Allograft Biopsies Research Articles

The Immunohistochemical Expression of the Von Willebrand Factor: A Potential Tool to Predict Kidney Allograft Outcomes.

Few reports assessed endothelial activation biomarkers in kidney allograft biopsies using immunohistochemistry. This retrospective cohort study evaluated the association between posttransplant outcomes and the immunohistochemistry expression of Caveolin-1, Von Willebrand Factor (Vwf), and T-Cadherin in for-cause biopsies diagnosed as interstitial fibrosis and tubular atrophy of unknown etiology. Samples with antibody-mediated changes were excluded. The patients were followed for 3 years after the biopsy or until graft loss/death. Seventy-one (71) samples from 66 patients were included. Eighteen (25.4%) patients lost their grafts, mainly due to chronic rejection (33.3%). Caveolin-1 and T-Cadherin were not associated with graft loss. Vwf had good accuracy in predicting graft failure (AUC 0.637, 95% CI 0.486 to 0.788 P =0.101). The presence of more than 10% of Vwf positivity in the microvasculature (Vwf >10%) was associated with reduced death-censored graft survival (58.2% vs. 85.4% P =0.006), and this result was also observed in the subgroup presenting mild interstitial fibrosis (ci=1) (65.7% vs. 88.6% P =0.033). The multivariate analysis showed that Vwf >10% was an independent risk factor for graft loss (HR=2.88, 95% CI 1.03 to 8.02 P =0.043). In conclusion, Vwf might be an additional tool to predict allograft outcomes in kidney transplant recipients with interstitial fibrosis and tubular atrophy of unknown etiology, probably reflecting immune endothelial activation.

The cellular landscape of the normal kidney allograft: Main players balancing the alloimmune response.

Despite recent advances made in short-term outcomes; minimal improvements have been observed in long-term kidney transplantation outcomes. Due to an imbalance between organ transplant availability and patient waiting list, expanding kidney allograft longevity is a critical need in the field. Prior studies have either focused on early ischemic and immunological conditions affecting kidney allografts (e.g., delayed graft function, acute rejection) or late stage chronic injury when interventions are no longer feasible. However, studies characterizing kidney allografts with normal function by its cellular distribution, cell-cell interactions, and associated molecular pathways are lacking. Herein, we used single nuclei RNA-sequencing to uncover the cellular landscape and transcriptome of the normal kidney allograft. We profiled 40,950 nuclei from seven human kidney biopsies (normal native, N = 3; normal allograft, N = 4); normal allograft protocol biopsies were collected ≥15-months post-transplant. A total of 17 distinct cell clusters were identified with proximal tubules (25.70 and 21.01%), distal tubules (15.22 and 18.20%), and endothelial cells (EC) (4.26 and 9.94%) constituting the major cell populations of normal native and normal allograft kidneys, respectively. A large proportion of cycling cells from normal native kidneys were in G1-phase (43.96%) whereas cells from normal allograft were predominantly in S-phase (32.69%). This result suggests that transcriptional differences between normal native and normal allograft biopsies are dependent on the new host environment, immunosuppression, and injury-affliction. In the normal allograft, EC-specific genes upregulated metabolism, the immune response, and cellular growth, emphasizing their role in maintaining homeostasis during the ongoing alloreactive stress response. Immune cells, including B (2.81%), macrophages (24.96%), monocytes (15.29%), natural killer (NK) (12.83%), neutrophils (8.44%), and T cells (14.41%, were increased in normal allografts despite lack of histological or clinical evidence of acute rejection. Phenotypic characterization of immune cell markers supported lymphocyte activation and proinflammatory cytokines signaling pathways (i.e., IL-15, IL-32). The activation of B, NK, and T cells reveals potential immune cells underlying subclinical inflammation and repair. These single nuclei analyses provide novel insights into kidney and immune cell associated signaling pathways that portray kidney grafts with normal allograft function beyond 2-years post-transplant, revealing a novel perspective in understanding long-term allograft graft survival.

Making the Cut: Parathyroidectomy Before or After Kidney Transplantation?

Hyperparathyroidism is common in patients with end-stage kidney disease and may persist after kidney transplantation (KT). Parathyroidectomy (PTx) is curative, but whether PTx should be performed before or after KT remains controversial. There is concern that PTx can adversely affect renal allograft function if performed post-KT and result in persistent hypocalcemia. This study evaluated outcomes and postoperative complications of PTx before and after KT at our institution. We performed a retrospective review of patients at our center (1/2012-2/2019) who had PTx either pre-KT or post-KT. Data on patient demographics, surgical outcomes, and postoperative complications of PTx were collected. Ninety-eight patients were included in this study, with 23 patients undergoing PTx before KT and 75 after KT. The length of follow-up after KT was 67.7 ± 25.5months. In post-KT PTx patients, 30-day allograft function was unchanged after PTx. Calcium oxalate and phosphate crystals were less common on allograft biopsies in pre-KT PTx patients (10.0% vs. 34.8%, p = 0.038). Patients in the pre-KT group required more calcium supplementation after PTx than the post-KT group (p < 0.001). At one-year post-PTx, 17 (19.1%) patients required > 1000mg elemental calcium per day and 7 (7.9%) patients required > 2000mg/day. There was no difference in surgical success or postoperative complications between the two groups. Parathyroidectomy before or after kidney transplantation does not adversely affect allograft function. The incidence of persistent hypocalcemia was low. Parathyroidectomy is safe and effective either before or after kidney transplantation.

S1906 A Rare Cause of Delayed Life-Threatening Hemorrhage in a Patient With Pancreas Transplant

Introduction: Complications of vascular anastomosis usually occur early after pancreas transplant (PTx) and carry a substantial risk of graft loss and mortality. Hemorrhage may result from arterial anastomosis failure, but more commonly from superior mesenteric or splenic vessel ligation. We present a challenging case of GI hemorrhage from a pseudoaneurysm (PA) in the implanted vascular Y graft 7 years after PTx. Case Description/Methods: A 45-year-old male 7 years post simultaneous pancreas-kidney transplant presented with painless rectal bleeding. EGD and Colonoscopy showed blood throughout the duodenum and colon, but the source could not be localized. Push enteroscopy showed blood extending into the proximal jejunum, and no active bleeding source could be identified. CT angiogram showed active extravasation into a small bowel loop near the PTx. Mesenteric angiography with IR revealed active extravasation into jejunum from a PA which had developed off the SMA Y graft anastomosis, eroding into the adjacent duodenal stump. Successful coil embolization was performed. 2 days later, he developed rectal bleeding requiring massive transfusion. Tagged RBC scan and CT angiography could not localize the bleeding source. Emergent mesenteric angiography showed contrast leakage into the anastomotic stump beyond the previously placed coil (Figure A). A covered stent was placed into the right external iliac artery arresting the bleeding into the SMA anastomotic stump (Figure B). Bleeding and shock resolved, and he was discharged with transplant surgery follow-up. Discussion: Pseudoaneurysms, AV fistulae, arterial dissection and stenosis represent rare vascular complications of PTx. PAs, if early, may be the result of surgical technique, infection, severe pancreatitis, or allograft biopsy. Arterial anastomotic PAs can be asymptomatic or present with symptoms signaling impending rupture such as abdominal pain, pulsatile mass, or endocrine failure. Endoscopic localization and management of bleeding from Y graft PAs are challenging due to altered anatomy and brisk bleeding. Multidisciplinary management is essential and often involves endovascular hemostasis with IR, possibly with surgical exploration. Stent placement is usually preferred over embolization due to the ability to deploy with precision while maintaining end-organ perfusion. Although guidelines are lacking, it would be reasonable to consider periodic Doppler US to screen for post-operative PAs for early detection and preventative endovascular treatment.Figure 1.: A. Pre-stent mesenteric angiogram with contrast leaking through coil into SMA anastomotic stump; B. Post stent mesenteric angiogram with no contrast leaking into the SMA anastomotic stump.

Lack of Efficacy and Safety of Eculizumab for Treatment of Antibody-Mediated Rejection Following Renal Transplantation

We evaluated the efficacy and safety of eculizumab in comparison with plasmapheresis and intravenous immunoglobulin therapy in renal transplant recipients diagnosed with antibody-mediated rejection (AMR). This was a multicenter, open-label, prospective, randomized analysis. The patients were randomized by therapy type (eg, eculizumab infusions or standard of care [SOC]: plasmapheresis/intravenous immunoglobulin). The patients (ie, eculizumab arm: 7 patients, SOC arm: 4 patients) were evaluated for the continued presence of donor-specific antibodies (DSAs) and C4d (staining on biopsy), as well as histologic evidence, using repeat renal biopsy after treatment. The allograft biopsies revealed that eculizumab did not prevent the progression to transplant glomerulopathy. Only 2 patients in the SOC arm experienced rejection reversal, and no graft losses occurred in either group. After AMR treatment, the DSA titers generally decreased compared to titers taken at the time of AMR diagnosis. There were no serious adverse effects in the eculizumab arm. Eculizumab alone cannot treat AMR effectively and does not prevent acute AMR from progressing to chronic AMR or transplant glomerulopathy. However, it should be considered as a potential alternative therapy because it may be associated with decreased DSA levels.

Combining donor-derived cell-free DNA and donor specific antibody testing as non-invasive biomarkers for rejection in kidney transplantation

Donor specific anti-HLA antibodies (DSA) and donor-derived cell-free DNA (dd-cfDNA) have lead to substantial progress in the non-invasive monitoring of the renal allograft by being able to detect or rule out subclinical rejection and guide immunosuppressive changes. In this study we sought to analyze the clinical, de novo DSA (dnDSA) and histological determinants of dd-cfDNA levels. The study included a cohort of stable renal function kidney transplant (KT) recipients who underwent anti-HLA dnDSA and dd-cfDNA testing between September 2017-December 2019. Statistical models were constructed to detect association with predictors of dd-cfDNA levels and other clinical characteristics. 171 renal allograft recipients were tested for dd-cfDNA and dnDSA at a median 1.06 years posttransplant (IQR: 0.37–4.63). Median dd-cfDNA was 0.25% (IQR: 0.19–0.51), 18.7% of patients having a dd-cfDNA ≥ 1%. In a multivariate linear regression model the presence of dnDSA MFI ≥ 2500 was the best independent determinant of dd-cfDNA level (p < 0.001). Among patients tested, 54 had concurrent dd-cfDNA determination at the time of an allograft biopsy. dd-cfDNA had an AUC of 0.82 (95% CI 0.69–0.91; p < 0.001) and of 0.96 (95% CI 0.87–0.99) to discriminate any rejection and ABMR, respectively. After multivariate adjustment, the models that included ABMR (R = 0.82, R2 = 0.67, p < 0.001), or ptc (R = 0.79, R2 = 0.63, p < 0.001) showed the best correlation with dd-cfDNA level. We are confirming a strong association of dd-cfDNA with dnDSA and underlying alloimmune-mediated injury in renal allograft recipients in a cohort of patients with unsuspecting clinical characteristics for rejection and excellent allograft function. Our findings support the need for noninvasive biomarker surveillance in KT recipients and we propose that dd-cfDNA may complement dnDSA screening.

216.7: The Immunohistochemical Expression of Endothelial Activation Biomarkers Is a Potential Tool to Predict Kidney Allograft Outcomes

Background and Objective: Few reports assessed biomarkers of endothelial activation in kidney allograft biopsies using immunohistochemistry (IHC). This study aimed to analyze the IHC expression of Caveolin-1 (Cav), Von Willebrand Factor (Vwf), and T-Cadherin (Cad) in for-cause allograft biopsies diagnosed as Interstitial Fibrosis and Tubular Atrophy (IF/TA) of unknown etiology and evaluate its association with graft loss. Methods: This was a retrospective single-center cohort study including kidney biopsies performed in a single center between January 2013 and December 2017, whose histological, laboratory and clinical analysis concluded for IF/TA of unknown etiology. Samples with antibody-mediated changes were excluded. Patients were followed for three years after the biopsy or until graft loss/death. Results: Seventy-one (71) samples from 66 patients were included. Eighteen (25.4%) patients lost their grafts, mainly due to chronic rejection (33.3%). Cav and Cad were not associated with graft loss. Vwf had good accuracy to predict graft failure (AUC 0.637, 95%CI 0.486-0.788, p = 0.101). The presence of more than 10% of Vwf positivity in the microvasculature (Vwf > 10%) was associated with reduced death-censored graft survival (58.2% versus 85.4%, p = 0.006). The multivariate analysis showed that Vwf > 10% was an independent risk factor for graft loss (HR = 2.88, 95%CI 1.03-8.02, p = 0.043). Conclusion: Vwf might be an additional tool to predict allograft outcomes in kidney transplant recipients with IF/TA of unknown etiology, probably reflecting immune endothelial activation.

216.3: Enhanced Histological Yield and Actionable Findings When Biopsy Is Guided by Donor-derived Cell-free DNA (dd-cfDNA)

Introduction: Implementation of molecular biomarkers in kidney transplantation has expanded the amount of information available to clinicians before deciding topursue biopsy. We evaluated how use of donor-derived cell-free DNA testing (dd-cfDNA) impacts histologic biopsy yield. Methods: 1663 kidney transplant (KTx) recipients enrolled in the Kidney allograft Outcomes AlloSure Registry (KOAR, NCT03326076) were followed with dd-cfDNA post-transplant; testing was obtained either as part of a surveillance strategy or for-cause; indications for biopsy and histologic diagnoses were captured. This analysis included only for-cause biopsies. Results: 65 biopsies (from 59 pts) driven by elevated dd-cfDNA levels were compared to 540 biopsies (392 pts) obtained for causes other than elevated dd-cfDNA. Patient age (55 vs 56 years), percent deceased donor (84.75% vs 78.83%), and biopsy timing post-transplant (123 vs 105.5 days) did not differ betweengroups (Table 1). Among dd-cfDNA-guided biopsies, yield was enriched for rejection (48% vs 29%) and included fewer cases of ATI/ATN (12% vs 22%)compared to biopsies obtained for other causes (p<0.05) (Figure 1). Among dd-cfDNA-guided biopsies with rejection (ABMR, TCMR, or Mixed), median dd-cfDNA was 1.46% (IQR: 1.15 - 2.87). Among biopsies not performed due to elevated dd-cfDNA but with paired results available (n = 267), median dd-cfDNA was0.24%. Within this group, patients with rejection had median dd-cfDNA of 0.82% and demonstrated an increase of 133% from the preceding result. Conclusions: Dd-cfDNA surveillance enhances the histologic yield for actionable results in for-cause allograft biopsies. A low dd-cfDNA result can obviate the need for biopsy even in the presence of other clinical factors that are routinely used to guide the biopsy decision.

401.7: Non-Invasive Assessment of Renal Allograft Fibrosis by Shearwave Elastography: A Radiological – Pathological Correlation Analysis.

Background & objectives: Currently, renal allograft 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 non-invasive tool to detect renal allograft fibrosis is needed. This study sought to evaluate the usefulness of SWE in the quantitative measurement of renal allograft fibrosis and to correlate shear-wave sonoelastography measurements with histological findings, resistive index, serum creatinine level and estimated glomerular filtration rate (eGFR) obtained with the CKD-EPI. Methods: A cross-sectional study of 110 renal transplant patients. Patients were classified as having stable allograft and allograft dysfunction based on clinical parameters. Only patients with allograft dysfunction underwent renal allograft biopsy. SWE assessment was done by a single radiologist using the same ultrasound machine to quantify allograft fibrosis in kPa. Renal allograft biopsies were interpreted by the same pathologist according to the 2019 Banff classification. The potential correlation between SWE scores and Banff classification was performed in allograft dysfunction group. The receiver operating characteristic curves were drawn to evaluate the likelihood of differentiation. Results: 66 patients had allograft dysfunction and 44 patients had stable allograft function. Mean parenchymal stiffness measured values in stable allograft and allograft dysfunction were 14.20±4.50 kPa and 16.54±5.42 kPa respectively.Mean parenchymal stiffness showed a significant negative correlation with eGFR (r= -0.317, p= 0.001) and a positive correlation with serum creatinine level (r= 0.256; p= 0.007). There was a significant correlation between the Banff grade of IFTA and the mean SWE score, (p= 0.003).The correlation of resistive index was insignificant when compared to the mean SWE score (r= 0.058, p= 0.547). The area under the curve was higher and significant at IFTA grading ≥ 2 (AUC- 0.801, p < 0.0001) when compared it with IFTA grading 0 (AUC–0.523, p= 0.831) and 1 (AUC-0.551, p= 0.431).The sensitivity was 86% and specificity was 73% in the differentiation of stable graft from graft dysfunction with moderate to severe fibrosis (cut-off value, 16.15 kPa). The sensitivity was 85% and specificity was 67% in the differentiation of graft dysfunction with grade 2/3 IFTA from graft dysfunction with grade 0/1 IFTA (cut-off value, 16 kPa). This means that shear wave elastography effectively detects moderate to severe fibrosis in the allograft. The severity of fibrosis increased with the increase in transplant vintage with statistical significance (p <0.001). Conclusions: Renal allograft parenchymal stiffness quantified by shear-wave elastography showed a significant correlation with allograft fibrosis graded by Banff classification. SWE can differentiate between mild fibrosis and moderate-to-severe fibrosis with high accuracy. The inverse correlation of parenchymal stiffness with eGFR and positive correlation with serum creatinine level show that shear-wave sonoelastography may reflect the functional status of the renal allograft. Therefore, shear wave elastography can be used as a noninvasive tool to assess renal allograft fibrosis and is better than the resistive index. Key words: elastography, SWE, renal allograft biopsy, renal transplant, allograft fibrosis.

235.3: Allograft Vasculopathy in Kidney Transplant Patients: Clinical and Histological Associated Factors

Introduction: Allograft vasculopathy (AV) defined by arterial intimal fibrosis with mononuclear cell inflammation in fibrosis and formation of neointima is one of the main limiting factors in renal graft survival. The etiopathogenesis is multifactorial, including immune mediated mechanism (chronic active or chronic ABMR or mixed ABMR/TCMR), inmune independent factors (e.g.,donnor age) and the endothelial to mesenchymal transition (ETM).The aim of this study is to evaluate the presence of transplant vasculopathy in kidney transplant biopsies, and the risk factors associated. Methods: This retrospective study included 400 allograft biopsies for cause in 294 kidney transplant recipient from 2014 to 2018. All biopsies, with and without transplant vasculopathy, donor, recipient and transplant variables were analyzed. We classified the biopsies in Early (< 3 months) and late(> 3 months).Clinical variables of the recipient were analyzed: age, antiHLA sensitization; from the donor: age, alive -deceased; optimal-marginal and transplant process: TIF, mismatch, delayed graft function (DGF); histological features: rejections, microcirculation injury, C4d. Statistical analysis: Categorical variables are reported as percentages and continuous variables as mean ± standard deviation, median or interquartile range, according to distribution. Chi square test was used to compare categorical variables and T test for continuous variables. Logistic regression models were applied to determine the association of clinical and histological factors with vasculopathy. Odds ratios (OR) with 95% CI were estimated. A p <0.05 was considered significant. The statistical package Medcalc 3.0 was used. Results: Transplant vasculopathy was found in 51,7 % (207/400) sample biopsies, 40% in the early and 60 % in the late transplant biopsies. Recipient age 46(18-76) years, 57 % men, 11,1 % sensitized and 12,2 % hypersensitized. 19,3 % had unacceptable antigens and 16,6%(49/294) developed DSA. All patients received induction and triple immunosuppression therapy. Donor age was 46 (10-73) years, 51 % males, 54,4 % deceased donors and 16, 6%(49) met expanded criteria. Mean Ischemic time was 16,7 ± hours, MM 3.5(1.4), DGF 62,3 %. Vasculopathy in early biopsies was associated with ABMR in patients with DSA. In univariate analysis transplant vasculopathy was correlated with male recipient gender (p=0,0354), staining c4d +(p=0,029) and chronic glomerulopathy (p=0,0116). Late vasculopathy was associated with c4d + p=0,0048; MVI p=0,0018; late rejection p<0.0001 and cellular rejection p=0.03. In multivariate analysis recipient gender (p=0,04) and time of rejection(p=0,03) were statistically significant. Conclusion: Transplant vasculopathy was correlated with male recipient gender, staining c4d +, and chronic glomerulopathy. Late vasculopathy was associated with c4d +, MVI, late and cellular rejection.

347.1: Unraveling the Stage-Shift of Acute Rejection in Renal Allografts

Introduction: The lack of accuracy in current allograft monitoring methodologies for allograft monitoring underscores a critical unmet need for more accurate immunosurveillance to uncover the flux in alloimmunity. QSant – a non-invasive, urine based multi-analyte diagnostic test – was clinically validated to prognosticate injury, risk of evolution, and resolution of acute rejection. QScore—the composite score across measurements of DNA markers: the amount of cell-free DNA, fraction of methylated cfDNA, protein and metabolic biomarkers in the QSant assay—enables this risk prognostication. This study now explores the clinical utility of QSant across the alloimmunity gradient of 32–100 for the early diagnosis of allograft injury and rejection. Methods: Utilizing a large real-world dataset of contemporaneous kidney allograft biopsy and QSant data from 11 US kidney transplant centers, the QScore offers a dynamic view into the changing state of allograft injury and rejection in response to alterations in the patients’ immunosuppression regimen. First analysis included Q-Score distributions in the biopsy-paired validation (n = 162) and prediction (n = 91) data from the Yang cohort, which were collected between 2010 and 2018 [10]. Second was a new analysis of a real-world data (RWD) cohort, comprising of prospectively collected urine samples (n = 235). Results: The RWD cohort (n = 235) of both adult and pediatric renal transplant patients demonstrated a spectrum across the Q-Score distribution. Specifically, 37.9% of the samples were enriched for immune quiescence (Q-Score < 32); the remaining 62.1% were enriched for the injury/rejection spectrum (Q-Score ≥ 32). The QScore <32 classifies stable allograft recipients; QScore between 32-55 exposes a state of alloimmunity flux; and the score between 55-100 provides advanced alloimmunity. Serial monitoring by QSant across a subset of 32 patients (RWD cohort,) over a period of two consecutive quarters (timepoints 1 and 2), captures the essence of the alloimmunity flux (p-value: 0.086 by non-parametric Wilcoxon test). Patients in the immunoquiescent domain (31% [10/32]) by and large continue to be stable (with Q-Scores that track at <32) 22% [7/32]). However, 9.4% (3/32) of patients show Q-Score drifts above 32 with evidence of early rejection. Patients in the alloimmune injury domain follow three trajectories: i) a planar gradient in QScore across time, showing persistence of alloimmunity; ii) an increasing gradient, delineating patients moving into the mature acute rejection domain; iii) a decreasing gradient, delineating patients moving into the immunoquiescent domain, putatively in response to immunomodulation following assessment of active rejection (Figure 1). Conclusion: The observed alloimmunity flux suggested precision monitoring with QSant has the potential to differentiate higher-grade acute rejection episodes from sub-clinical rejection, thereby improving a transplant recipient’s’ quality of life.

POS-045 HISTOPATHOLOGICAL SPECTRUM OF INFECTIONS IN RENAL ALLOGRAFT BIOPSIES: 13 YEARS OF EXPERIENCE FROM INDIA

With the development of new immunosuppressive agents the graft and patient survival in kidney transplantation (KT) has improved. But the infectious complications (community acquired as well as opportunistic infections) still pose a great challenge in their course. Infections are the commonest cause of allograft dysfunction and may lead to high mortality and morbidity especially in the tropical countries. Our analysis aims to study the profile of infections in renal allograft biopsies.

427.4: Combining Donor Derived Cell-Free DNA Fraction and Quantity To Detect Kidney Transplant Rejection Using Molecular Diagnoses

Background: Donor-derived cell-free DNA (dd-cfDNA) fraction and quantity have both been shown to be associated with allograft rejection. The present study examined the relative predictive power of each of these variables to the combination of the two and developed an algorithm incorporating both variables to detect active rejection in renal allograft biopsies. Methods: The first 426 sequential indication biopsy samples collected from the Trifecta study (ClinicalTrials.gov # NCT04239703) with microarray-derived gene expression and dd-cfDNA results were included. After exclusions to simulate intended clinical use, 367 samples were analyzed. Biopsies were assessed using the Molecular Microscope Diagnostic System (MMDxTM) and histology (Banff 2019). Logistic regression analysis examined whether combining dd-cfDNA fraction and quantity adds predictive value to either alone. The first 149 sequential samples were used to develop a two-threshold algorithm, and the next 218 to validate the algorithm. Results: In regression, the combination of dd-cfDNA fraction and quantity was found to be significantly more predictive than either variable alone (p-value 0.009 and <0.0001). In the test set, the AUC of the two-variable system was 0.88 and performance of the two-threshold algorithm showed sensitivity 83.1% and specificity 81.0% using molecular diagnoses, and sensitivity 73.5% and specificity 80.8% using histology diagnoses. Conclusions: This prospective, biopsy-matched, multi-site dd-cfDNA study in kidney transplant patients found that the combination of dd-cfDNA fraction and quantity was more powerful than either dd-cfDNA fraction or quantity alone, and validated a novel two-threshold algorithm incorporating both variables.

P8.007: Single Nuclei RNA-Sequencing Unravels Complex Renal And Immune Cells Signaling Driving Fibrosis in Kidney Allografts

Introduction: Chronic allograft dysfunction (CAD), characterized by interstitial fibrosis and tubular atrophy (IFTA), is the main cause of late graft loss in kidney transplantation. This study dissects the molecular and cellular heterogeneity of the human kidney stroma and immune cells driving CAD. Methods: Using single-nuclei RNA sequencing, 41,893 nuclei were evaluated from 8 rare kidney allograft biopsies (6 CAD and 3 normal patients). These needle biopsies were collected on average at ≥15-months posttransplanation and were stored in the cryopreservative, RNAlater. Following, integrative analysis was applied including pathway and enrichment analysis, pseudotime trajectories, and ligand receptor analyses. XY chromosome linked gene expression analysis was also used to determine the origin of both immune and nonimmune cells in sex-mismatch transplants. Results: Two states of fibrosis were discovered (low and high ECM) that differ based on significant alterations in kidney subclusters, immune cell proportions and phenotypes, and intercellular communication. Pseudotime trajectories revealed a mixed tubule cluster, a key intermediate of injured proximal tubular cells, that transitioned to an activated fibroblast cluster also enriched in myofibroblasts markers. Such differentiation of proximal tubular cells was the main driver of fibrosis. Unique to high ECM, MT1 performed replicative repair evidenced by dedifferentiation and nephrogenic transcriptional signatures. Conversely, low ECM did not show a strong replicative repair signature. Differences in immune cell populations highlighted the dynamic nature of immune responses, some of which, promoted severe metabolic dysfunction and limited repair in low ECM or perpetual tissue regeneration and sustained injury in high ECM. Low ECM was characterized by increased dendritic cells (cDCs, pDCs), mast cells, and monocytes (MO1, MO2) whereas high ECM was characterized by increased B cells, T cells (CD8+ T cells, NKs, and Tregs), and plasma cells. Moreover, 65 ligand-receptor pairs between kidney cells and donor-derived macrophages (previously unidentified after several years post-transplantation) played a novel role in injury propagation. Conclusion: This work improves our understanding of targetable cell types and regulatory mechanisms involved in tissue repair. Interventions aimed at graft fibrogenesis likely require a more targeted approach based on the unique molecular pathways characterizing each condition. National Institutes of Health grant R01DK109581. National Institutes of Health grant R01DK122682 (VRM).

240.4: Cytomegalovirus-Induced Thrombotic Microangiopathy After Renal Transplant: Case Report and Review of the Literature

Introduction: CMV infection as a triggering factor for de novo post-transplantation TMA has long been suggested. We herein report a case of CMV and BKPyV co-infection in a kidney transplant recipient and CMV-induced TMA manifested by severe AKI and thrombocytopenia. Case report: A 48-year-old man with end stage kidney disease secondary to diabetes mellitus underwent a 4 HLA-mismatched, deceased donor renal transplant from a 40-year-old donor who died of a cerebrovascular accident. Cold ischemia time and warm ischemia time were 31 and 18 minutes respectively. CMV IgG was positive in donor and negative in recipient. Intravenous methylprednisolone and antithymocyte globulin were used for induction for anticipated delayed graft function. He was discharged on postoperative day 6 with a serum creatinine (SCr) of 8.1 mg/dL. His allograft function continued to improve, achieving a SCr of 1.67 mg/dL at 1-month post-transplantation. His SCr had since fluctuated between 1.4-1.7 mg/dL. At 4-month post-transplantation, he was found to have low grade BK DNAemia (900 copies/mL) managed with mycophenolate dose reduction alone. He did well until 14-month post-transplantation when he presented with non-oliguric AKI with a SCr of 15.6 mg/dL (baseline 1.5 mg/dL) requiring dialysis initiation. Peripheral smear demonstrated thrombocytopenia but no schistocytes. A renal allograft biopsy (figure 3) was performed which demonstrated BK nephropathy, TMA, and no evidence of rejection. Labs revelaed CMV DNAemia of >140,000 copies/ml and BK DNAemia of 17,000 copies/ml. Donor-specific antibodies, HIV RNA polymerase chain reaction (PCR), hepatitis C RNA PCR, anticardiolipin antibody, stool shiga toxin PCR, and stool and blood cultures were all negative. Intravenous ganciclovir was initiated with improvement in CMV DNAemia and concomitant improvement in renal function. He was transitioned to oral valganciclovir after 10 days of intravenous therapy with complete resolution of CMV DNAemia and steady improvement in renal function. His SCr continued improve and nadired at 1.7 mg/dL. At 2-year follow-up, his SCr was 1.74 mg/dL. Discussion: Initiation of IV ganciclovir and reduction in CMV DNAemiwith decrease in SCr and a transition of the patient off hemodialysis suggested that CMV is the causative factor for TMA. CMV-induced renal injury can manifest as various histopathological patterns, commonly as a CMV glomerulopathy with viral inclusions usually seen in the glomerular endothelial cells, acute tubulointerstitial nephritis and rarely as TMA.Improvement in renal function despite continuation of tacrolimus in current patient suggested that calcineurin-inhibitor-induced TMA was unlikely.Although the precise mechanism whereby CMV viremia may cause TMA is currently not fully understood, it is speculated that infection can initiate endothelial cell damage and inflammation which triggers the thrombotic cascade leading to TMA.

Extracellular vesicle-bound DNA in urine is indicative of kidney allograft injury.

Extracellular vesicle‐bound DNA (evDNA) is an understudied extracellular vesicle (EV) cargo, particularly in cancer‐unrelated research. Although evDNA has been detected in urine, little is known about its characteristics, localization, and biomarker potential for kidney pathologies. To address this, we enriched EVs from urine of well‐characterized kidney transplant recipients undergoing allograft biopsy, characterized their evDNA and its association to allograft injury. The SEC‐based method enriched pure EVs from urine of kidney transplant recipients, regardless of the allograft injury. Urinary evDNA represented up to 29.2 ± 8% (mean ± SD) of cell‐free DNA (cfDNA) and correlated with cfDNA in several characteristics but was less fragmented (P < 0.001). Importantly, using DNase treatment and immunogold labelling TEM, we demonstrated that evDNA was bound to the surface of urinary EVs. Normalised evDNA yield (P = 0.042) and evDNA copy number (P = 0.027) significantly differed between patients with normal histology, rejection injury and non‐rejection injury, the later groups having significantly larger uEVs (mean diameter, P = 0.045) and more DNA bound per uEV. ddDNA is detectable in uEV samples of kidney allograft recipients, but its quantity is highly variable. In a proof‐of‐principle study, several evDNA characteristics correlated with clinical and histological parameters (P = 0.040), supporting that the potential of evDNA as a biomarker for kidney allograft injury should be further investigated.

232.1: Donor-derived Cell-Free DNA Testing in Kidney Transplantation: Do One-month Values Have Any Prognostic Significance?

Introduction: Percutaneous renal allograft biopsy is the current standard for detecting acute rejection after kidney transplantation (KT). Noninvasive tests such as plasma-based donor-derived cell-free DNA (dd-cfDNA) assays have been proposed as an alternative to biopsy. We sought to examine the value of dd-cfDNA testing at one month following KT in different donor and recipient categories. Methods: We performed a single center retrospective review of all adult KT patients (pts) who underwent dd-cfDNA testing between January 2018 and August 2021. The diagnosis of acute rejection was based on histopathology obtained from percutaneous, ultrasound-guided needle biopsies. Our center’s standard practice included dd-cfDNA testing and performing surveillance kidney biopsies at 1 month following KT. The dd-cfDNA test was considered elevated if >1.0%. Results: During the study period, 3157 dd-cfDNA tests were performed in 695 de novo pts. Higher mean 1-month dd-cfDNA levels were noted in the following categories: Retransplants (n=30, 1.8±2.1); highly sensitized pts (PRA 98-100%, n=27, 1.6±2.0; for highly sensitized retransplants, n=18, 2.0±2.3; for highly sensitized primary KTs, n=9, 0.7±0.5); and pts who underwent early reoperations (n=16, 0.85±0.9). In all other KT categories, mean 1-month dd-cfDNA levels were lower and similar: DCD donor KTs (n=53, 0.56±0.4), living donor KTs (n=51, 0.47±0.4); acute kidney injury (AKI) donor KTs (n=23, 0.47±0.6); standard criteria donor (SCD) KTs (n=91, 0.47±0.4); expanded criteria donor (ECD) KTs (n=44, 0.46±0.5); dual KTs (n=16, 0.45±0.4); and pts with delayed graft function (DGF, n=50, 0.46±0.4). In 58 primary KT pts with negative 1-month surveillance biopsies, corresponding mean 1-month dd-cfDNA levels were 0.49±0.4. In 10 pts with positive 1-month surveillance biopsies, corresponding mean 1-month dd-cfDNA levels were 1.5±1.8. In 26 pts with a 1-month dd-cfDNA level >1.0, the subsequent incidence of acute rejection was 27%; in 22 pts with a 1-month dd-cfDNA level ≥2.0, the subsequent incidence of acute rejection was 50% (mean follow-up 14 months). Conclusions: We noted a bimodal distribution of 1-month dd-cfDNA levels. Higher mean 1-month dd-cfDNA levels (range 0.72-2.2) were associated with retransplants, high PRA pts, and pts with early reoperations; 22-44% of these pts had 1-month dd-cfDNA levels >1.0. Conversely, primary KT alone pts (DCD, living donor, AKI donor, SCD, ECD, dual KT, those with DGF) had lower 1-month dd-cfDNA levels (range 0.45-0.56) and only 7-14% had 1-month dd-cfDNA levels >1.0. One-month surveillance biopsy results correlated with corresponding dd-cfDNA levels. Elevated 1-month dd-cfDNA levels occurred in pts at a higher risk for acute rejection, suggesting that these pts need to be monitored more closely.

Long-term immunological outcomes of early subclinical inflammation on surveillance kidney allograft biopsies

The long-term impact of early subclinical inflammation (SCI) through surveillance biopsy has not been well studied. To do this, we recruited a prospective observational cohort that included 1000 sequential patients who received a kidney transplant from 2013-2017 at our center. A total of 586 patients who underwent a surveillance biopsy in their first year post-transplant were included after excluding those with clinical rejections, and those who were unable to undergo a surveillance biopsy. Patients were classified based on their biopsy findings: 282 with NSI (No Significant Inflammation) and 304 with SCI-T (SCI and Tubulitis) which was further subdivided into 182 with SC-BLR (Subclinical Borderline Changes) and 122 with SC-TCMR (Subclinical T Cell Mediated Rejection, Banff 2019 classification of 1A or more). We followed the clinical and immunological events including Clinical Biopsy Proven Acute Rejection [C-BPAR], long-term kidney function and death-censored graft loss over a median follow-up of five years. Episodes of C-BPAR were noted at a median of two years post-transplant. Adjusted odds of having a subsequent C-BPAR was significantly higher in the SCI-T group [SC-BLR and SC-TCMR] compared to NSI 3.8 (2.1-7.5). The adjusted hazard for death-censored graft loss was significantly higher with SCI-T compared to NSI [1.99 (1.04-3.84)]. Overall, SCI detected through surveillance biopsy within the first year post-transplant is a harbinger for subsequent immunological events and is associated with a significantly greater hazard for subsequent C-BPAR and death-censored graft loss. Thus, our study highlights the need for identifying patients with SCI through surveillance biopsy and develop strategies to prevent further alloimmune injuries.

Biliary Epithelial Senescence in Cellular Rejection Following Live Donor Liver Transplantation

As with the hepatocytes, cholangiocyte senescence can also easily be detected in damaged small bile ducts and bile ductules during liver disease affecting the biliary system and cholangiocytes. Despite cellular senescence being a feature of chronic progressive cholangiopathies in adults, only a few studies have investigated its role in liver transplant rejection. Transplant biopsies displaying features of rejection were reviewed and classified based on the type of rejection and the time since transplantation. An immunohistochemistry panel has been applied for 3 senescent cell markers (p53, p21, p16). Immunohistochemical expression analysis for the biliary senescence markers (53 biopsies) was done in the post-transplantation periods (Group 1-4) for the cases with the histologically proven diagnosis of rejection. In post-transplant group 1 (<3 months), group 2 (3-6 months), group 3 (6-12 months) and group 4 (>12 months), any 2 senescent markers' positivity was noted in 5/14 (35.7%), 8/13 (61.5%), 16/17 (94.1%) and 9/9 (100%) biopsies respectively and were comparable in all four groups (P= 0.001). A comparison of early biopsies (Group1; 3 months) and late biopsies (Group 2,3&4; >3 months) revealed significantly higher expression in late biopsies (>3 months) (P= 0.001 for any two markers). In ACR, LAR, ECR, and CR/DR any two senescent markers were positive in 14/28 (50%), 12/13 (92.3%) cases, 9/9 (100%), and 3/3cases (100%). Senescent markers (any two) were comparable in all four histological groups (P < 0.001).LAR group had increased expression (P= 0.009 for any two markers and 0.001 for all three markers) and has increased progression to CR (P= 0.019) as compared to ACR. This study on a large number of LDLT allograft biopsies demonstrates the role of biliary senescence in rejection and suggests a pathobiological role for senescence in the poor prognosis seen in late acute cellular rejection and chronic rejection.

Development of a Bak gene based standard curve for absolute quantification of BK virus in real time quantitative PCR assay and noninvasive diagnosis of BK virus nephropathy in kidney allograft recipients

BackgroundBK virus nephropathy (BKVN) is a frequent and serious post-transplant complication and undermines realization of the full benefits of kidney transplantation. We developed a Bak amplicon-based standard curve for absolute quantification of BKV VP1 mRNA copy number in the real time quantitative PCR (RT-qPCR) assay and investigated the performance characteristics of this novel assay. MethodsWe determined analytical specificity, sensitivity, and precision of our 73 bp mouse Bak amplicon based standard curve for absolute quantification of BKV VP1 mRNA in RT-qPCR assays. The diagnostic accuracy of the Bak standard curve in the RT-qPCR assay for the noninvasive diagnosis of BKVN in human kidney allograft recipients was investigated by quantification of BKV VP1 mRNA copy number in 192 urine samples matched to 192 kidney allograft biopsies from 155 unique kidney allograft recipients. Intraclass correlation coefficients (ICC) were calculated for the threshold cycles (Ct) and BKV VP1 mRNA copy number observed in the RT-qPCR assay with the Bak standard curve or the BKV standard curve. ResultsPerformance characteristics of the Bak amplicon-based RT-qPCR assay were exceptional with a slope of −3.291, Y-intercept of 38.60, R2 value of 1.00, efficiency of 101% and error of 0.014. Amplification was specific for the Bak amplicon. Intra assay standard deviation (SD) was 0.08 or less and inter assay SD was 0.11 or less for 31 cycles or less of amplification of the Bak amplicon. Receiver operating characteristic (ROC) curve analysis of BKV VP1 mRNA copy number in 192 biopsy matched urines yielded an area under the ROC of 0.982 (95% CI, 0.964 to 0.999, P < 0.0001) for discriminating patients with BKVN biopsies from patients without BKVN biopsies. The striking identity in the measurement of BKV VP1 mRNA copy numbers in the Bak amplicon-based RT-qPCR assay and in the BKV amplicon-based RT-qPCR assay was shown by an ICC of 1.00 when the Cts were compared, and an ICC of 0.99 when the log10 BKV VP1 mRNA copy numbers were compared. ConclusionsBak standard curve for absolute quantification of BKV VP1 mRNA copy number in the RT-qPCR assay demonstrated high efficiency, short and long-term precision and analytical specificity. BKVN was diagnosed with high accuracy. Our new findings, viewed in the light of our earlier demonstration that absolute quantification of a panel of mRNAs encoding immunoregulatory proteins is feasible with the Bak amplicon-based RT-qPCR assays, suggest that the Bak standard curve could serve as a universal calibrator for absolute quantification of transcripts in RT-qPCR assays and help reduce the workload, costs and eliminate contamination of genes of interest by repeated amplification of gene specific standard curves.