid). Subclinical inflammation, adjusted for confounders, was associated with low TAC-C0 in the early (OR: 0.75, 95%CI: 0.61-0.92; p=0.006) and late biopsies (OR: 0.69, 95%CI: 0.50-0.95; p=0.023) from Barcelona. In the Oslo cohort, it was associated with low MMF in early biopsies (OR: 0.90, 95%CI: 0.83-0.98; p=0.0101) and with low TAC-C0 in late biopsies (OR: 0.77, 95%CI: 0.61-0.97; p=0.0286). MMF dose was significantly reduced in Oslo between early and late biopsies. IF/TA was not associated with TAC-C0 or MMF dose in the multivariate analysis. Our data suggest that in TAC and MMF based regimens, TAC-C0 levels are associated with subclinical inflammation in patients receiving reduced MMF dose. This research has been accepted and published in Transplant International. © 2017 Wiley

Histologic diagnosis of T cell-mediated rejection is flawed by subjective assessments, nonspecific lesions and arbitrary rules. This study developed a molecular test for T cell-mediated rejection. We used microarray results from 403 kidney transplant biopsies to derive a classifier assigning T cell-mediated rejection scores to all biopsies, and compared these with histologic assessments. The score correlated with histologic lesions of T cell-mediated rejection (infiltrate, tubulitis). The accuracy of the classifier for the histology diagnoses was 89%. Very high and low molecular scores corresponded with unanimity among three pathologists on the presence or absence of T cell-mediated rejection, respectively. The molecular score had low sensitivity (50%) and positive predictive value (62%) for the histology diagnoses. However, histology showed similar disagreement between pathologists--only 45-56% sensitivity of one pathologist with diagnoses of T cell-mediated rejection by another. Discrepancies between molecular scores and histology were mostly when histology was ambiguous ("borderline") or unreliable, e.g. in cases with scarring or inflammation induced by tissue injury. Vasculitis (isolated v-lesion TCMR) was particularly discrepant, with most cases exhibiting low TCMR scores. We propose new rules to integrate molecular tests and histology into a precision diagnostic system that can reduce errors, ambiguity and interpathologist disagreement.

We prospectively studied kidney transplants that progressed to failure after a biopsy for clinical indications, aiming to assign a cause to every failure. We followed 315 allograft recipients who underwent indication biopsies at 6 days to 32 years posttransplant. Sixty kidneys progressed to failure in the follow-up period (median 31.4 months). Failure was rare after T-cell-mediated rejection and acute kidney injury and common after antibody-mediated rejection or glomerulonephritis. We developed rules for using biopsy diagnoses, HLA antibody and clinical data to explain each failure. Excluding four with missing information, 56 failures were attributed to four causes: rejection 36 (64%), glomerulonephritis 10 (18%), polyoma virus nephropathy 4 (7%) and intercurrent events 6 (11%). Every rejection loss had evidence of antibody-mediated rejection by the time of failure. Among rejection losses, 17 of 36 (47%) had been independently identified as nonadherent by attending clinicians. Nonadherence was more frequent in patients who progressed to failure (32%) versus those who survived (3%). Pure T-cell-mediated rejection, acute kidney injury, drug toxicity and unexplained progressive fibrosis were not causes of loss. This prospective cohort indicates that many actual failures after indication biopsies manifest phenotypic features of antibody-mediated or mixed rejection and also underscores the major role of nonadherence.

Assessment of kidney transplant biopsies relies on nonspecific inflammatory lesions: Interstitial infiltrates (i), tubulitis (t) and intimal arteritis (v). We studied the relationship between inflammation and prognosis in biopsies for clinical indications from 314 patients (median follow-up 25 months). We used a modified Banff classification, separately assessing inflammation (i-) in nonscarred (i-Banff), scarred (i-IFTA) and whole cortex (i-total), plus tubulitis and intimal arteritis. In early biopsies (1 year), i- and t-lesions had no association with graft survival. In late (1 year) biopsies, all i-scores correlated with progression to failure, due to the association of these infiltrates with progressive diseases: antibody-mediated rejection (ABMR) and glomerulonephritis. Tubulitis in nonscarred areas had no impact on survival. Severe tubulitis including scarred areas (tis3) was associated with worse survival, but reflected polyoma virus nephropathy or ABMR, not T-cell-mediated rejection. Intimal arteritis (v-lesions) had no association with allograft loss in early or late biopsies. In multivariate analysis, outcome was better predicted by the presence of progressive disease than by inflammation. Thus inflammation in late kidney transplants has no inherent prognostic impact, but predicts reduced survival because inflammation indicates actively progressing diseases. The most important predictor of outcome is the diagnosis of a progressive disease.

To explore the mechanisms of antibody-mediated rejection (ABMR) in kidney transplants, we studied the transcripts expressed in clinically indicated biopsies from patients with donor-specific antibody (DSA). Comparison of biopsies from DSA-positive versus DSA-negative patients revealed 132 differentially expressed transcripts: all were associated with class II DSA but none with class I DSA. Many transcripts were expressed in DSA-positive ABMR but were also expressed in T-cell-mediated rejection (TCMR), reflecting shared molecular features. Removal of shared transcripts created 23 DSA selective transcripts (DSASTs). Some DSASTs (6/23) showed selective high expression in NK cells, whereas others (8/23) were expressed in endothelium or in endothelium plus other cell types (7/23). Of 145 biopsies ranked by DSAST expression, the 25 with highest DSAST expression primarily consisted of ABMR (22/25, 88%), either C4d-positive or C4d-negative. By immunostaining, CD56+ and CD68+ cells in peritubular capillaries, but not CD3+ cells, were increased in ABMR compared to TCMR, compatible with a role for NK cells, as well as macrophages, as effectors in endothelial injury during ABMR. Thus, the strategy of using DSASTs in the biopsy to identify mechanism-related transcripts in biopsies from patients with clinical phenotypes indicates the selective involvement of NK cells in ABMR.

In a reference set of 403 kidney transplant biopsies, we recently developed a microarray-based test that diagnoses antibody-mediated rejection (ABMR) by assigning an ABMR score. To validate the ABMR score and assess its potential impact on practice, we performed the present prospective INTERCOM study (clinicaltrials.gov NCT01299168) in 300 new biopsies (264 patients) from six centers: Baltimore, Barcelona, Edmonton, Hannover, Manchester and Minneapolis. We assigned ABMR scores using the classifier created in the reference set and compared it to conventional assessment as documented in the pathology reports. INTERCOM documented uncertainty in conventional assessment: In 41% of biopsies where ABMR features were noted, the recorded diagnoses did not mention ABMR. The ABMR score correlated with ABMR histologic lesions and donor-specific antibodies, but not with T cell-mediated rejection lesions. The agreement between ABMR scores and conventional assessment was identical to that in the reference set (accuracy 85%). The ABMR score was more strongly associated with failure than conventional assessment, and when the ABMR score and conventional assessment disagreed, only the ABMR score was associated with early progression to failure. INTERCOM confirms the need to reduce uncertainty in the diagnosis of ABMR, and demonstrates the potential of the ABMR score to impact practice.

We previously developed a microarray-based test for T cell-mediated rejection (TCMR) in a reference set of 403 biopsies. To determine the potential impact of this test in clinical practice, we undertook INTERCOM, a prospective international study of 300 indication biopsies from 264 patients (ClinicalTrials.gov NCT01299168). Biopsies from six centers-Baltimore, Barcelona, Edmonton, Hannover, Manchester and Minneapolis-were analyzed by microarrays, assigning TCMR scores by an algorithm developed in the reference set and comparing TCMR scores to local histology assessment. The TCMR score correlated with histologic TCMR lesions-tubulitis and interstitial infiltration. The accuracy for primary histologic diagnoses (0.87) was similar to the reference set (0.89). The TCMR scores reclassified 77/300 biopsies (26%): 16 histologic TCMR were molecularly non-TCMR; 15 histologic non-TCMR were molecularly TCMR, including 6 with polyoma virus nephropathy; and all 46 "borderline" biopsies were reclassified as TCMR (8) or non-TCMR (38). Like the reference set, discrepancies were primarily in situations where histology has known limitations, for example, in biopsies with scarring and inflammation/tubulitis potentially from other diseases. Neither the TCMR score nor histologic TCMR was associated with graft loss. Thus the molecular TCMR score has potential to add new insight, particularly in situations where histology is ambiguous or potentially misleading.

Reproducibility and predictive value on outcome are the main criteria to evaluate the utility of histological scores. Here we analyze the reproducibility of donor biopsy assessment by different on-call pathologists and the retrospective evaluation by a single renal pathologist blinded to clinical outcomes. We also evaluate the predictive value on graft outcome of both evaluations. A biopsy was performed in donors with any of the following: age≥55 years, hypertension, diabetes, creatinine1.5 mg/dl, or stroke. Glomerulosclerosis, interstitial fibrosis, tubular atrophy, intimal thickening, and arteriolar hyalinosis evaluated according to the Banff criteria were added to obtain a chronic score. Biopsies were classified as mild (≥3), intermediate (4-5), or advanced (6-7) damage, and unacceptable (≥8) for transplantation of 127 kidneys biopsied. Weighted κ value between both readings was 0.41 (95% CI: 0.28-0.54). Evaluation of biopsies by the renal pathologist was significantly and independently associated with estimated 12-month glomerular filtration rate and a significant composite outcome variable, including death-censored graft survival and time to reach an estimated glomerular filtration rate30 ml/min per 1.73 m2. Thus, there was no association between readings of on-call pathologists and outcome. The lack of association between histological scores obtained by the on-call pathologists and graft outcome suggests that a specific training on renal pathology is recommended to optimize the use of kidneys retrieved from expanded criteria donors.

Antibody-mediated rejection is the major cause of kidney transplant failure, but the histology-based diagnostic system misses most cases due to its requirement for C4d positivity. We hypothesized that gene expression data could be used to test biopsies for the presence of antibody-mediated rejection. To develop a molecular test, we prospectively assigned diagnoses, including C4d-negative antibody-mediated rejection, to 403 indication biopsies from 315 patients, based on histology (microcirculation lesions) and donor-specific HLA antibody. We then used microarray data to develop classifiers that assigned antibody-mediated rejection scores to each biopsy. The transcripts distinguishing antibody-mediated rejection from other conditions were mostly expressed in endothelial cells or NK cells, or were IFNG-inducible. The scores correlated with the presence of microcirculation lesions and donor-specific antibody. Of 45 biopsies with scores >0.5, 39 had been diagnosed as antibody-mediated rejection on the basis of histology and donor-specific antibody. High scores were also associated with unanimity among pathologists that antibody-mediated rejection was present. The molecular score also strongly predicted future graft loss in Cox regression analysis. We conclude that microarray assessment of gene expression can assign a probability of ABMR to transplant biopsies without knowledge of HLA antibody status, histology, or C4d staining, and predicts future failure.

Little is known regarding the molecular phenotype of kidneys with AKI because biopsies are performed infrequently. However, all kidney transplants experience acute injury, making early kidney transplants an excellent model of acute injury, provided the absence of rejection, because donor kidneys should not have CKD, post-transplant biopsies occur relatively frequently, and follow-up is excellent typically. Here, we used histopathology and microarrays to compare indication biopsies from 26 transplants with acute injury with 11 pristine protocol biopsies of stable transplants. Kidneys with acute injury showed increased expression of 394 transcripts associated with the repair response to injury, including many epithelium-like injury molecules tissue, remodeling molecules, and inflammation molecules. Many other genes also predicted the phenotype, including the acute injury biomarkers HAVCR1 and IL18. Pathway analysis of the injury-repair transcripts revealed similarities to cancer, development, and cell movement. The injury-repair transcript score in kidneys with acute injury correlated with reduced graft function, future renal recovery, brain death, and need for dialysis, but not with future graft loss. In contrast, histologic features of acute tubular injury did not correlate with function or with the molecular changes. Thus, the transcripts associated with repair of injury suggest a massive coordinated response of the kidney parenchyma to acute injury, providing both an objective measure for assessing the severity of injury in kidney biopsies and validation for many biomarkers of AKI.

NK cell transcripts are increased in biopsies with antibody-mediated rejection, whereas T cell transcripts are increased in T cell-mediated rejection. However, NK and T cells share many features, creating potential ambiguity. Therefore to estimate the NK- versus T cell transcript burdens separately, we defined nonoverlapping transcripts selective for NK cells (N = 4) or T cells (N = 5). We compared NK- versus T cell transcript burdens in microarrays from 403 kidney transplant biopsies (182 early, 221 late). In late biopsies, high NK-cell transcript expression was associated with antibody-mediated rejection, correlating with microvascular inflammation and donor specific HLA antibody. However, some early biopsies with T cell-mediated rejection had high NK-cell transcript expression, as well as T cell transcripts, without evidence of antibody-mediated rejection or DSA, correlating with interstitial inflammation and tubulitis. Both NK-cell and T cell transcripts were moderately increased in many kidneys with inflammation secondary to injury or atrophy scarring. These results support the distinct role of NK cells in late antibody-mediated rejection, but indicate a role for NK-transcript expressing cells (NK cells or T cells with NK features) both in T cell-mediated rejection and in inflammation associated with injury and atrophy scarring.

Background Subclinical rejection and interstitial fibrosis and tubular atrophy (IF/TA) in protocol biopsies are associated with outcome. We study the relationship between histologic lesions in early protocol biopsies and histologic diagnoses in late biopsies for cause. Materials and methods Renal transplants with a protocol biopsy performed within the first 6 months posttransplant between 1988 and 2006 were reviewed. Biopsies were evaluated according to Banff criteria, and C4d staining was available in biopsies for cause. Results Of the 517 renal transplants with a protocol biopsy, 109 had a subsequent biopsy for cause which showed the following histological diagnoses: chronic humoral rejection (CHR) (n=44), IF/TA (n=42), recurrence of the primary disease (n=11), de novo glomerulonephritis (n=7), T-cell-mediated rejection (n=4), and polyoma virus nephropathy (n=1). The proportion of retransplants (15.9% vs. 2.3%, P=0.058) and the prevalence of subclinical rejection were higher in patients with CHR than in patients with IF/TA (52.3% vs. 28.6%, P=0.0253). Demographic donor and recipient characteristics and clinical data at the time of protocol biopsy were not different between groups. Logistic regression analysis showed that subclinical rejection (relative risk, 2.52; 95% confidence interval, 1.1-6.3; P=0.047) but not retransplantation (relative risk, 6.7; 95% confidence interval, 0.8-58.8; P=0.085) was associated with CHR. Conclusion Subclinical rejection in early protocol biopsies is associated with late appearance of CHR.

In kidney transplantation, many inflamed biopsies with changes insufficient to be called T-cell-mediated rejection (TCMR) are labeled "borderline", leaving management uncertain. This study examined the nature of borderline biopsies as a step toward eventual elimination of this category. We compared 40 borderline, 35 TCMR and 116 nonrejection biopsies. TCMR biopsies had more inflammation than borderline but similar degrees of tubulitis and scarring. Surprisingly, recovery of function after biopsy was similar in all categories, indicating that response to treatment is unreliable for defining TCMR. We studied the molecular changes in TCMR, borderline and nonrejection using microarrays, measuring four published features: T-cell burden; a rejection classifier; a canonical TCMR classifier; and risk score. These reassigned borderline biopsies as TCMR-like 13/40 (33%) or nonrejection-like 27/40 (67%). A major reason that histology diagnosed molecularly defined TCMR as borderline was atrophy-scarring, which interfered with assessment of inflammation and tubulitis. Decision tree analysis showed that i-total >27% and tubulitis extent >3% match the molecular diagnosis of TCMR in 85% of cases. In summary, most cases designated borderline by histopathology are found to be nonrejection by molecular phenotyping. Both molecular measurements and histopathology offer opportunities for more precise assignment of these cases after clinical validation.

Data-driven approaches to deteriorating kidney transplants, incorporating histologic, molecular and HLA antibody findings, have created a new understanding of transplant pathology and why transplants fail. Transplant dysfunction is best understood in terms of three elements: diseases, the active injury–repair response and the cumulative burden of injury. Progression to failure is mainly attributable to antibody-mediated rejection, nonadherence and glomerular disease. Antibody-mediated rejection usually develops late due to de novo HLA antibodies, particularly anti-class II, and is often C4d negative. Pure treated T cell-mediated rejection does not predispose to graft loss because it responds well, even with endothelialitis, but it may indicate nonadherence. The cumulative burden of injury results in atrophy-fibrosis (nephron loss), arterial fibrous intimal thickening and arteriolar hyalinosis, but these are not progressive without ongoing disease/injury, and do not explain progression. Calcineurin inhibitor toxicity has been overestimated because burden-of-injury lesions invite this default diagnosis when diseases such as antibody-mediated rejection are missed. Disease/injury triggers a stereotyped active injury–repair response, including de-differentiation, cell cycling and apoptosis. The active injury–repair response is the strongest correlate of organ function and future progression to failure, but should always prompt a search for the initiating injury or disease.

Microarray studies of kidney transplant biopsies pro-vide an opportunity to define the molecular pheno-type. To facilitate this process, we used experimen-tal systems to annotate transcripts as members ofpathogenesis-based transcript sets (PBTs) represent-ing biological processes in injured or diseased tissue.Applyingthisannotationtomicroarrayresultsrevealedthat changes in single molecules and PBTs reflected alarge-scalecoordinatedisturbance,stereotypedacrossvarious diseases and injuries, without absolute speci-ficity of individual molecules or PBTs for rejection.Nevertheless, expression of molecules and PBTs wasquantitatively specific: IFNG effects for rejection; T celland macrophage transcripts for T cell-mediated re-jection; endothelial and NK transcripts for antibody-mediated rejection. Various diseases and injuries in-duced the same injury–repair response, undetectableby histopathology, involving epithelium, stroma andendothelium, with increased expression of develop-mental, cell cycle and apoptosis genes and decreasedexpression of differentiated epithelial features. Tran-scripts reflecting this injury–repair response were thebest correlates of functional disturbance and risk offuture graft loss. Late biopsies with atrophy-fibrosis,reflecting their cumulative burden of injury, displayedmore transcripts for B cells, plasma cells and mastcells. Thus the molecular phenotype is best describedin terms of three elements: specific diseases, includingrejection; the injury–repair response and the cumula-tive burden of injury.Keywords: Antibody-mediated rejection, kidney biop-sies, microarrays, rejection, T cells, transplantsAbbreviations: BFC, biopsies for clinical indication;TCMR, T cell-mediated rejection; ABMR, antibody-mediated rejection; ATN, acute tubular necrosis;PBTs, pathogenesis-based transcript sets; SLC, solutecarrier.Received 14 June 2010, revised 19 July 2010 andaccepted for publication 27 July 2010