Renal Allograft Biopsies Research Articles

DNA methylation modulates allograft survival and acute rejection after renal transplantation by regulating the mTOR pathway

Acute rejection (AR) can lead to allograft dysfunction following renal transplantation, despite immunosuppressive treatments. Accumulating evidence points out a role for epigenetic modification in immune responses. However, the mechanism and contribution of DNA methylation in allograft survival remain unclear. In this study, we followed up patients who successively experienced end‐stage renal disease, renal transplantation with allograft function or dysfunction, and hemodialysis. Peripheral blood mononuclear cells were collected at different time points for analysis of the DNA methylation. Epigenetic modifier analysis was also performed to explore its effect of methylation in a mouse model of AR. Compared with the allograft‐stable cohort, patients who experienced AR‐induced allograft dysfunction demonstrated more changes in methylation patterns. Pathway analysis revealed that the hypermethylated areas in the allograft dysfunction group were associated with genes related to the mechanistic target of rapamycin (mTOR) signaling pathway. Moreover, in the mouse AR model, treatment with the DNA methyltransferase inhibitor—decitabine regulated the Th1/2/17/regulatory T cell (Treg cell) immune response via its demethylating role in the suppressing the activity of the mTOR pathway, which ultimately ameliorated renal allograft‐related inflammatory injuries. These results revealed that changes in methylation accompany AR‐induced allograft dysfunction after renal transplantation. Epigenetics may provide new insights into predicting and improving allograft survival.

The protective function of miR-378 in the ischemia-reperfusion injury during renal transplantation and subsequent interstitial fibrosis of the renal allograft.

Survival time of kidney transplant patients is primarily threatened by chronic allograft dysfunction (CAD), whose typical feature is interstitial fibrosis and tubular atrophy (IF/TA). CAD could be caused by ischemia-reperfusion injury (IRI) during renal transplantation. MiR-378 is correlated with multiple kidney diseases and implicated in CAD. To clarify the underlying mechanism of miR-378 on renal allograft, we utilize renal unilateral IRI mice and H/R NRK52E cells. To evaluate the acute tubular damage, we determined the apoptotic rate by TUNEL assay and examined mice kidney sections of H&E staining 1day after IRI. To assess the chronic renal interstitial inflammation and fibrosis, we detected the infiltration rates of CD45R+ leukocytes and Ly6b+ neutrophils by immunohistochemistry, examined mice kidney sections of picrosirius staining and measured the mRNA level of Col1a1 14days after IRI. To investigate the H/R injury of NRK52E cells, MTT assay was performed to detect cell viability, TUNEL assay was performed to determine apoptotic rate and luciferase reporter assay was carried out to demonstrate the potential target of miR-378. Moreover, we determined the levels of miR-378 of renal allograft biopsies in healthy patients and patients diagnosed with IF/TA. We found agomir-378 treatment significantly reduced the apoptotic rate and tubular damage scores assessed by H&E staining 1day after IRI. Agomir-378 treatment also decreased infiltration rates of both CD45R+ leukocytes and Ly6b+ neutrophils and fibrosis examined by picrosirius staining and by the mRNA level of Col1a1 14days after IRI. Experiments in vitro revealed that miR-378 increased cell viability and decreased apoptotic rate of NRK52E cells subjected to H/R. Additionally, luciferase reporter assay confirmed that caspase 3 was targeted by miR-378 directly. Furthermore, we found the levels of miR-378 were significantly lower in renal allografts of patients with IF/TA than those of healthy controls. Taken together, we have found that miR-378 has potential protective effects on renal allografts to prevent IRI during kidney transplantation and following IF/TA of renal allografts.

Investigating the threshold for early renal allograft biopsy: A South African single-centre perspective.

The most common clinical indication for renal biopsy in the early post-transplant period is early graft dysfunction (EGD), which may present either as delayed graft function (DGF) or acute graft dysfunction. Even though it is a valuable diagnostic tool, renal allograft biopsy is not without risk of major complications. Recent studies have suggested that, with modern immunosuppressive induction regimens and more accurate ways to determine high immunological risk transplants, early acute rejection (AR) is uncommon and routine biopsy for EGD does not result in a change in management. To describe the histological findings and complications of renal allograft biopsies for EGD in our setting, and to determine whether our current threshold for biopsy is appropriate. This study was a retrospective audit that included all patients who underwent renal allograft biopsy within the first 30 days of transplantation at Groote Schuur Hospital, Cape Town, South Africa, from 1 June 2010 to 30 June 2018. The indication for biopsy was any patient who showed significant EGD, characterised by acute graft dysfunction or DGF with dialysis dependence. During the study period, 330 patients underwent renal transplantation, of whom 105 (32%) had an early biopsy and were included in the study. The median age of recipients was 39 (range 17 - 62) years, with 65% males and 35% females. The majority of donors were deceased donations after brain death (70%), with an overall median cold ischaemic time of 9 hours (interquartile range (IQR) 4 - 16). The average number of human leukocyte antigen mismatches was 5 (IQR 4 - 7). A donor-specific antibody was recorded for 18% of recipients and a panel-reactive antibody score of >30% was recorded for 21%. The median duration after transplant for biopsy was 8 (IQR 6 - 10) days. During the first month of EGD, AR was diagnosed in 42% of patients who underwent biopsies. In 21% of these patients, there was acute cellular rejection, in 16% antibody-mediated rejection, and in 5% both of these. Acute tubular necrosis was the primary finding in 32%, with acute interstitial nephritis in 8%, and acute calcineurin toxicity in 4% of cases. A significant biopsy-related complication was recorded in 3 patients: 1 small-bowel perforation repaired via laparotomy, and 2 vascular injuries successfully embolised by interventional radiology. Considering the relative safety and high rate of detection of AR, a liberal approach to renal biopsy for EGD remains justifiable in our setting.

P0127ROUTINE USE OF COMBINATION CD3/PAS STAIN IN EVALUATING RENAL ALLOGRAFT BIOPSIES FOR REJECTION RESULTS IN MORE ACCURATE BANFF SCORES FOR TUBULITIS

Abstract Background and Aims The BANFF Classification of renal allograft rejection consists of scores for cellular rejection which rely primarily on the evaluation of lymphocytic infiltration into tubules (t), non-atrophic interstitial inflammation (i), total inflammation (ti), and endotheliatis (v.) CD3/PAS is a combination stain that highlights T lymphocytes (CD3) and basement membrane (PAS). The original BANFF scoring was developed using a hematoxylin and eosin (H&E) stain only. This study compares scores for (t) using H&E versus CD3/PAS. Method 50 consecutive renal allograft biopsies were retrieved and on each case H&E and CD3/PAS combination stains (Ventana/Roche tissue Diagnostics Clone 2GV6 for CD3) were performed. One blinded group of matching CD3/PAS and H&E slides were evaluated and reviewed independently by two renal pathologists (Reviewer 1 and 2). Each case was graded in an identical manner following criteria designated for (t) as previously described in the Banff Classification for Renal Allograft Biopsies. Briefly (t)=0 no lymphocytes in 10 tubule cross section (tcx), (t)=1 between 1-4 lymphocytes per tcx, (t)=2 5-10 lymphocytes per tcx and (t)=3 greater than 10 lymphocytes per tcx. Cases were not graded for (v) as there were too few cases with endotheliatis to allow for statistical evaluation. The difference between CD3/PAS versus H&E scores were tested for significance using the Wilcoxon Signed Rank test. All p values were two-tailed and values of <0.05 were considered statistically significant. Results Mean age was 44 years with a range of 9 -75 years. Standard deviation (SD) for age was 18 years. Gender distribution was 20% female and 80% male. 28 cases were graded as (t) = 0 in H&E versus 18 in the CD3/PAS group (59% versus 36%). 17 cases were graded as (t)=1 in H&E group versus 19 in the CD3/PAS group (36% versus 38% respectively). 2 cases were graded (t)=>2 in H&E versus 12 cases in CD3/PAS groups (4% versus 24% respectively). In all categories of (t) there was a higher (t) score when using a CD3/PAS with 42% showing a one digit score increase and 8% showing a 2 digit increase. The difference in scoring for (t) between the H&E set versus the CD3/PAS set for both Reviewers 1 and 2 were statistically significant (p<0.0001). Conclusion This study showed statistically significant higher tubulitis scores when using a CD3/PAS combination stain to grade allograft biopsies. Since (t) scores directly affect the diagnosis of cellular rejection, we propose routine use of CD3/PAS in addition to H&E when reviewing renal allograft biopsies. Compared with H&E, CD3/PAS makes recognition and quantification of lymphocytes crossing the tubular basement membrane easier to detect and therefore more accurate.

P0093IMPROVED INTEROBSERVER AGREEMENT IN BANFF SCORING OF RENAL ALLOGRAFT BIOPSIES WHEN ROUTINELY USING CD3/PAS STAIN

Abstract Background and Aims The BANFF Classification of renal allograft rejection consists of scores for cellular and antibody mediated rejection. The scores for tubulitis (t), interstitial inflammation (i), glomerulitis (g), total inflammation (ti), and endotheliatis (v) have direct prognostic and therapeutic significance as they contribute to a diagnosis of renal allograft rejection. The original BANFF scoring was developed using hematoxylin and eosin (H&E) stain only. This study examines the level of interobserver agreement using H&E and a combination CD3/PAS stain that highlights T lymphocytes and basement membranes. Method 50 consecutive renal allograft biopsies were retrieved and on each case H&E and CD3/PAS stains (Ventana/Roche Tissue Diagnostics clone 2GV6 for CD3) were performed. CD3/PAS and H&E slides were matched and blinded for two reviewers [reviewer (R1) and reviewer 2 (R2)]. Each case was graded in an identical manner following criteria designated for (t),(i),(g) and (ti) as previously described in the Banff Classification for Renal Allograft Biopsies. Cases were not graded for (v) as there were too few cases with endothelialitis to allow for statistical evaluation. The matched scores were analyzed using weighted Kappa statistics for interobserver agreement within each group. Agreement levels were characterized using Landis and Koch descriptions for level of agreement. All p values were two-tailed, values of <0.05 were considered statistically significant. Statistical analyses was performed using SAS Version 9.4, Cary NC. Results Mean age was 44 years with a range of 9 -75 years. Standard deviation (SD) for age was 18 years. Gender distribution was 20% female and 80% male. Agreement between R1 and R2 for (t) scores was moderate (Weighted Kappa 0.5276) as opposed to fair for H&E group (Weighted Kappa 0.4189). Similarly, interobserver agreement was moderate for scores of (i) and (ti) in the CD3/PAS group and fair in the H&E group. For both CD3/PAS and H&E interrater evaluations, agreement was fair to poor for (g) with weighted Kappa of 0.3928 and 0.3559 respectively. Conclusion This study showed increased levels of interobserver agreement when using a CD3/PAS combination stain to score allograft biopsies for (t), (i) and (ti). Since (t), (i) and (ti) scores directly affect a diagnosis of rejection, we propose routine use of CD3/PAS in addition to H&E when reviewing renal allograft biopsies. Glomerulitis (g) shows poor interobserver agreement regardless of the stain used and further studies to refine the criteria for (g) in Banff revisions of the classification may be of value.

SO030HISTOLOGICAL CHARACTERIZATION OF RENAL BIOPSIES FROM HCV-INFECTED DONORS: A VALUABLE SOURCE OF KIDNEY ALLOGRAFTS

Abstract Background and Aims The introduction of effective direct-acting antiviral agents (DAAs) has allowed safe transplantation of kidneys from HCV positive (HCV+) donors into HCV negative (HCV-) recipients. Some studies demonstrated an increased risk for rejection as well as polyomavirus infection in these situations (Molnar et al, Am J Transplant. 2019 Nov;19(11):3046-3057), possibly due to the development of an inflammatory milieu in the setting of HCV seroconversion and persistent viremia (Durand et al, Am J Transplant. 2019 Nov;19(11):2969-2970). With this concern for HCV-associated renal injury and increased risk for rejection, this retrospective study explores the histologic findings of allograft biopsies from HCV+ donors. Method Fifty renal allograft biopsies from 28 recipients of HCV+ donors evaluated at Vanderbilt University Medical Center between September 2018 and December 2019 were retrospectively reviewed with recording of clinical and histological features, and final diagnoses. All recipients were transplanted under a clinical protocol and were treated with DAA therapy. In the same period of time, 30 renal allograft biopsies from 24 recipients of HCV- donors were selected having the same post-transplant interval as the study cohort. Categorical variables are expressed in percentage and compared using Pearson’s chi-squared test. Continuous variables are expressed as mean±SD and compared used Student t-test. A p-value of <0.05 was considered as statistically significant. Results The complete results of the comparison between the two groups (HCV+ and HCV- donors) are demonstrated in Figure. The differences reaching statistical significance include higher frequency of males and tubulointerstitial fibrosis (IFTA) in the HCV+ group (p = 0.038 and 0.044, respectively) and the higher incidence of DSA positivity and acute tubular injury in the control group (p = 0.001 and 0.029). Notably, no significant differences were noted in terms of antibody-mediated events (microcirculation inflammation, transplant glomerulopathy, C4d positivity, or diagnostic ABMR), or T-cell mediated events including borderline or diagnostic T-cell mediated rejection (Banff 2013). The two groups also demonstrated the same incidence of polyomavirus nephropathy and de novo/recurrent glomerulonephritis. Interestingly, two of the HCV+ cases showed mesangial IgA deposits on immunofluorescence and electron microscopy on the first biopsy, of which one showed persistence of deposits, 18 and 44 days after transplant respectively. Once case demonstrated a de novo focal proliferative IgM-dominant glomerulonephritis on the 7th biopsy, 266 days after transplantation. Conclusion Kidneys from HCV+ donors represent a valuable resource in the era of DAA therapy. This reported experience demonstrates similar frequency of rejection and viral infection affecting allografts from HCV positive and negative donors. The presence of immune complex deposits and glomerulonephritis in some of these cases could represent either a donor-derived injury or an acquired post-seroconversion feature. In conclusion, HCV+ donors appear to be a safe resource for transplantation. Further studies with post-transplant intervals will provide additional characterization of their impact.

BK Virus RNA in Renal Allograft Biopsies.

BK polyomavirus-associated nephropathy (BKpyVAN) remains a cause of graft loss in kidney transplant recipients on immunosuppressive therapy. Its diagnosis relies on the identification of BK virus (BKV) in the renal allograft biopsy by positive immunohistochemical (IHC) stain for the viral SV40 large T antigen, although in situ hybridization (ISH) for viral DNA is used in some centers. We examined tissue detection of BKV RNA by RNAscope, a novel, automated ISH test, in 61 allograft biopsies from 56 patients with BKpyVAN. We found good correlation between the estimate of BKV tissue load by RNAscope ISH and SV40 IHC (R2 = 0.65, p<0.0001). RNAscope ISH showed 88% sensitivity and 79% specificity and, as an alternative test, could confirm the presence of BKV tissue in presumed BKpyVAN and rule out BKV as the causative agent in JC virus nephropathy. We also used tissue BK viral load estimates by both RNAscope ISH and SV40 IHC to examine the relation between tissue and plasma BK levels and found significant correlation only between BK viremia and tissue BK measured by RNAscope ISH. Our findings suggest that the RNAscope ISH assay could be a reliable test for BKV detection in allograft biopsies.

Diagnosis of renal transplant rejection: Banff classification and beyond.

Diagnosis of renal transplant rejection is dependent on interpretation of renal allograft biopsies. The Banff Classification of Allograft Pathology, which was developed as a standardized working classification system in 1991, has contributed to the standardization of definitions for histologic injuries resulting from renal allograft rejections and provided a universal grading system for assessing these injuries. It has also helped to provide insight into the underlying pathogenic mechanisms that contribute to transplant rejection. In addition to histological and immunologic parameters, molecular tools are now being used to facilitate the diagnosis of rejection. In this review, I will discuss morphologic features of renal transplant rejections as well as major revisions and pitfalls of the Banff classification system, and provide future perspectives.

Automated detection algorithm for C4d immunostaining showed comparable diagnostic performance to pathologists in renal allograft biopsy.

A deep learning-based image analysis could improve diagnostic accuracy and efficiency in pathology work. Recently, we proposed a deep learning-based detection algorithm for C4d immunostaining in renal allografts. The objective of this study is to assess the diagnostic performance of the algorithm by comparing pathologists' diagnoses and analyzing the associations of the algorithm with clinical data. C4d immunostaining slides of renal allografts were obtained from two different institutions (100 slides from the Asan Medical Center and 86 slides from the Seoul National University Hospital) and scanned using two different slide scanners. Three pathologists and the algorithm independently evaluated each slide according to the Banff 2017 criteria. Subsequently, they jointly reviewed the results for consensus scoring. The result of the algorithm was compared with that of each pathologist and the consensus diagnosis. Clinicopathological associations of the results of the algorithm with allograft survival, histologic evidence of microvascular inflammation, and serologic results for donor-specific antibodies were also analyzed. As a result, the reproducibility between the pathologists was fair to moderate (kappa 0.36-0.54), which is comparable to that between the algorithm and each pathologist (kappa 0.34-0.51). The C4d scores predicted by the algorithm achieved substantial concordance with the consensus diagnosis (kappa = 0.61), and they were significantly associated with remarkable microvascular inflammation (P = 0.001), higher detection rate of donor-specific antibody (P = 0.003), and shorter graft survival (P < 0.001). In conclusion, the deep learning-based C4d detection algorithm showed a diagnostic performance similar to that of the pathologists.

Molecular Analysis of Renal Allograft Biopsies: Where Do We Stand and Where Are We Going?

A renal core biopsy for histological evaluation is the gold standard for diagnosing renal transplant pathology. However, renal biopsy interpretation is subjective and can render insufficient precision, making it difficult to apply a targeted therapeutic regimen for the individual patient. This warrants a need for additional methods assessing disease state in the renal transplant. Significant research activity has been focused on the role of molecular analysis in the diagnosis of renal allograft rejection. The identification of specific molecular expression patterns in allograft biopsies related to different types of allograft injury could provide valuable information about the processes underlying renal transplant dysfunction and can be used for the development of molecular classifier scores, which could improve our diagnostic and prognostic ability and could guide treatment. Molecular profiling has the potential to be more precise and objective than histological evaluation and may identify injury even before it becomes visible on histology, making it possible to start treatment at the earliest time possible. Combining conventional diagnostics (histology, serology, and clinical data) and molecular evaluation will most likely offer the best diagnostic approach. We believe that the use of state-of-the-art molecular analysis will have a significant impact in diagnostics after renal transplantation. In this review, we elaborate on the molecular phenotype of both acute and chronic T cell-mediated rejection and antibody-mediated rejection and discuss the additive value of molecular profiling in the setting of diagnosing renal allograft rejection and how this will improve transplant patient care.

SUN-353 A comprehensive review of 50 cases of kidney transplants performed over 10 months a single centre experience from an African country

Access to kidney transplantation has always been a problem in the African countries with many patients having to travel to other medically advanced countries in Asia, Europe and America. This involves unnecessary excessive expenditure and the travails of journey and stay in an unknown foreign land. To ease this situation and provide affordable Renal transplant services in their home land, we have made an effort to start the transplant services at our medical facility and have successfully carried out about 50 transplants in a period of 10 months from December 2018 till September 2019. A comprehensive overview of the cases has been presented forthwith. All the Kidney transplants done between the period Nov 2018- Sept 2019 ( 50 cases) were included in the analysis. All the transplants were performed at a single center and the data were collected progressively during their Pre transplant evaluation, perioperative course and post op follow up. All the laboratory and radiological tests were done locally at the center except the HLA cross matches and typing which were outsourced to Transplant immunology labs outside the country. All the patients underwent laparoscopic donor nephrectomy except in patients with technical difficulties. A series of 50 kidney transplants were performed over a period of 10 months [ DEC 2018- september 2019] at a private hospital successfully. All the cases were live donor kidney transplants with majority of the donors being 1st or 2nd degree relatives or spousal donors. There were no cases of mortality and all the patients had good immediate graft function except in 6 patients, who had delayed graft function; 5 of them improving over 2 – 6 weeks and one patient not in follow-up at the time of submission of this abstract]. Renal allograft biopsy was done in 5 patients. In 4 patients biopsy findings were suggestive of ATN, and the other, report awaited at the time of submission of this abstract. Of all patients, 2 were HBsAg +ve, and 2 were HIV +Ve. Two patients required simultaneous nephrectomy at the time of transplant, one for ADPKD and the other for VUR. Other postoperative problems included; one patient had sub-acute intestinal obstruction due to adhesions requiring laparoscopic intervention and the other segmental consolidation with cough and fever. All the patients received induction with rabbit Thymoglobulin, and IV methyl prednisolone. 12 patients required plasma exchange and IVIG for positive X match { 11 pre op’ and 1 post op’}. Overall patient survival was 100% and graft survival over 98% {49/50}. Our experience shows that kidney transplantation is a viable and practical option for the treatment of End stage kidney disease and can be performed even in resource constrained centers in third world countries.

SAT-378 De-novo and recurrent glomerulonephritis in renal allograft recipients – a single centre experience

Glomerulonephritis (GN) in the renal allograft recipient, especially recurrence of the native kidney disease can affect the long-term graft outcome. Data from our country is scant. When as many as 54.4% have Chronic Kidney Disease stage 5 (CKD 5) as the initial presentation of renal illness, the native kidney disease is frequently unknown. We studied the pattern of GN in the renal allograft recipients from our centre over a six year period. Renal allograft recipients over a six year period (2003 to 2008) were retrospectively evaluated to look for appearance of GN in the follow-up period. There were 548 patients who underwent transplantation during this period, of which, the native kidney disease (NKD) as GN was known in 83 patients (15.2%). The most common NKD was IgA nephropathy (33.7%) followed by focal and segmental glomerulosclerosis (FSGS) in 27.6%. The mean time of onset of renal illness to CKD 5 was 41.8 (± 46.36) months, ranging from one month to 16 years. 36 patients (6.4 %) developed GN, of which eleven had recurrence of GN, eight had de-novo GN while sixteen had a de-novo GN where NKD was unknown. The most common GN were IgA nephropathy (9, 28.1%) and FSGS (9, 28.1%). The most common recurrent GN was IgA nephropathy in five patients (62.5%). The most common de novo GN in those with a known NKD was calcineurin (CNI) induced thrombotic microangiopathy (TMA) in five patients (62.5%) while the most common de novo GN in those with unknown NKD were IgA nephropathy (4, 26.7%) and FSGS (4, 26.7%). Renal allograft biopsy was done mostly for proteinuria (18, 56.3%) followed by deterioration in renal function (9, 28.1%). Biopsy was done 17.5 (±15) months post-transplant with the minimum duration being as low as six days. The mean period of follow up of patients with denovo/recurrent GN (from the time of diagnosis) was 25.8(±22.3) months. In patients with denovo CNI induced TMA, graft function improved after the withdrawal of CNI. Excluding the above subgroup of patients, the rate of decline in GFR in patients with recurrent/denovo GN over the mean follow up period was 0.39ml/min/month. Graft loss was observed in 8 patients with recurrent/denovo GN. Glomerulonephritis (GN) in the renal allograft recipient is not uncommon. Of the 548 patients, 6.4 % developed GN of which the most common was IgA nephropathy. The most common recurrent GN was IgA nephropathy while the most common de novo GN in those with a known NKD was calcineurin induced thrombotic microangiopathy while the most common de novo GN in those with unknown NKD were IgA nephropathy and FSGS.

Bleeding Complications and Adverse Events After Desmopressin Acetate for Percutaneous Renal Transplant Biopsy

Percutaneous renal biopsy remains critical for the workup of renal allograft dysfunction but is associated with the risk of bleeding. Prophylactic intravenous desmopressin has been proposed to reduce bleeding risk in native renal biopsies, but its efficacy in the renal transplant population is unclear and adverse events such as severe hyponatraemia have been reported. We conducted a single-centre retrospective cohort study involving adult (≥21 years old) renal transplant recipients with impaired renal function (serum creatinine ≥150 µmol/L) who underwent ultrasound-guided renal allograft biopsies from 2011‒2015 to investigate the effect of prebiopsy desmopressin on the risk of bleeding and adverse events. Desmopressin was administered to 98 of 195 cases who had lower renal function, lower haemoglobin and more diuretic use. Postbiopsy bleeding was not significantly different between the 2 groups (adjusted odds ratio [OR] 0.79, 95% confidence interval [CI] 0.26‒2.43, P = 0.68) but desmopressin increased the risk of postbiopsy hyponatraemia (sodium [Na] <135 mmol/L) (adjusted OR 2.24, 95% CI 1.10‒4.59, P = 0.03). Seven cases of severe hyponatraemia (Na <125 mmol/L) developed in the desmopressin group, while none did in the non-desmopressin group. Amongst those who received desmopressin, risk of hyponatraemia was lower (OR 0.26, 95% CI 0.09‒0.72, P = 0.01) if fluid intake was <1 L on the day of biopsy. Prophylactic desmopressin for renal allograft biopsy may be associated with significant hyponatraemia but its effect on bleeding risk is unclear. Fluid restriction (where feasible) should be recommended when desmopressin is used during renal allograft biopsy. A randomised controlled trial is needed to clarify these outcomes.

Quantitative Proteomics for Monitoring Renal Transplant Injury.

This study is aimed at developing a molecular diagnostics platform to enhance the interpretation of renal allograft biopsies using quantitative proteomic profiling of formalin-fixed and paraffin-embedded (FFPE) specimens. A quantitative proteomics platform composed of 1) an optimized FFPE protein sample preparation method, 2) a tandem mass tag TMT10-plex-based proteomic workflow, and 3) a systematic statistical analysis pipeline to reveal differentially expressed proteins has been developed. This platform is then tested on a small sample set (five samples per phenotype) to reveal proteomic signatures that can differentiate T-cell mediated rejection (TCMR) and polyomavirus BK nephropathy (BKPyVN) from healthy functionally stable kidney tissue (STA). Among 2798 quantified proteins, the expression levels of 740 BKPyVN and 638 TCMR associated proteins are significantly changed compared to STA specimens. Principal component analysis demonstrated good segregation of all three phenotypes investigated. Protein detection and quantitation are highly reproducible: replicate comparative analyses demonstrated 71-84% overlap of detected proteins, and the coefficient of variation for protein measurements is <15% in triplicate liquid chromatography-tandem mass spectrometry runs. Quantitative proteomics can be applied to archived FFPE specimens to differentiate different causes of renal allograft injury.

Immune Cell Infiltrate in Chronic-Active Antibody-Mediated Rejection.

Background: Little is known about immune cell infiltrate type in the kidney allograft of patients with chronic-active antibody-mediated rejection (c-aABMR).Methods: In this study, multiplex immunofluorescent staining was performed on 20 cases of biopsy-proven c-aABMR. T-cell subsets (CD3, CD8, Foxp3, and granzyme B), macrophages (CD68 and CD163), B cells (CD20), and natural killer cells (CD57) were identified and counted in the glomeruli (cells/glomerulus) and the tubulointerstitial (TI) compartment [cells/high-power field (HPF)].Results: In the glomerulus, T cells and macrophages were the dominant cell types with a mean of 5.5 CD3+ cells/glomerulus and 4 CD68+ cells/glomerulus. The majority of T cells was CD8+ (62%), and most macrophages were CD68+CD163+ (68%). The TI compartment showed a mean of 116 CD3+ cells/HPF, of which 54% were CD8+. Macrophage count was 21.5 cells/HPF with 39% CD68+CD163+. CD20+ cells were sporadically present in glomeruli, whereas B-cell aggregates in the TI compartment were frequently observed. Natural killer cells were rarely identified. Remarkably, increased numbers of CD3+FoxP3+ cells in the TI compartment were associated with decreased graft survival (p = 0.004).Conclusions: Renal allograft biopsies showing c-aABMR show a predominance of infiltrating CD8+ T cells, and increased numbers of interstitial FoxP3+ T cells are associated with inferior allograft survival.

Usefulness of real-time sonoelastography for assessment of renal allograft fibrosis

Background: Currently, allograft renal biopsy is the only reliable tool available to detect fibrosis in the transplanted kidney. However, it is an invasive procedure and is associated with complications. Therefore, a noninvasive tool to detect renal allograft fibrosis is needed. Aims: The aim of the study was to evaluate the usefulness of real-time sonoelastography (RTS) in the diagnosis of renal allograft fibrosis. Subjects and Methods: We studied 15 renal allograft recipients who had chronic allograft nephropathy. RTS was performed by an experienced radiologist to semi-quantitatively determine cortical and medullary stain ratio. These parameters were compared with the degree of fibrosis as assessed by allograft renal biopsy. For comparison, patients were divided into two groups based on the degree of fibrosis: those with mild fibrosis (interstitial fibrosis and tubular atrophy [IFTA] 25%). A receiver operating characteristic (ROC) curve analysis was performed to evaluate the accuracy of cortical strain ratio to discriminate between patients with mild fibrosis versus patients with moderate-to-severe fibrosis. Results: The mean cortical strain ratio was significantly higher in those who had mild fibrosis as compared to those who had moderate-to-severe fibrosis (2.46 ± 0.55 vs. 1.78 ± 0.15, P = 0.01), while the medullary strain ratio was comparable between the two groups. The diagnostic accuracy of cortical strain ratio, as evaluated by area under the curve of ROC analysis, was 0.96. Conclusion: RTS can differentiate between mild fibrosis and moderate-to-severe fibrosis with high accuracy.

Tubular Ectasia in Renal Allograft Biopsy: Associations With Occult Obstructive Urological Complications.

Urological obstructive complications (UOC) affect up to 15% of kidney transplants (KTX). Most cases are excluded by ultrasonography (US); however, accuracy may be limited in the early transplant phase. Features of acute tubular injury (ATI) in KTX biopsy may be informative but histological features indicating UOC are ill defined. Tubular ectasia (TE) was shown to be associated with UOC in experimental data. We evaluated the association of histomorphological features, particularly TE, with occult (=without relevant hydronephrosis in US) UOC and renal outcomes. We included all recipients with an early indication biopsy (976 of 1537 consecutive KTX). The biopsy finding of TE classified as "suspicious of UOC" was compared with the following endpoints: delayed graft function, estimated glomerular filtration rate, and occult UOC. Additionally, histopathological features of ATI were reevaluated by a single pathologist to increase diagnostic accuracy. Fifty-eight (5.9%) patients presented with TE, which was not related to delayed graft function or estimated glomerular filtration rate. Forty percent of patients had a UOC (most frequently ureteral stenosis) close to biopsy. Comparing these biopsies to matched controls, TE was significantly associated with UOC (odds ratio 2.69; P = 0.018). After histopathological reevaluation of these biopsies including additional features of ATI, we developed a final multivariate model with a highly significant relationship to UOC (Receiver operating characteristic-area under the curve: 0.77; P = 0.001). The model provides a specificity of 78% and negative predictive value of 73%. TE together with additional signs of ATI indicates occult UOC. This histological phenotype should trigger more detailed evaluation for UOC when there is no evidence of relevant hydronephrosis in the ultrasonography.

Post Renal Transplant Collapsing Glomerulopathy is Associated with Poor Outcomes.

Introduction:Collapsing glomerulopathy (CG) is a distinct morphologic pattern of proliferative renal parenchymal injury. It differ from focal segmental glomerulosclerosis (FSGS) by clinicopathologic pattern and its adverse outcome. The clinical significance of CG in renal allograft biopsies is not yet clear due to scant data and less occurrence of CG in renal transplant recipients. We conducted this single-center retrospective study to evaluate the prevalence, clinicopathological features, and outcome of post renal transplant CG.Subjects and Methods:We studied 127 renal allograft biopsies performed over a period of 45 months (Jan 2015–Oct 2018). A diagnosis of CG was made if at least one glomerulus demonstrated global or segmental collapse of the glomerular capillary walls, associated marked hyperplasia, and hypertrophy of the overlying visceral epithelial cells. We analyzed clinical, biochemical, and pathological characteristics and its impact on renal allograft outcome. Statistical analysis was performed and continuous variables were expressed as means ± standard deviation (SD) or medians (interquartile range and noncontinuous data were expressed in percentage and numerical values.Results:The prevalence of CG was 5.3% (7/127) of allograft biopsies. Out of the seven patients, six patients had undergone live donor transplant and one patient had undergone deceased donor renal transplant. The native kidney disease was unknown in these patients except one (IgA nephropathy). The median duration of diagnosis for CG was 17 months after transplantation (range 5–132months). Presenting symptoms were pedal edema and hypertension in 71.4% (5) patients each. All patients had proteinuria of more than 1 gm and renal allograft dysfunction and median serum creatinine of 3.05 mg/dl (1.5–4.8 mg/dl). All patients received standard triple immunosuppression. Over a period of 2–20 months, 57.14% (4) patients developed a graft failure and 43% (3) of the other patients had functioning grafts with serum creatinine of 1.5–4.2 mg/dl.Conclusions:CG presents with moderate to severe proteinuria and may lead to rapid graft dysfunction and subsequent graft failure in most of the patients.

Use of elastographic techniques as noninvasive tools in assessment of renal allograft fibrosis

Context Chronic allograft dysfunction is still the main cause of late allograft loss in kidney transplantation. Increased serum creatinine could be an indicator of progressive damage of the renal allograft and tubular atrophy and interstitial fibrosis, which need to be confirmed by the invasive, sometimes hazardous renal allograft biopsy. Aims We aimed to assess the possibility of using transient elastography (fibroscan) either based on ultrasound or MRI as noninvasive tools for evaluation of renal allograft fibrosis and chronic allograft index (CAI). Setting and design A prospective cohort study was conducted. Patients and methods The study included 15 patients with renal allograft. Pelvi-abdominal sonar, renal Doppler, and fibroscan have been performed for the included patients. Serum urea and creatinine have been measured, and estimated glomerular filtration rate (eGFR) has been calculated for all patients. Renal biopsies were done in seven cases. Statistical analysis Statistical package for the social sciences (version 13.0) was used for statistical analysis. Results Stiffness was significantly correlated with interstitial fibrosis (P Conclusion Parenchymal renal allograft stiffness by TE is an effective method for identifying patients with CAI indicative for biopsy and modification of the immunosuppressive regimen.

Thrombotic microangiopathy in a renal allograft: Single-center five-year experience

Thrombotic microangiopathy (TMA) is devastating for renal transplantation (RT) causing graft/ patient loss. We present 5-year experience of TMA in RT in retrospective study of indicated renal allograft biopsies with TMA. Patient-donor demographics and associated histological findings with respect to transplants under tolerance induction protocol (Group 1) were compared with patients transplanted under triple immunosuppression (Group 2). Statistical analysis was performed using IBM SPSS Statistics version 20. Sixty-one (4.1%) of 1520 biopsies [Group 1:17 (1.9%)/882, Group 2:44 (6.9%)/638] revealed TMA. Tacrolimus trough levels were normal. There was no evidence of systemic involvement in any patient. Mean age was 36.8 years with 70.6% males, HLA-match, 2.6/6, and the most common original disease unknown (41.2%) in Group 1, and 35.9 years with 86.4% males, HLA-match, 2.1/6, and the most common original disease unknown (50%) in Group 2. Biopsies were performed at mean 5.1-year posttransplant in Group 1 and 2.3 years in Group 2. Acute TMA constituted 47% Group 1 and 43.2% Group 2 biopsies; of these, antibody-mediated rejections were observed in 58.8%, T-cell mediated rejections in 11.8%, tacrolimus toxicity in 76.5%, and other findings in 35.3% Group 1; and 61.4%, 25%, 50%, and 18.2%, respectively, in Group 2 biopsies. Higher rejection activity scores were more in Group 2. Postbiopsy 1- and 5- year patient survival was 94.1%, 86.9% in Group 1 and 92.1%, 88.3% in Group 2; 1- and 4-year graft survival was 52.9%, 15.9% in Group 1 and 20.3%, 5.4% in Group 2. TMA was poor prognosticator for RT, especially under triple immunosuppression. Antibody- mediated rejection and tacrolimus toxicity were more prone to TMA.