BACKGROUND Acute interstitial nephritis (AIN) is a common cause of acute kidney injury (AKI), but the diagnosis may be missed as kidney biopsies are rarely obtained when acute tubular injury (ATI) is suspected.METHODS The Kidney Precision Medicine Project is a cohort study that obtains kidney biopsies from individuals with AKI, which undergo pathologic and molecular interrogation. We compared ATI and AIN cases among the first 60 AKI participants.RESULTS On clinicopathologic adjudication, 30 patients (50%) had a primary adjudicated diagnosis of ATI, 13 (22%) patients had AIN, 9 (15%) had diabetic nephropathy, and 3 (5%) had other conditions. There were increased interstitial white blood cells and tubulitis (P < 0.05 for both) in AIN compared with ATI. Prior to biopsy, the treating clinician suspected ATI in 83% of the cases with adjudicated ATI, while the treating clinician suspected AIN in 54% of the cases with AIN. Tissue transcriptomic signatures showed enrichment of proinflammatory signaling and increased expression of CXCL9, a chemokine induced by IFN-γ, in myeloid cells of participants with AIN. CXCL9 localized to inflammatory infiltration in spatial transcriptomic data.CONCLUSION Adjudication of kidney biopsies revealed distinct pathologic and molecular profiles between ATI and AIN. Kidney biopsy should be considered more frequently in AKI, as AIN is clinically underrecognized.TRIAL REGISTRATION ClinicalTrials.gov NCT04334707.FUNDING National Institute of Diabetes and Digestive and Kidney Diseases grants U01DK133081, U01DK133091, U01DK133092, U01DK133093, U01DK133095, U01DK133097, U01DK114866, U01DK114908, U01DK133090, U01DK133113, U01DK133766, U01DK133768, U01DK114907, U01DK114920, U01DK114923, U01DK114933, U24DK114886, UH3DK114926, UH3DK114861, UH3DK114915, and UH3DK114937.

Building upon a foundational Human Kidney resource, we present a comprehensive multi-modal atlas that defines spatially resolved versus unresolved repair states and mechanisms in human kidney disease. Homeostatic interactions between injured kidney epithelium and its surrounding milieu determine successful repair outcomes, while pathogenic signaling promotes unresolved inflammation and fibrosis leading to chronic disease. We integrated multiple single-cell and spatial modalities across ~700 samples from >350 patients (~250 research biopsies), analyzing ~1.7 million cells alongside complementary mouse multi-omic profiles spanning acute-to-chronic injury and aging (>300,000 cells) and spatial transcriptomic analysis of >150 human biopsies. This cross-species atlas delineates functional pathways and druggable targets across the nephron and defines gene regulatory networks and chromatin landscapes governing tubular, fibroblast, and immune cell transitions from injury to either recovery or failed repair states. We identified distinct cellular states associated with specific pathological features that show dynamic distributions between acute kidney injury (AKI) and chronic kidney disease (CKD), organized within unique spatial niches that reveal progression mechanisms from early injury to unresolved disease. Gene regulatory analyses prioritized key transcription factor activities (SOX4, SOX9, NFKB1, REL, KLFs) and their target networks establishing disease states and tissue microenvironments. These regulatory programs were directly linked to clinical outcomes, identifying molecular signatures of recovery and secreted biomarkers predictive of AKI-to-CKD progression, providing a key resource for therapeutic development and precision medicine approaches in kidney disease.

Rationale & Objective:The Kidney Precision Medicine Project is obtaining kidney biopsies from people with chronic kidney disease (CKD) and acute kidney injury for comprehensive clinical, histopathological, and molecular characterization. Here, we describe histopathological findings from a subset of kidney biopsies from adults with CKD. Study Design:Descriptive case series of histopathology findings from adjudicated CKD biopsies. Setting & Participants:Kidney Precision Medicine Project enrolled adults with CKD and diabetes (DKD) and/or hypertension (HCKD) with persistent eGFR 30-59 mL/min/1.73 m2, urine albumin-creatinine ratio ≥30 mg/g, or urine protein-creatinine ratio ≥150 mg/g. Clinicopathological adjudication by study nephrologists and kidney pathologists was completed as part of a pilot program for 39 participants enrolled 2019-2022. Clinicians completed surveys to assess impacts of biopsies on diagnosis, prognosis, and management. Exposures:This is a descriptive study without defined exposures. Outcomes:Characterization of glomerular, tubulointerstitial, and vascular histopathological features across CKD biopsies. Analytical Approach:Continuous variables were summarized as mean (standard deviation) or median (interquartile range); categorial variables were summarized as count (percentage). Results:Participants' mean age was 59 years, 59% were female. Mean eGFR was 55 mL/min/1.73 m2; median urine albumin-creatinine ratio and urine protein-creatinine ratio were 81 mg/g and 211 mg/g, respectively. Among DKD-enrolled participants (N = 28), 15 (54%) had a primary diagnosis of diabetic nephropathy, 3 (11%) had hypertension-associated nephropathy, 2 (7%) had other glomerular diseases, and 8 (29%) had nonspecific findings. Among HCKD-enrolled participants (N = 11), 5 (46%) had hypertension-associated nephropathy and 6 (55%) had nonspecific findings. A range of glomerular, tubulointerstitial, and vascular findings was observed. 26% of clinicians stated results were different than expected; 77% stated results affected prognostic discussions. Limitations:Small sample size and lack of longitudinal data limit generalizability. Conclusions:Kidney biopsies in people with common causes of CKD demonstrate a broad range of histopathology and may have clinical utility. Unsuspected disease processes and unexpected and nonspecific findings precluding a definitive diagnosis are often present.

Transplantation using kidneys from deceased donors with marginal clinical characteristics is increasing to maximize access to transplant. Preimplantation biopsies of such kidneys are often obtained to determine the degree of chronic changes as an objective supplement to donor clinical-based assessment of organ quality, particularly in the United States. There is conflicting evidence and heterogenous practice about how preimplantation biopsies should be performed, interpreted, and incorporated into organ disposition decisions. In 2022, the Banff Foundation for Allograft Pathology tasked its Time-Zero Biopsy Working Group with reviewing existing literature on preimplantation biopsies to prepare consensus recommendations on situations in which these biopsies should be performed and suggest best practices for improving reproducibility of biopsy readings. This workgroup report tabulates the Working Group's recommendations and identifies gaps that need to be filled to advance preimplantation biopsy clinical practice in donor selection. The major recommendations are that a preimplantation biopsy should be performed only when clinical assessment tools indicate that the kidney is unsuitable for utilization, that the clinical and preimplantation biopsy findings should be interpreted together for organ utilization decisions, and that digital whole slide images be promoted to facilitate setting up reporting networks of nephropathologists. Additional prospective studies using predictive modeling approaches are needed to optimize preimplantation biopsy criteria, sampling, interpretation, and incorporation into kidney allocation decisions.

Donor-recipient mismatches in histocompatibility antigens recognized by lymphoid cells have been demonstrated to adversely affect allograft outcomes. In contrast, it remains unclear whether mismatches sensed by innate myeloid cells have a similar effect. We investigated the consequences of mismatch in the polymorphic gene encoding signal regulatory protein α (SIRPα) on kidney allograft pathology and survival in mice and humans. We found that SIRPα variants elicit monocyte activation by binding to CD47 and that eliminating SIRPα mismatch or recipient CD47 expression prevented chronic allograft pathology in mice receiving major histocompatibility complex (MHC)–mismatched renal allografts. Human genomic analysis identified two haplotype categories, A and B, encoding SIRPα variants with distinct CD47 binding interfaces. In kidney transplant recipients ( N = 455), SIRPα mismatch was associated with increased acute rejection and graft fibrosis in the first posttransplant year, and A recipients of B kidneys had reduced long-term graft survival (hazard ratio, 3.2; 95% confidence interval, 1.5 to 6.9; P = 0.002), a finding that was confirmed in an independent validation cohort ( N = 258). Moreover, monocytes in these graft recipients had an activated phenotype. The effects of SIRPα mismatch were independent of ancestry, human leukocyte antigen mismatch, donor-specific antibodies, and delayed graft function. Therefore, these data demonstrate that a donor-recipient mismatch that causes innate immune activation is a determinant of kidney transplantation outcomes.

Transplantation using kidneys from deceased donors with marginal clinical characteristics is increasing to maximize access to transplant. Preimplantation biopsies of such kidneys are often obtained to determine the degree of chronic changes as an objective supplement to donor clinical-based assessment of organ quality, particularly in the United States. There is conflicting evidence and heterogenous practice about how preimplantation biopsies should be performed, interpreted, and incorporated into organ disposition decisions. In 2022, the Banff Foundation for Allograft Pathology tasked its Time-Zero Biopsy Working Group with reviewing existing literature on preimplantation biopsies to prepare consensus recommendations on situations in which these biopsies should be performed and suggest best practices for improving reproducibility of biopsy readings. This workgroup report tabulates the Working Group's recommendations and identifies gaps that need to be filled to advance preimplantation biopsy clinical practice in donor selection. The major recommendations are that a preimplantation biopsy should be performed only when clinical assessment tools indicate that the kidney is unsuitable for utilization, that the clinical and preimplantation biopsy findings should be interpreted together for organ utilization decisions, and that digital whole slide images be promoted to facilitate setting up reporting networks of nephropathologists. Additional prospective studies using predictive modeling approaches are needed to optimize preimplantation biopsy criteria, sampling, interpretation, and incorporation into kidney allocation decisions.

OBJECTIVES:Pathologists interpreting kidney allograft biopsies using the Banff system usually start by recording component scores (eg, i, t, cg) using histopathologic criteria committed to memory. Component scores are then melded into diagnoses using the same manual/mental processes. This approach to complex Banff rules during routine sign-out produces a lack of fidelity and needs improvement.METHODS:We constructed a web-based "smart template" (software-assisted sign-out) system that uniquely starts with upstream Banff-defined additional diagnostic parameters (eg, infection) and histopathologic criteria (eg, percent interstitial inflammation) collectively referred to as feeder data that is then translated into component scores and integrated into final diagnoses using software-encoded decision trees.RESULTS:Software-assisted sign-out enables pathologists to (1) accurately and uniformly apply Banff rules, thereby eliminating human inconsistencies (present in 25% of the cohort); (2) document areas of improvement; (3) show improved correlation with function; (4) examine t-Distributed Stochastic Neighbor Embedding clustering for diagnosis stratification; and (5) ready upstream incorporation of artificial intelligence-assisted scoring of biopsies.CONCLUSIONS:Compared with the legacy approach, software-assisted sign-out improves Banff accuracy and fidelity, more closely correlates with kidney function, is practical for routine clinical work and translational research studies, facilitates downstream integration with nonpathology data, and readies biopsy scoring for artificial intelligence algorithms.

Solid organ transplantation remains the life-saving treatment for end-stage organ failure, but chronic rejection remains a major obstacle to long-term allograft outcomes and has not improved substantially. Tertiary lymphoid organs (TLOs) are ectopic lymphoid structures that form under conditions of chronic inflammation, and evidence from human transplantation suggests that TLOs regularly form in allografts undergoing chronic rejection. In this study, we utilized a mouse renal transplantation model and manipulation of the lymphotoxin αβ/lymphotoxin β receptor (LTαβ/LTβR) pathway, which is essential for TLO formation, to define the role of TLOs in transplantation. We showed that intragraft TLOs are sufficient to activate the alloimmune response and mediate graft rejection in a model where the only lymphoid organs are TLOs in the allograft. When transplanted to recipients with a normal set of secondary lymphoid organs, the presence of graft TLOs or LTα overexpression accelerated rejection. If the LTβR pathway was disrupted in the donor graft, TLO formation was abrogated, and graft survival was prolonged. Intravital microscopy of renal TLOs demonstrated that local T and B cell activation in TLOs is similar to that observed in secondary lymphoid organs. In summary, we demonstrated that immune activation in TLOs contributes to local immune responses, leading to earlier allograft failure. TLOs and the LTαβ/LTβR pathway are therefore prime targets to limit local immune responses and prevent allograft rejection. These findings are applicable to other diseases, such as autoimmune diseases or tumors, where either limiting or boosting local immune responses is beneficial and improves disease outcomes.

BACKGROUND:Microvascular inflammation (MVI) can occur in biopsies showing T-cell mediated rejection (TCMR), but it is not well established that T-cells can directly mediate microvascular injury (TCMR-MVI). METHODS:This was a cross sectional RNAseq based Banff Human Organ Transplant (BHOT) gene expression (GE) analysis. The objective of this study was to probe the molecular signature of TCMR-MVI in comparison with C4d+, DSA+ antibody mediated rejection (ABMR), stable renal function (STA), and TCMR without MVI. Transcriptome analysis utilized CLC genomic workbench and R-studio software. RESULTS:No gene set was specific for any diagnostic category, and all were expressed at low levels in STA biopsies. BHOT gene set scores could differentiate ABMR from TCMR and TCMR-MVI, but not TCMR from TCMR-MVI. TCMR-MVI underexpressed several genes associated with ABMR including DSATs, ENDAT, immunoglobulin genes, ADAMDEC1, PECAM1 and NK cell transcripts (MYBL1, GNLY), but overexpressed C3, NKBBIZ, and LTF. On the other hand, there was no significant difference in the expression of these genes in TCMR-MVI versus TCMR. This indicates that the GE profile of TCMR MVI aligns more closely with TCMR than ABMR. The limitations of classifying biopsies using the binary ABMR-TCMR algorithm, and the occurrence of common pathogenesis mechanisms amongst different rejection phenotype was highlighted by the frequent presence of molecular mixed rejection. CONCLUSIONS:T-cell mediated mechanisms play a significant role in the pathogenesis of MVI. GE was broadly different between rejection phenotypes, but molecular scores varied substantially between biopsies with the same Banff grade. It was not always possible to achieve precise molecular score-based diagnostic categorization of individual patients.

The Banff Working Group on Liver Allograft Pathology met in September 2022. Participants included hepatologists, surgeons, pathologists, immunologists, and histocompatibility specialists. Presentations and discussions focused on the evaluation of long-term allograft health, including noninvasive and tissue monitoring, immunosuppression optimization, and long-term structural changes. Potential revision of the rejection classification scheme to better accommodate and communicate late T cell-mediated rejection patterns and related structural changes, such as nodular regenerative hyperplasia, were discussed. Improved stratification of long-term maintenance immunosuppression to match the heterogeneity of patient settings will be central to improving long-term patient survival. Such personalized therapeutics are in turn contingent on a better understanding and monitoring of allograft status within a rational decision-making approach, likely to be facilitated in implementation with emerging decision-support tools. Proposed revisions to rejection classification emerging from the meeting include the incorporation of interface hepatitis and fibrosis staging. These will be opened to online testing, modified accordingly, and subject to consensus discussion leading up to the next Banff conference.

Rejection is a primary cause of allograft dysfunction after kidney transplantation. The diversity of immune subpopulations involved in the different endotypes of rejection remains to be delineated at single-cell resolution. In a cohort of 76 kidney transplant recipients, we conducted high-dimensional immune phenotyping of blood CD4 T and B cells, single-cell RNA and T/B cell receptor sequencing, and plasma cytokine profiling. Phenotypic, transcriptional, and clonal states of CD4T and B cells could significantly distinguish stable allograft states from rejection. Patients undergoing T cell-mediated rejection displayed accumulation of clonally expanded cytotoxic T helper (Th)1 cells and Th17-like cells, associated with predominant naive B cell responses. In contrast, antibody-mediated rejection was characterized by clonal expansion of Th1-polarized T follicular helper cells and effector T-bet+ memory B cells, both of which strongly expressed interleukin 12 and tumor necrosis factor-signaling pathways. Plasma cytokine analysis confirmed mixed Th1/Th17 and Th1/T follicular helper cell-driven inflammatory profiles distinguishing T cell-mediated rejection and antibody-mediated rejection, respectively. CD4T and B cell subpopulations and signatures were validated using bulk RNA-seq analysis of matched kidney allografts and using an independent single-cell RNA-seq data set. These data improve mechanistic understanding of the immune pathogenesis of rejection and support the development of more specific immunosuppressive therapies to treat allograft rejection.

Histologic separation of injury, T cell-mediated rejection, or antibody-mediated rejection in allograft heart biopsies is difficult. A critical review of publications was performed to evaluate the caveats of using molecular diagnostics (MDX) to distinguish between these entities. Typically, only 1 to 2 fragments of unknown histologic appearance are evaluated. Archetype and molecular classifier analyses use gene lists derived from histologic labels and associated reproducibility issues influence the accuracy of the derived MDX classes. Archetypes A1, A2, and A3 archetypes created by bioinformatics were renamed no rejection, T cell-mediated rejection, and antibody-mediated rejection despite as little as 40% concordance with histologic diagnoses and overlapping archetype scores. Additional archetypes S4 and minor injury were created using arbitrary cutoffs based on visual examination of principal component analysis plots. Therapeutic implications of the numerous discrepancies with histology remain unexplored. Many MDX-derived observations are ambiguous and open to alternate logical explanations. Better molecular methods and more rigorous validation studies are needed to advance the field. Ideally, these methods should analyze all available biopsy fragments to minimize sampling issues. It is also desirable to incorporate spatial transcriptomics into the workflow, so that gene expression data can be directly compared with the underlying histology lesions.

Natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC) is a major mechanism of humoral allograft injury. FCGR3A V176/F176 polymorphism influences ADCC activity. Additionally, NK cell FcγRIIc expression, dictated by the Q13/STP13 polymorphism, was never investigated in kidney transplantation. To assess the clinical relevance of FCGR2C Q13/STP13 polymorphism in conjunction with FCGR3A V176/F176 polymorphism, 242 kidney transplant recipients were genotyped. NK cell Fc gamma receptor (FcγR) expression and ADCC activity were assessed. RNA sequencing was performed on kidney allograft biopsies to explore the presence of infiltrating FcγR+ NK cells. The FCGR2C Q13 allele was enriched in antibody-mediated rejection patients. FcγRIIc Q13+ NK cells had higher ADCC activity than FcγRIIc Q13- NK cells. In combination with the high-affinity FCGR3A V176 allele, Q13+V176+ NK cells were the most functionally potent. Q13+ was associated with worse microvascular inflammation and a higher risk of allograft loss. Among V176- patients, previously described in the literature as lower-risk patients, Q13+V176- showed a lower graft survival than Q13-V176- patients. In antibody-mediated rejection biopsies, FCGR2C transcripts were enriched and associated with ADCC-related transcripts. Our results suggest that FCGR2C Q13 in addition to FCGR3A V176 is a significant risk allele that may enhance NK cell-mediated ADCC and contribute to allograft injury and poor survival.

Systematic screening for BKPyV-DNAemia has been advocated to aid prevention and treatment of polyomavirus associated nephropathy (PyVAN), an important cause of kidney graft failure. The added value of performing a biopsy at time of BKPyV-DNAemia, to distinguish presumptive PyVAN (negative SV40 immunohistochemistry) and proven PyVAN (positive SV40) has not been established. Therefore, we studied an unselected cohort of 950 transplantations, performed between 2008-2017. BKPyV-DNAemia was detected in 250 (26.3%) transplant recipients, and positive SV40 in 91 cases (9.6%). Among 209 patients with a concurrent biopsy at time of first BKPyV-DNAemia, 60 (28.7%) biopsies were SV40 positive. Plasma viral load showed high diagnostic value for concurrent SV40 positivity (ROC-AUC 0.950, 95% confidence interval 0.916-0.978) and the semiquantitatively scored percentage of tubules with evidence of polyomavirus replication (pvl score) (0.979, 0.968-0.988). SV40 positivity was highly unlikely when plasma viral load is below 4 log10 copies/ml (negative predictive value 0.989, 0.979-0.994). In SV40 positive patients, higher plasma BKPyV-DNA load and higher pvl scores were associated with slower viral clearance from the blood (hazard ratio 0.712, 95% confidence interval 0.604-0.839, and 0.327, 0.161-0.668, respectively), whereas the dichotomy positivity/negativity of SV40 immunohistochemistry did not predict viral clearance. Although the pvl score offers some prognostic value for viral clearance on top of plasma viral load, the latter provided good guidance for when a biopsy was unnecessary to exclude PyVAN. Thus, the distinction between presumptive and proven PyVAN, based on SV40 immunohistochemistry, has limited clinical value. Hence, management of BKPyV-DNAemia and immunosuppression reduction should be weighed against the risk of occurrence of rejection, or exacerbation of rejection observed concomitantly.

ABSTRACT Arteriolar hyalinosis in kidneys is an independent predictor of cardiovascular disease, the main cause of mortality in chronic kidney disease (CKD). The underlying molecular mechanisms of protein accumulation in the subendothelial space are not well understood. Using single cell transcriptomic data and whole slide images from kidney biopsies of patients with CKD and acute kidney injury in the Kidney Precision Medicine Project, the molecular signals associated with arteriolar hyalinosis were evaluated. Co-expression network analysis of the endothelial genes yielded three gene set modules as significantly associated with arteriolar hyalinosis. Pathway analysis of these modules showed enrichment of transforming growth factor beta / bone morphogenetic protein (TGFβ / BMP) and vascular endothelial growth factor (VEGF) signaling pathways in the endothelial cell signatures. Ligand-receptor analysis identified multiple integrins and cell adhesion receptors as over-expressed in arteriolar hyalinosis, suggesting a potential role of integrin-mediated TGFβ signaling. Further analysis of arteriolar hyalinosis associated endothelial module genes identified focal segmental glomerular sclerosis as an enriched term. On validation in gene expression profiles from the Nephrotic Syndrome Study Network cohort, one of the three modules was significantly associated with the composite endpoint (> 40% reduction in estimated glomerular filtration rate (eGFR) or kidney failure) independent of age, sex, race, and baseline eGFR, suggesting poor prognosis with elevated expression of genes in this module. Thus, integration of structural and single cell molecular features yielded biologically relevant gene sets, signaling pathways and ligand-receptor interactions, underlying arteriolar hyalinosis and putative targets for therapeutic intervention.