Transplant Biopsies Research Articles

Association of Oral Tobacco-Free Nicotine Delivery Product with Acute Renal Tubular Necrosis

Usage of novel non-tobacco oral nicotine delivery products (ONDPs) has been increasing among adolescents in the United States. It is presumed that they are less toxic than their tobacco-containing counterparts, but that has not been examined in controlled studies. Most of the studies have focused on non-renal manifestations of tobacco consumption via different means. The renal manifestations of non-tobacco ONDPs are not very well known, especially in immunocompromised patients. A 19-year-old male transplant recipient presented with flank pain and a few days’ history of intake of ZYNR pouches. Immunosuppression was with tacrolimus, mycophenolate, and steroids. Baseline serum creatinine was 1.1–1.3 mg/dL. Laboratory evaluation showed elevated C-reactive protein, increased serum creatinine and blood urea nitrogen, leukocytosis, neutrophilia, and increased lactate dehydrogenase (LDH). Infectious disease work-up was negative. A kidney transplant biopsy showed severe acute tubular injury/necrosis (ATN) without evidence of rejection. Donor-specific antibodies were negative. Other etiologies of hemolysis were negative. He did not require renal replacement therapy. Kidney function and LDH improved gradually. The most recent follow-up eight months after presentation showed a serum creatinine level of 2.6 mg/dL with stable electrolytes, with eGFR of 35 mL/min/1.73 m2. Here, we describe a 19-year-old adolescent with a kidney transplant who sustained ATN leading to advanced chronic disease apparently following the usage of non-tobacco ONDP. Further larger studies are needed to study ATN as a possible renal manifestation of these next-generation products to raise awareness among the public.

Single-cell RNA-sequencing of BK polyomavirus replication in primary human renal proximal tubular epithelial cells identifies specific transcriptome signatures and a novel mitochondrial stress pattern.

BK polyomavirus (BKPyV) contributes to premature renal failure in 10%-20% of kidney transplant recipients. Current treatment relies on reducing immunosuppression to regain BKPyV-specific immune control. Subsequently, declining allograft function may result from persisting viral cytopathology, BKPyV-specific immune reconstitution, or alloimmunity/rejection, all being poorly distinguishable by current histological or molecular approaches. To reduce the complexity encountered in BKPyV-replicating kidneys, we analyzed differentially expressed genes (DEGs) in primary human renal proximal tubular epithelial cells at 24 and 48 h post-infection (hpi) using single-cell RNA-sequencing (10x-Genomics-3´ kit). At 24 hpi, viral transcript reads predominantly mapped to the early viral gene region (EVGR) and shifted to >100-fold higher late viral gene region (LVGR) levels at 48 hpi, matching the sequential bi-directional viral protein expression from the circular double-stranded BKPyV-DNA genome. Besides expected coverage "hills" at viral 3´-poly-A sites, unexpected "spike" and "pulse" reads resulted from off-target TSO priming. "Spike" and "pulse" patterns were rare for the mostly unidirectional reads mapping to the circular mitochondrial genome. Bioinformatic curation removed "spikes" and "pulses" and reclassified 10% of DEGs in renal proximal tubular epithelial cells (RPTECs). Up-regulated gene ontologies included S and G2/M phase, double-stranded DNA repair, proximal tubulopathy, and renal tubular dysfunction, whereas allograft rejection, antigen presentation, innate immunity, translation, and autophagy were down-regulated. BKPyV-LVGR expression induced a novel mitochondrial cell stress pattern consisting of discordant up-regulation and down-regulation of mitochondria-encoded and nucleus-encoded mitochondrial genes, respectively. We explored which top-scoring gene sets of late-phase BKPyV-replicating RPTECs can identify BKPyV-associated nephropathy in kidney transplant biopsies. The results should facilitate distinguishing BKPyV-associated pathology from other entities in kidney transplant biopsies.IMPORTANCEBK polyomavirus (BKPyV) infects more than 90% of the general population and then persists in the reno-urinary tract. Subsequently, low-level urinary shedding is seen in 10% of healthy BKPyV-seropositive persons, indicating that BKPyV replication occurs despite the presence of virus-specific cellular and humoral immunity. Notably, transplantation of donor kidneys with low-level BKPyV replication is a risk factor for progression to high-level BKPyV viruria, new-onset BKPyV-DNAemia and biopsy-proven BKPyV nephropathy. Here, we identify a short list of robust up- and down-regulated nucleus-encoded differentially expressed genes potentially allowing to discriminate viral from allograft immune damage. By carefully curating viral and mitochondrial transcriptomes, we identify a novel virus-associated mitochondrial stress pattern of up-regulated mitochondria-encoded and down-regulated nucleus-encoded mitochondrial transcripts which heralds the BKPyV-agnoprotein-mediated immune escape by breakdown of the mitochondrial membrane potential and network and mitophagy. The results may prove useful when assessing the role of BKPyV replication in kidney transplant patients with suspected acute rejection and/or BKPyV nephropathy.

Quantifying measurement uncertainty in renal transplant biopsy assessment.

Renal transplant biopsies provide insights into graft health and support decision making. The current evidence on links between biopsy scores and transplant outcomes suggests there may be numerous factors affecting biopsy scores. Here we adopt measurement science approach to investigate the sources of uncertainty in biopsy assessment and suggest techniques to improve its robustness. Histological assessments, Remuzzi scores, biopsy processing and clinical variables are obtained from 144 repeat biopsies originating from 16 deceased-donor kidneys. We conducted sensitivity analysis to find the morphometric features with highest discriminating power and studied the dependencies of these features on biopsy and stain type. The analysis results formed a basis for recommendations on reducing the assessment variability. Most morphometric variables are influenced by the biopsy and stain types. The variables with the highest discriminatory power are sclerotic glomeruli counts, healthy glomeruli counts per unit area, percentages of interstitial fibrosis and tubular atrophy as well as diameter and lumen of the worst artery. A revised glomeruli adequacy score is proposed to improve the robustness of the glomeruli statistics, whereby a minimum of 104 µm2 of cortex tissue is recommended to keep type 1 and type 2 error probabilities below 0.15 and 0.2. The findings are transferable to several biopsy scoring systems. We hope that this work will help practitioners to understand the sources of statistical uncertainty and improve the utility of renal biopsy.

Active immunologic participation and metabolic shutdown of kidney structural cells during kidney transplant rejection

Contrary to immune cells, the response of the kidney structural cells in rejection is less established. We performed single-cell RNA sequencing of 18 kidney transplant biopsies from 14 recipients. Single-cell RNA sequencing identified cells from the major compartments of the kidney, next to infiltrated immune cells. Endothelial cells from the glomerulus, peritubular capillaries, and vasa recta showed upregulation of class I and II human leukocyte antigen genes, adhesion molecules, cytokines, and chemokines, suggesting active participation in the alloimmune process, with compartment-specific differences. Epithelial cells including proximal tubular, loop of Henle, and collecting duct cells, also showed increased expression of immune genes. Strikingly, in proximal tubule cells, a strong downregulation of energy metabolism upon inflammation was observed. There was a large overlap between the cell-specific expression changes upon alloimmune inflammation and those observed in 2 large microarray biopsy cohorts. In conclusion, the kidney structural cells, being the main target of the alloimmune process, appear to actively contribute herein, enhancing the damaging effects of the infiltrating immune cells. In epithelial cells, a profound shutdown of metabolism was seen upon inflammation, which is associated with poor kidney function. These observations highlight the critical role of the graft in triggering and sustaining rejection after transplantation.

Safety and Diagnostic Yield of Kidney Transplant Biopsies in an Interventional Nephrology Program: A Retrospective Single-Center Study

Safety and Diagnostic Yield of Kidney Transplant Biopsies in an Interventional Nephrology Program: A Retrospective Single-Center Study

Accurate Visualization of C4d Complement Fragment in Immunohistochemistry by C-Terminal Linear Neoepitope-Specific Antibodies

C4d is the end degradation product of activated complement component C4b that appears during the early steps of the classical and lectin complement pathways. Within the primary sequence of C4d, there is a reactive thioester group that binds covalently to nearby surfaces, thus labeling the locations of complement activation. This feature makes C4d a target for immunohistochemical staining aimed to aid the diagnosis of, among others, the antibody-mediated rejection of transplanted organs, membranous glomerulonephritis, bullous pemphigoid, or inflammatory myopathies. However, the credibility of C4d immunostaining is debatable, as a high background in surrounding tissues and body fluids and diffused patterns of deposits in target structures are experienced with some of the available anti-C4d antibodies. Herein, we present an improved version of a rabbit anti-C4d antibody, originally raised against the C-terminal linear neoepitope of this complement fragment. Minor cross-reactivity with C4b and native C4 proteins, measured by ELISAs, as well as relatively low concentrations necessary for obtaining a specific signal in immunohistochemical analyses of formalin-fixed paraffin-embedded material, makes the improved antibody superior to commercially available rabbit monoclonal anti-C4d antibody SP91 dedicated to ex vivo diagnostics, as demonstrated by the staining of a panel of kidney transplant biopsies.

Early and Late Microvascular Inflammation Have Differing Etiological Causes and Clinical Expression.

Microvascular inflammation (MVI) is an important pathological feature of antibody-mediated rejection (AMR). How posttransplant time affects its clinicopathological expression is little understood. This retrospective, single-center study screened 3398 kidney transplant biopsies and dichotomized 202 MVI ≥ 2 (Banff glomerulitis + peritubular capillaritis ≥ 2) samples by 9-mo median incidence time for comparison. The prevalence of MVI ≥ 2 was 12.4% in transplant kidneys, which failed more frequently than propensity-matched normal controls (n = 202; P < 0.001). Epidemiological risk factors for early MVI ≥ 2 were delayed graft function, prior AMR, and circulating donor-specific antibodies (DSAs+). Prior recipient sensitization occurred in 72.3%. Early MVI ≥ 2 was classified AMR in 65.3% and cellular rejection in 34.7%, and demonstrated excellent functional recovery and graft survival comparable to normal control kidneys. Late MVI ≥ 2 was predicted by younger (18 = 29 y) age, female recipient, living-donation, prior methylprednisolone, cyclosporine (versus tacrolimus, levels <5 ng/mL), absent antiproliferative therapy, and DSA+ using multivariable epidemiological modeling. Nonadherence caused 49.5%, with iatrogenic minimization responsible for 47.5%, usually for recipient infection. Late MVI ≥ 2 was because of AMR in 93.1%, and characterized by greater interstitial fibrosis, tubular atrophy, complement degradation split-product 4d (C4d) staining of peritubular capillaries+, endothelial C4d staining of glomerular capillaries+, transplant glomerulopathy and vasculopathy scores, DSA strength, and graft failure than early MVI ≥ 2 or normal transplant kidneys. Death-censored graft survival in 149 unique MVI ≥ 2 kidneys was independently determined by nonadherence, serum creatinine, proteinuria, DSA+, Banff C4d staining of peritubular capillaries+, and chronic interstitial fibrosis scores. MVI score and time lost significance using multivariable Cox regression. The changing expression of MVI ≥ 2 over time is best explained by differences in underimmunosuppression and microvascular injury from AMR impacting allograft function and survival.

Tubulointerstitial nephropathy is the predominant finding in men in a review of more than 3000 renal biopsies over a 10-year period from Sri Lanka

BackgroundChronic kidney disease (CKD) is a significant clinical challenge in Sri Lanka. The present study presents histopathological diagnoses from native renal biopsies in Kandy District, 2011–2020.MethodsReports of 5,014 renal biopsies principally performed at Kandy Teaching Hospital over 2011–2020 were reviewed. After exclusions for post-kidney transplant biopsies (1,572) and those without evident pathology (347), 3,095 biopsies were included. The predominant histopathological entities were grouped and categorised according to diagnosis and stratified by age and sex.ResultsThe main histopathological entities (all biopsies) were tubulointerstitial nephropathy (TIN) 25% (n = 760), glomerulonephritis (GN) 15% (467), lupus nephropathy 14% (429), focal segmental glomerular sclerosis (FSGS) 10% (297), and IgA nephropathy (IgAN) 8% (242). For adult women ≥ 15 years, the main histopathological entities were lupus nephropathy 24% (325), TIN 17% (228), and GN 16% (217). For adult men ≥ 15 years, the main histopathological entities were TIN 34% (449), GN 14% (180), and IgAN 10% (125). The proportion of TIN in the present study was higher than international studies of a similar size.ConclusionThis is the largest study of renal biopsies reported from Sri Lanka to date. TIN was the most common diagnosis in adults ≥ 15 years at 25%. Notable sex differences showed TIN was the most common histopathology in men (34%) but not in women (17%). No previously published similar study of this size has found TIN as the predominant diagnosis amongst renal biopsies in men. Further research is required into the possible causes of these observations in Sri Lanka.Clinical Trial Number.Not applicable.

Incidence, Nature and Natural History of Additional Histological Findings in Preimplantation and Implantation Kidney Transplant Biopsies.

The quality assurance provided by preimplantation biopsy quantification of chronic damage may allow greater use of kidneys from expanded criteria donors, and thereby expand the deceased donor pool. Preimplantation biopsy may, however, identify additional acute or chronic pathologies not considered in the scoring of chronic damage, and these may influence the decision to implant or discard the kidney. This single-centre retrospective cohort study of a contemporary UK donor population systematically characterised the nature of additional findings in 1,046 preimplantation and implantation biopsies over an eight-year period. A diverse range of findings were identified in 111/1,046 (11%) organs; most frequently diabetic glomerulopathy, focal segmental glomerulosclerosis, (micro)thrombi, neutrophil casts, and immunoglobulin/complement staining. Seventy (63%) of these were transplanted, with subsequent biopsy in 41 (58%) cases confirming that 80% of the initial acute changes had spontaneously resolved, while there was no progression of diabetic glomerulopathy, and the lesions of focal segmental glomerulosclerosis were not identified. Over 75% of assessable grafts with additional histological findings at the time of transplant showed adequate function at one-year following transplant. In conclusion, most histological abnormalities that may be identified in addition to chronic scarring in preimplantation kidney biopsies would not preclude transplantation nor predict poor graft function.

Clinical analysis of seven cases of primary hyperoxaluria type 1

We retrospectively analyzed the clinical data of seven patients (four men and three women) with primary hyperoxaluria (PH) type 1 (PH1) in the Department of Nephrology of Zhongda Hospital, Southeast University from January 2018 to October 2023. The mean age at disease onset was 32.1 (range: 26-42) years. The mean age at diagnosis was 40.6 (range: 28-51) years. All patients initially had kidney stones, and three patients were found to have renal insufficiency at the time of disease onset. Among them, two patients underwent hemodialysis immediately. Symptoms at the first visit included bone pain (n=7), joint pain or deformity (n=5), fatigue (n=5), hypotension (n=3), and subcutaneous nodules (n=2). Four patients had a family history of PH. All patients had varying degrees of anemia (60-114 g/L), significant hypoalbuminemia (16.5-32.1 g/L), and hypercoagulable state (D-dimer: 2 230-12 781 μg/L). Seven patients received maintenance hemodialysis; their mean age was 37.7 (range: 26-50) years. The mean duration from disease onset to hemodialysis was 5.6 (range: 0-20) years. Five patients repeatedly experienced dialysis access dysfunction. Three patients underwent kidney transplantation before a diagnosis was made, and all transplanted kidneys lost function due to oxalate deposition. The mean follow-up duration was 14.43 (range: 4-38) months. Unfortunately, one patient died. All seven patients underwent computed tomography of the abdomen. All patients suffered skeletal abnormalities, bilateral nephrolithiasis, and nephrocalcinosis. Six patients carried AGXT gene mutations, including four compound heterozygous mutations and two pure homozygous mutations.The mutation sites included: c.823-824dup.AG (p.S275Rfs*38)(exon 8), c.815-816ins.GA (p.S275Rfs*38)(exon 8), c.595G>A (p.G199S) (exon 5), c.32C>G (p.P11R) (exon 1), and c.638C>T (p.A213V)(exon 6). According to the American College of Medical Genetics and Genomics guidelines, two loci were identified as likely pathogenic variants, seven were identified as pathogenic variants, and one locus was identified as having uncertain significance. In addition, patients 1 and 4 underwent skin biopsy, patient 2 underwent renal transplant biopsy, and patient 3 underwent bone marrow biopsy. Interestingly, significant oxalate deposition was found in the tissues. Therefore, PH1 is a rare autosomal recessive inherited disease. This study not only enhanced the understanding of the clinical characteristics of PH1 patients but also had great significance in early diagnosis and treatment of the disease.

Interstitial Foci Expression of Indoleamine 2,3-Dioxygenase 1: A Potential Biomarker for Kidney Transplant Rejection.

(1) Background: Kidney transplantation is the best therapy for patients with end-stage renal disease, but the risk of rejection complicates it. Indoleamine 2,3-dioxygenase 1 (IDO1), an enzyme involved in immune response modulation, has been suggested to play a role in transplant immunological injury. The aim of the study was to explore the expression of IDO1 in the interstitial foci of transplanted kidneys and its potential association with rejection episodes. (2) Methods: This retrospective study analysed kidney transplant biopsies from 121 patients, focusing on IDO1 expression in interstitial foci. Immunohistochemistry was used to detect IDO1, and patients were categorised based on IDO1 presence (IDO1-IF positive or negative). The incidence of rejection was compared between these groups. (3) Results: Patients with IDO1 expression in interstitial foci (IDO1-IF(+)) exhibited higher incidences of rejection 46/80 (57.5%) vs. 10/41 (24.34%) patients compared to IDO1-IF(-) patients, which was statistically significant with p = 0.0005. The analysis of antibody-mediated rejection showed that IDO1-IF(+) patients developed AMR at 12/80 (15%), while only 1 IDO1-IF(-) negative patient did (2,44%), with p = 0.035. T-cell-mediated rejection was also more common in IDO1-IF(+) patients 43/80 (53.75%) than in IDO1-IF(-) patients 7/41 (17.07%), with p = 0.0001. (4) Conclusions: IDO1 expression in interstitial foci of renal transplant biopsies is associated with a higher incidence of rejection, suggesting that IDO1 could serve as a potential biomarker for transplant rejection. These findings highlight the importance of IDO1 in immune regulation and its potential utility in improving the management of kidney transplant recipients.

The Effects of COVID-19 in Kidney Transplantation: Evidence From Tissue Pathology.

The biological effects of SARS-CoV-2 infection in transplanted kidneys are uncertain with little pathological information. This single-center, prospective observational study evaluated kidney transplant biopsies from recipients of deceased donors with COVID-19, current recipients contracting SARS-CoV-2 Omicron variant in 2022, against prior BK virus (BKV) infection and uninfected (without SARS-CoV-2 or BKV) samples, as respective positive and negative comparators (n = 503 samples). We demonstrated nonvirus tubular injury in implanted tissue from infected donors and prevalent recipients with mild acute COVID-19 and acute kidney injury, excluding direct viral infection as a cause of kidney damage. COVID particles were absent in 4116 ultrastructural images of 295 renal tubules from 4 patients with acute COVID-19. No viral cytopathic effect, viral allograft nephropathy, or SARS-CoV-2 RNA was detected in acute tissues, nor in 128 sequential samples from infected donors or recipients with COVID-19. Following recipient COVID-19 (mean 16.8 ± 12.0 wk post-infection), the biopsy-prevalence of rejection was 33.0% (n = 100 biopsies) versus 13.4% for contemporaneous uninfected controls (n = 337; P < 0.001). Prior COVID-19 was an independent risk factor for incident rejection using multivariable generalized estimating equation adjusted for competing risks (odds ratio, 2.195; 95% confidence interval, 1.189-4.052; P = 0.012). Landmark and matched-pair analyses confirmed an association of SARS-CoV-2 with subsequent transplant rejection, with a similar pattern following BKV infection. Transplantation from COVID-19+ deceased donors yielded good recipient outcomes without evidence of viral tissue transmission. Acute kidney injury during COVID-19 was mediated by archetypical tubular injury and infection correlated with an increased risk of subsequent rejection.

Antibody Mediated Rejection and T-cell Mediated Rejection Molecular Signatures Using Next-Generation Sequencing in Kidney Transplant Biopsies.

Recently, interest in transcriptomic assessment of kidney biopsies has been growing. This study investigates the use of NGS to identify gene expression changes and analyse the pathways involved in rejection. An Illumina bulk RNA sequencing on the polyadenylated RNA of 770 kidney biopsies was conducted. Differentially-expressed genes (DEGs) were determined for AMR and TCMR using DESeq2. Genes were segregated according to their previous descriptions in known panels (microarray or the Banff Human Organ Transplant (B-HOT) panel) to obtain NGS-specific genes. Pathway enrichment analysis was performed using the Reactome and Kyoto Encyclopaedia of Genes and Genomes (KEGG) public repositories. The differential gene expression using NGS analysis identified 6,141 and 8,478 transcripts associated with AMR and TCMR. While most of the genes identified were included in the microarray and the B-HOT panels, NGS analysis identified 603 (9.8%) and 1,186 (14%) new specific genes. Pathways analysis showed that the B-HOT panel was associated with the main immunological processes involved during AMR and TCMR. The microarrays specifically integrated metabolic functions and cell cycle progression processes. Novel NGS-specific based transcripts associated with AMR and TCMR were discovered, which might represent a novel source of targets for drug designing and repurposing.

IDO+ Endothelial Cells in Glomeruli of Kidney Transplantation Patients With Glomerulitis.

Kidney transplantation is the preferred treatment option for patients with end-stage renal disease. However, long-term graft survival remains a challenge. The enzyme indoleamine 2,3 dioxygenase (IDO) has been reported to have immunomodulatory effects with IDO transcripts being elevated in both antibody-mediated rejection and T cell-mediated rejection. A metal-conjugated antibody panel for the staining of kidney biopsies was developed, allowing the visualization of 41 structural and immune markers on a single tissue slide to gain in-depth insight into the composition and localization of the immune cell compartment. Staining was applied to week 4 and 24 protocol biopsies of 49 patients as well as on 15 indication biopsies of the TRITON study and 4 additional transplantation biopsies with glomerulitis. A highly distinctive and specific glomerular IDO expression was observed in biopsies from 3 of 49 patients in imaging mass cytometry. Immunohistochemistry confirmed IDO expression in glomeruli of 10 of 10 cases with glomerulitis. IDO was found to be expressed by CD31+ glomerular endothelial cells, accompanied by the presence of granzyme-B+Tbet+CD7+CD45RA+ natural killer cells and CD68+ macrophages. Furthermore, a proportion of both the immune cells and endothelial cells expressed Ki-67, indicative of cell proliferation, which was not observed in control glomeruli. Our results show glomerular IDO expression in transplanted kidneys with glomerulitis, which is accompanied by increased numbers of natural killer cells and macrophages and likely reflects local immune activation.

Changes in Numbers of Glomerular Macrophages Between Two Consecutive Biopsies and the Association With Renal Transplant Graft Survival.

Macrophages are involved in kidney transplants. The aim of the study was to investigate if changes exist in the levels of glomerular macrophage index (GMI) between two consecutive kidney transplant biopsies, and if so to determine their potential impact on graft survival. Two consecutive biopsies were performed on the same renal graft in 623 patients. GMI was categorized into three GMI classes: ≤1.8 Low, 1.9-4.5 Medium, and ≥4.6 High. This division yielded nine possible switches between the first and second biopsies (Low-Low, Low-Medium, etc.). Cox-regressions were used and hazard ratios (HR) with 95% confidence interval (CI) are presented. The worst graft survival was observed in the High-High group, and the best graft survival was observed in the Low-Low and High-Low groups. Compared to the High-High group, a reduction of risk was observed in nearly all other decreasing groups (reductions between 65% and 80% of graft loss). After adjustment for covariates, the risk for graft-loss was lower in the Low-Low (HR = 0.24, CI 0.13-0.46), Low-Medium (HR = 0.25, CI 0.11-0.55), Medium-Low (HR = 0.29, CI 0.11-0.77), and the High-Low GMI (HR = 0.31, CI 0.10-0.98) groups compared to the High-High group as the reference. GMI may change dynamically, and the latest finding is of most prognostic importance. GMI should be considered in all evaluations of biopsy findings since high or increasing GMI levels are associated with shorter graft survival. Future studies need to consider therapeutic strategies to lower or maintain a low GMI. A high GMI besides a vague histological finding should be considered as a warning sign requiring more frequent clinical follow up.

#1985 Tocilizumab in chronic active antibody-mediated rejection: a randomized controlled open-label multi-center trial (INTERCEPT study)

Abstract Background and Aims Chronic active antibody mediated graft rejection (caAMR) caused by immune-mediated injury is the leading cause of long-term graft loss in kidney transplant recipients. Nevertheless, no effective treatment exists for caAMR to date, highlighting a great unmet need for evaluation of novel treatment options. Recently, tocilizumab (TCZ), a monoclonal antibody directed against the human interleukin-6 (IL-6) receptor, by antagonizing IL-6-triggered inflammation and B cell/plasma cell-driven immune activation, has shown promise in the treatment of caAMR. However, TCZ has not been assessed systematically in a randomized study. The INTERCEPT study aims to evaluate the efficacy of addition of TCZ to standard of treatment (SOC) as compared to SOC alone in reducing the decline of graft function from baseline at 24 months after start of treatment as assessed by estimated glomerular filtration rate (eGFR) in kidney transplant recipients with caAMR. Method This ongoing randomized controlled open-label multi-center investigator-initiated trial plans to include a total of 50 adult kidney transplant recipients with biopsy-proven caAMR using Banff 2019 criteria at least 12 months after transplantation and eGFR ≥20 ml/min/1.73 m2. Any subject who underwent kidney transplantation in Sweden or is currently living in Sweden and being followed up at any of the Swedish transplant centers or their affiliated regional centers can be considered for participation. Participants are randomized to receive either TCZ (n=25) added to our standard of care (SOC) maintenance treatment or SOC alone (n=25) for a period of 24 months and followed for additional 12 months after cessation of the study medication. After the inclusion transplant biopsy at baseline, protocol biopsies are performed at 12 and 24 months. The study design flow-chart is shown in the Figure. The primary endpoint is the slope of eGFR at 24 months after start of treatment. The eGFR is assessed using Modification of Diet in Renal Disease (MDRD) formula. The secondary endpoints include assessment of the following at 12, 24 and 36 months: composite risk score iBox, safety, evolution and characteristics of donor-specific antibodies (DSA), graft histology, proteinuria, kidney function assessed by measured GFR (mGFR), patient- and death-censored graft survival, patient-reported outcomes that include transplant-specific well-being, adherence to immunosuppressive medications and perceived threat of the risk of graft rejection. Results We expect that addition of TCZ to SOC will slow down the decline of graft function in kidney transplant recipients with caAMR. Conclusion Currently there is no effective treatment for caAMR. Based on the hypothesis that inhibition of IL-6 by TCZ will reduce antibody production and reduce antibody-mediated damage, our randomized trial has a potential to provide evidence for a novel treatment strategy for caAMR. If TCZ is found to be effective, our study has the potential to improve long-term graft survival and quality of life, reduce morbidity and mortality in kidney transplant patients as well as lower the health care costs.

#2566 Clinicopathological and molecular characteristics of plasma cell rich rejection in renal transplant biopsies

Abstract Background and Aims Plasma cell rich rejection (PCRR) is an uncommon, ill-defined type of renal allograft rejection in the literature considered as a subtype of T cell-mediated rejection (TCMR). PCRR has poorer clinical outcome and is often refractory to classic immunosuppressive therapy. Our study analyzed In this study, we analyzed PCRR in a large cohort of patients who underwent renal transplantation at the Amsterdam UMC. Our objective was to study clinical, histological and molecular signatures of PCRR in comparison to other types of rejection, especially late presentations of TCMR and ABMR. We hypothesized PCRR could be a specific subtype of allograft rejection with different molecular and cellular characteristics compared to (late) TCMR and ABMR with differences in clinical outcome. Method We retrospectively scored and reclassified the last known biopsy of 263 renal transplant recipients, morphologically classified as rejection according to the 2019 Banff classification. mRNA expression analysis was performed using the Nanostring B-HOT panel on a subset of cases. PCRR was compared to (late) TCMR, ABMR and mixed rejection for renal function follow-up and graft survival. Results mRNA analysis revealed uniquely expressed genes in PCRR including LOX, CPA3, IL4, IL17F, and MMP12. PCRR is enriched for genes related to mast cells, memory B- and T-cells and transcripts involved in NK cells and allograft fibrosis with heterogeneity in gene expression in biopsies with PCRR. PCRR might be a late event compared to late TCMR and ABMR, with a higher degree of total inflammation and fibrosis. Graft survival and kidney function was similar to late TCMR and ABMR during a 5-year follow-up period after renal biopsy Conclusion PCRR represents a distinct late-onset stage of inflammation displaying diverse gene expression patterns, with presence of mainly mast cells, NK cells and transcripts involved in renal allograft fibrosis. Such insights could enable a better and more tailored treatment for these patients and ultimately enhance long-term graft survival for patients affected by PCRR. Clinical outcomes in patients with PCRR appeared similar to late TCMR and ABMR.

#537 Proliferative glomerulonephritis with monoclonal immunoglobulin deposits: effects of pretreatment on posttransplant outcomes

Abstract Background and Aims Proliferative glomerulonephritis with monoclonal immunoglobulin deposits (PGNMID) has a poor renal prognosis and a high rate of recurrence after kidney transplantation, up to 90%, typically within the first-year post-transplant. Nearly 50% of patients lose their renal allograft within 3 years of recurrence. It has been suggested that pretreatment therapy (with B cell depleting or plasma-cell directed therapy) can help prevent recurrence or modulate the course of disease post-transplant. The aim of our study is to evaluate the effect of pretreatment therapy given within 6 months prior to kidney transplant on outcomes including PGNMID recurrence and all cause graft loss. Method Between 1996 and 2022, a total of 28 patients with biopsy-proven PGNMID as the cause of end stage kidney disease received a total of 31 kidney transplants. All patients had at least one year of follow-up data, for all but one patient this included surveillance kidney transplant biopsies completed at our center. Recurrence was defined histologically by immunofluorescence finding of monoclonal immunoglobulin deposits, independent of other histological or clinical parameters. The incidence of recurrence and all cause graft loss were analyzed by Kaplan-Meier plots. Results Median age of patients at transplant was 57 years, 87% were white, 65% were male, 74% received living donor transplant, 59% received thymoglobulin induction, 81% received maintenance with tacrolimus, mycophenolate, and prednisone. Median follow up time was 80 months. A total of 9 patients received pretreatment within 6 months of transplant, 8 received Rituximab and one received Daratumumab. The pretreatment group was generally younger at transplant (53 years vs 57 years) and had a shorter median follow up time (65 months vs 96.5 months), but neither were statistically significant. Overall recurrence rate was 78% for pretreatment and 91% for control, though difference in time to recurrence was not statistically significant (Fig. 1). The difference in all cause graft loss between groups was not statistically significant, however, none in the pretreatment group had graft loss by 60 months (Fig. 2). One patient who received daratumumab had no recurrence by 1 year post-transplant. Conclusion In kidney transplant recipients with PGNMID, pretreatment (primarily with rituximab) may be modulating the post-transplant course of disease. Further studies are needed to determine the extent of this effect and if plasma-cell directed vs B-cell directed therapy may be superior.

#1130 Biopsy-based transcript diagnostics identify early antibody-mediated rejection phenotypes in biopsies with subclinical donor-specific antibodies

Abstract Background and Aims Biopsy based transcript diagnostics using the molecular microscope diagnostic system (MMDx) have the potential to add diagnostic clarification in the antibody mediated rejection (AMR) continuum. While de novo donor specific antibody (dnDSA) or preformed DSA accompanied by allograft function deterioration are accepted indications for allograft biopsy with therapeutic implications, management strategy for subclinical DSA lacks consensus. MMDx might show clinical utility in this controverse scenario by helping with therapeutic decisions. Method In this single-center cohort of 326 indication kidney transplant biopsies assessed by histology and MMDx at the University Hospital Zurich, we analyzed 58 cases with subclinical DSA as biopsy indication. Cases with (n = 44) and without (n = 14) histopathologic features of AMR according to Banff 2022 were compared for molecular AMR rejection transcripts. Results When biopsied for subclinical DSA, all 14 cases without histopathologic features of AMR showed absence of molecular AMR. In contrast, molecular AMR was detected in 6/19 (32%) cases with histopathologic diagnosis of AMR and 2/25 (8%) with probable AMR (p = 0.059), Fig. 1. Microvascular inflammation below threshold with C4d negativity was significantly associated with absence of molecular AMR (p &amp;lt; 0.0001). Higher number of calculated epitope mismatch load for HLA class II as measured by PIRCHE II Score was significantly associated with molecular AMR (p = 0.01). In cases with molecular AMR, early AMR scores predominated (R4 vs R6, 0.0002 and R4 vs R5 0.051). Conclusion No molecular AMR was observed in absence of histopathologic AMR. Yet, in cases with histopathologic features of AMR, only 8/44 showed molecular AMR. Predominance of early AMR rejection phenotype in cases with molecular AMR transcripts allows hope for MMDx to identify treatment amenable cases with subclinical DSA. Discrepant cases call for re-biopsy to guide therapeutic decisions.

Computational Pathology Assessments of Cardiac Stromal Remodeling: Clinical Correlates and Prognostic Implications in Heart Transplantation.

Both overt and indolent inflammatory insults in heart transplantation can accelerate pathologic cardiac remodeling, but there are few tools for monitoring the speed and severity of remodeling over time. To address this need, we developed an automated computational pathology system to measure pathologic remodeling in transplant biopsy samples in a large, retrospective cohort of n=2167 digitized heart transplant biopsy slides. Biopsy images were analyzed to identify the pathologic stromal changes associated with future allograft loss or advanced allograft vasculopathy. Biopsy images were then analyzed to assess which historical allo-inflammatory events drive progression of these pathologic stromal changes over time in serial biopsy samples. The top-5 features of pathologic stromal remodeling most strongly associated with adverse outcomes were also strongly associated with histories of both overt and indolent inflammatory events. Our findings identify previously unappreciated subgroups of higher- and lower-risk transplant patients, and highlight the translational potential of digital pathology analysis.