Fibrotic remodeling of extracellular matrix is a central driver of autosomal dominant polycystic kidney disease (ADPKD) progression since early stages of the disease. Unfortunately, it cannot be assessed with currently available methodologies before irreversible structural and functional decline, creating a diagnostic blind spot that hinders accurate early risk stratification and management. Here, we report the development of Gd-hProCA32.Collagen, a collagen -targeted protein MRI contrast agent that enables precision molecular MRI (pMRI) of early fibrosis by directly imaging collagen type I deposition in vivo before conventional laboratory and imaging methods detect changes in kidneys and liver of Pkhd1 PCK/PCK (PCK) rats and Pkd2 mutant mice. Gd-hProCA32.Collagen, used at 10-fold lower dose, outperformed the widely clinically used agent gadobutrol (Gadovist), detecting approximately 2.8-fold greater total renal cyst volume (~8,500 vs ~3,000 mm3, p<0.0001) and 1.5-fold higher total cyst count (~245 vs ~160, p<0.0001), with superior T1W and T2W kidney AUC (p<0.01 and p<0.001) and preferential sensitivity to small and medium cysts. Signal enhancement in kidneys and liver correlated strongly with histological collagen burden quantified by Sirius red staining, whereas Gadovist showed no meaningful correlation. Gd-hProCA32.Collagen also enabled in vivo visualization of previously undetectable changes resembling radial striations at sites of microcyst cluster-adjacent microfibrosis and sustained delayed MRI enhancement due to specific collagen binding. These results reveal a previously inaccessible subclinical fibrotic phase of cystic kidney and liver disease and establish collagen-targeted pMRI as a strategy for early noninvasive detection and spatial mapping of multi-organ extracellular matrix remodeling when conventional biomarkers remain non-discriminating.

The current Banff report summarizes topics central to the pancreas session in the 2024 conference held in Paris (France). The focus of discussion was on diagnostic criteria of chronic active rejection, the indeterminate (IND) category, and the utility of immunostains in pancreas transplant biopsies. Concepts were validated in a retrospective PanTxBx cohort after the meeting. First, chronic active rejection criteria introduced in the 2022 report were confirmed, and recommendations were refined. The clinical relevance of this category, with an increased risk of graft loss, was proven in the retrospective cohort. Second, the so far very narrowly defined IND category was expanded from septal inflammation to also include subthreshold lobular changes and incomplete findings of antibody-mediated rejection. IND cases were evaluated in the context of the clinical findings and CD3 and CD68 immunostaining. This process helped in further subcategorizing the IND diagnoses into rejection and non-rejection events and, third, led to the general recommendations provided for utilization of CD3 and CD68 immunostains to facilitate the histologic diagnosis in unclear biopsies. The working group expects that the current recommendations and refinements will enhance the applicability and reproducibility of the histopathological pancreas schema. Additionally, an outlook is given regarding ongoing projects and future perspectives.

PURPOSE OF REVIEW:The degree to which computerized methods, such as artificial intelligence (AI), will aid in the assessment of kidney histopathology is undergoing intense study and application; and this is particularly true for interstitial fibrosis, which is often used as a surrogate measure of chronic kidney disease progression, since interobserver variability among human pathologists has been demonstrated in the assessment of interstitial fibrosis and other features. RECENT FINDINGS:Computerized assessment of interstitial fibrosis, including with AI, has been assessed alongside pathologists. Computerized methods such as AI have shown direct interstitial fibrosis measurement and indirect assessment through kidney compartment segmentation; however, some studies have shown lack of complete concordance among computerized methods and humans; and studies have still shown the persistent value of human assessment in many circumstances. SUMMARY:Computerized methods, including AI, are showing increased application in kidney pathology for a wide variety of clinical and histopathologic parameter assessment, including interstitial fibrosis; however, further studies are needed to characterize the performance of AI and handcrafted methods; and additional work is needed to fully integrate computerized methods into routine pathology practice. Ultimately, humans working with AI ("humans + AI") may provide enhanced analysis for more effective patient care.

The Banff Classification, established in 1991, provides a global standard for diagnosing and grading kidney transplant pathology, evolving through regular consensus meetings to incorporate new advances. Initially designed to address the lack of uniformity in renal allograft biopsy reporting, the Banff system now integrates semiquantitative scores for acute and chronic lesions. Recent progress in artificial intelligence (AI) and deep learning (DL) has accelerated quantitative analysis in kidney histology, particularly for interstitial fibrosis and tubular atrophy (IFTA) and inflammation, reducing interobserver variability and supporting prognostic assessment. Targeted algorithms such as positive pixel count (PPC) and DL-based segmentation models have demonstrated robust performance in identifying renal compartments and quantifying injury; and AI-assisted approaches for vascular lesions and immune cell profiling show promise but are limited in data diversity, stain standardization, and external validation. Additional metrics, such as nephron size, IFTA foci density, and mesangial expansion (ME), have been explored, with computational metrics and AI models showing potential for improved reproducibility and clinical relevance. Despite concerns about the implications of AI in pathology, optimism prevails regarding its ability to augment human expertise and enhance diagnostic precision. Ongoing work within the Banff Digital Pathology Working Group aims to extend automated assessment to additional Banff lesions, including glomerulitis, peritubular capillaritis, arteritis, and tubulitis. As these tools mature, they may enable more comprehensive quantitative characterization of transplant biopsies and facilitate exploration of histopathologic patterns that have been difficult to study. The Banff system’s iterative evolution and embrace of AI-driven digital pathology underscore its enduring impact and promise in kidney transplant medicine.

Background Diagnostic interpretation of kidney allograft biopsies using the Banff classification remains variable, but the determinants of this variability are not fully defined. We performed a global, fully digital multi-reader study to identify the principal drivers of disagreement in Banff-based assessment. Methods Thirty six kidney transplant biopsies were independently scored by 67 renal pathologists on a standardized digital platform. Readers assessed Banff lesions on hematoxylin and eosin, periodic acid Schiff, and Jones' silver stains; final diagnostic categories were assigned using prespecified Banff-based decision rules. Interobserver agreement was quantified with Gwet's agreement coefficient (AC) statistics. Determinants of diagnostic agreement were evaluated) using pairwise mixed-effects logistic regression, and reader similarity was examined by principal component analysis (PCA) with post hoc molecular annotation. Results Agreement for final diagnostic categories was moderate (Gwet's AC1, 0.55; 95% CI, 0.47 - 0.63). Lesion-level agreement varied substantially, with lowest agreement for selected threshold-dependent inflammatory or semi-quantitative lesions, including interstitial inflammation in areas of IFTA, peritubular capillaritis and arteriolar hyalinosis. Diagnostic concordance differed markedly across biopsies, indicating strong case-level heterogeneity. In pairwise models, differences in active inflammatory and vascular lesion scoring were the strongest correlates of diagnostic disagreement; reader experience and geography contributed minimally. Principal component analysis showed reader variation was organized along two dominant axes: a rejection-calling threshold axis linked mainly to tubulointerstitial inflammatory injury, and a T cell-mediated (TCMR/TI) and antibody-mediated/microvascular (AMR/MVI) inflammation-oriented phenotypic classification axis. Conclusion Interobserver variation in Banff-based kidney transplant biopsy assessment is structured rather than random and driven mainly by how readers threshold and integrate key inflammatory lesion compartments rather than experience or geographic location.

The XVIIth Banff meeting for transplant pathology was held in Paris, France, from September 16, 2024 to September 20, 2024, hosted by the Paris Institute for Transplantation & Organ Regeneration. The Banff 2024 meeting resulted in no changes to the Banff kidney classification. Important outputs of the meeting were a reaffirmation of the clinical usefulness of the clinical reasoning framework and flowchart for cases with microvascular inflammation/antibody-mediated rejection introduced at the Banff 2022 meeting and the introduction of a similar flowchart for tubulointerstitial inflammation and intimal arteritis (v lesion). The meeting highlighted the complexity of the immunologic processes (alloimmune and others) that lead to allograft inflammation and the need to strengthen the Banff system for differential diagnostic reasoning. Guidance is put in place for cases with incomplete/mixed phenotypes, which acknowledges the limits of our understanding. A proposal for potential future implementation of activity and chronicity indices was discussed, as well as digital and biopsy-based molecular tools that have the potential to help transform the classification into a probabilistic tool reflective of the underlying immunologic processes. Finally, guidelines for reporting of glomerular disease in the posttransplant setting were developed.

Background Since their inception, Delphi studies have been a key part of medical literature. They consist of an expert panel tasked with coming to consensus on answers to various questions where obtaining objective results is difficult or impossible, with ranked responses based on a Likert scale. The ability of artificial intelligence (AI), particularly large language models (LLMs), to perform this role traditionally assigned to a panel of experts has been scarcely explored in medicine. This study accordingly aimed to explore the feasibility of an “AI-run” Delphi study applied to the practice of pathology. Methods A prior human-based Delphi study (PMID: 36603288) employed to forecast the future role of AI in pathology was repeated, but this time with LLMs (Llama 3, ChatGPT-4, and ChatGPT-3.5 based on availability at the time of the study). This was done at various temperature settings (0, 0.7, and 1.0), a measurement of how much an LLM prioritizes determinism versus creativity. Low temperature caused the models to be more deterministic and focused, whereas high temperature increased creativity. “Delphi-GPT” was created to automate prompts that entailed 5 trials for 180 questions, leading to data that were compared to the original human expert panel. Findings All LLM and temperature combinations were able to reach consensus for a greater percentage of the 180 questions posed than human experts. Newer ChatGPT-4 and Llama 3 models performed better than ChatGPT-3.5. Whereas AI models and human experts did not always agree, the amount of agreement increased when the temperature setting was increased across all LLMs. Interpretation LLMs are shown here to successfully be able to simulate a Delphi study in medicine. The data show that generative AI models were consistently able to reach greater degrees of consensus than human experts in their responses to 180 prompts related to the future practice of pathology. This serves as a proof-of-concept that one day, pending further robust methodological validation, AI could even serve as a surrogate for de novo Delphi studies that ordinarily would have relied on feedback from a panel of experts. The reliability of consensus/concordance achieved will depend upon the combination of LLM and temperature setting selected.

Current desensitization and maintenance immunosuppression regimens for kidney transplantation in sensitized individuals show limited ability to control the posttransplant humoral response, resulting in high rates of antibody-mediated rejection (ABMR) and graft failure. Here, we showed that anti-CD154 monoclonal antibody (mAb)-based immunosuppression more effectively controlled allograft rejection and humoral rebound in a highly sensitized nonhuman primate kidney transplantation model compared with tacrolimus-based standard-of-care (SOC) immunosuppression. Desensitization with an anti-CD154 mAb (5C8) and a proteasome inhibitor led to decreased donor-specific antibodies (DSAs) and disruption of lymph node germinal centers with reduction of proliferating, memory, and class-switched B cells as well as T follicular helper cells. After transplant, the nonhuman primates maintained on 5C8-based immunosuppression had significantly better survival compared with those maintained on SOC immunosuppression (135.2 days versus 32.8 days, P = 0.013). The 5C8-treated group demonstrated better suppression of DSAs after transplant, more robust suppression of B cell populations, and better induction of regulatory T cells. Fewer infectious and welfare complications, including viral reactivation and weight loss, were also observed with 5C8-based immunosuppression compared with SOC immunosuppression. Therefore, anti-CD154 mAbs may improve kidney transplant outcomes through better control of posttransplant immune responses. The superior efficacy of anti-CD154 mAb-based immunosuppression over tacrolimus-based SOC seen in this highly sensitized NHP transplant model suggests that anti-CD154 mAbs could potentially be used to desensitize and treat highly sensitized patients receiving kidney transplantation.

INTRODUCTION:"Delayed" antibody-mediated xenograft rejection is one of the most important obstacles to clinical application of pig organ xenografts. The aim of this study was to assess the impact of a structured desensitization regimen including proteasome inhibition and next-generation costimulation blockade on xenoreactive antibodies. METHODS:Rhesus macaques with moderate-high pre-treatment xenoreactive antibody titers (N = 2) were selected. Recipients received twice-weekly carfilzomib (20 mg/m2), anti-CD154 (20 mg/kg) every other week, and CD4 and CD20 lymphocyte cell depletion. Bone marrow was acquired to assess plasma cell depletion in response to proteasome inhibition. A flow cytometry-based xenoreactive crossmatch assay was performed to assess levels of circulating xenoreactive antibodies. RESULTS:The desensitization regimen resulted in a >50% depletion of CD38+CD27+ bone marrow plasma cells; these changes were progressive over the duration of the desensitization treatment period. The desensitization strategy and plasma cell depletion resulted in a progressive reduction in anti-pig IgG antibodies. Following xenotransplantation, both desensitized recipients demonstrated superior graft survival to a highly xenoreactive recipient (MST 30 days vs. 6 days), but neither desensitized recipient experienced prolonged graft survival. CONCLUSIONS:A structured desensitization regimen including proteasome inhibition and costimulation blockade results in plasma cell depletion and resultant reduction in circulating xenoreactive anti-pig IgG antibodies, with a modest improvement in xenograft survival. This desensitization regimen has promise for pig-to-NHP xenotransplant models.

Kidney allograft rejection occurs in clinically stable patients, but its long-term significance in children is unknown. Previous studies demonstrated that subclinical (SC) inflammation is associated with an increased risk of rejection. However, the prevalence and significance of SC antibody-mediated rejection (AMR) and the impact of SC rejection phenotypes on graft survival remains to be assessed. We included children who underwent transplantation from 8 centers in France and the United States performing surveillance biopsies and compared the risk of acute rejection and graft loss stratified on surveillance biopsies' findings. In total, 1406 surveillance biopsies were performed in 776 kidney transplantation recipients including 134 (10%) SC borderline, 46 (3%) SC T cell-mediated rejection, 42 (3%) SC AMR, 9 (1%) SC mixed rejections. SC rejection was associated with acute rejection (5-year rejection-free survival of 88%, 78%, 68%, and 63% in the no rejection, SC borderline, SC T cell-mediated rejection, and SC AMR groups, respectively). Treatment of SC borderline lesions was not associated with a decreased incidence of clinical rejection. SC AMR was associated with a lower 5-year graft survival (P = .02). SC rejection is associated with acute rejection in stable pediatric kidney recipients, while SC AMR only is associated with an increased risk of allograft failure. Further studies evaluating the impact of treating these SC findings are needed.

The purpose of this white paper is to recommend the minimum reporting standards for pathologic characterization of kidney and heart xenografts in humans. This proposal is based on the current human classifications for kidney and heart allografts, with additions and caveats relevant to xenografts that are primarily based on non-human primate and a limited number of organ xenografts in decedent and living humans. Such recommendations should not be regarded as diagnostic criteria, given that many new pathologic patterns and mechanisms remain to be fully characterized. While xenograft transplantation continues to evolve, this report serves as groundwork and an initial step towards defining international standards in xenograft histopathology assessment and reporting. Note: The authors welcome comments from the international transplantation community through the AJT Banff Blog: https://amjtransplant.wixsite.com/ajtbanffblog

BACKGROUND:Skin transplantation is often used in nonhuman primate (NHP) transplant research for sensitization and to assess tolerance. However, the availability of donor animals for skin can be constrained after organ donation. In this study, we evaluated the feasibility of cryopreserving skin patches to address this issue. METHODS:A full-thickness dorsal skin section with a diameter of ∼3 cm was excised. Following defatting process, the skin graft was immersed in a skin-cryopreservation media and preserved in liquid nitrogen. Prior to transplantation, the frozen skin was rapidly thawed using a 37°C water bath and thoroughly washed with normal saline. Cryopreserved syngeneic skin transplantations were performed, monitored, and assessed histologically. RESULTS:Autologous skin grafts preserved in liquid nitrogen with prolonged storage time (>1 month) demonstrated successful engraftment in NHP recipients. The cryopreserved skin exhibited well-preserved epidermis and dermis, with minimal distinction compared to non-cryopreserved samples. In fully engrafted cryopreserved skin grafts, dermal fibrosis appeared slightly less distinct at 28 days after transplantation. These differences could be attributable to anatomical location of the samples. CONCLUSION:The cryopreserved skin showed well-preserved normal skin histology before and after skin transplantation. Here, we show that cryopreserved NHP skin can be stored for a prolonged time and grafted in a delayed manner. Cryopreserved skin could serve as a source when a living donor NHP is unavailable, including for testing donor-specific tolerance in the absence of the organ donor.

BACKGROUND:Visual scoring of tubular damage has limitations in capturing the full spectrum of structural changes and prognostic potential. Here, we investigated if computationally quantified tubular features can enhance prognostication and reveal spatial relationships with interstitial fibrosis. METHODS:Deep-learning and image-analysis approaches were employed on 254/266 Periodic acid Schiff-stained whole slide image (WSI) kidney biopsies from participants in the NEPTUNE/CureGN prospective observational cohort studies (135/153 with focal segmental glomerulosclerosis (FSGS) and 119/113 with minimal change disease (MCD)) to segment cortex, tubular lumen (TL), epithelium (TE), nuclei (TN), and basement membrane (TBM). One hundred four pathomic features were extracted from these segmented tubular substructures and aggregated at the patient level using summary statistics. In the NEPTUNE dataset, tubular features were quantified at the WSI level and in manually segmented regions of mature interstitial fibrosis and tubular atrophy (IFTA), pre-IFTA, and non-IFTA. Minimum Redundancy Maximum Relevance was then used to select features most associated with disease progression and proteinuria remission. Ridge-penalized Cox models evaluated their predictive discrimination compared to clinical/demographic data and visual-assessment. Models were evaluated in the CureGN dataset. RESULTS:Nine features were predictive of disease progression and/or proteinuria remission. Models with tubular features had high prognostic accuracy in both NEPTUNE and CureGN, and higher prognostic accuracy for both outcomes compared to conventional parameters alone in NEPTUNE. TBM thickness/area and TE flattening and/or reduced cell size progressively increased from non- to pre- and mature IFTA. CONCLUSIONS:Previously underrecognized computationally derived and quantifiable tubular characteristics may contribute to improving prognostic accuracy and risk stratification in patients with FSGS/MCD. Future studies are needed to test their generalizability across different diseases and populations before they can be deployed in clinical practice.

Frozen section biopsies stained with Hematoxylin & Eosin (H&E) are standard for assessing donor kidneys but are often interpreted by general pathologists with limited renal expertise, and hindered by freezing artifacts and poor tissue morphology. There is currently no rapid, cost-effective, and easily deployable method for generating virtual special stains and quantifying clinically significant histopathological features on frozen section H&E slides. This study evaluates the utility of DUET-generated virtual Periodic acid-Schiff (vPAS) stains produced from H&E-stained frozen kidney biopsies. DUET produces virtual PAS by combining pixel-registered brightfield and fluorescence images from the frozen section H&E-stained slide, overlaying extracted collagen masks to generate the virtual stain. Renal and general pathologists evaluated interstitial fibrosis/tubular atrophy (IF/TA), inflammation, and arteriosclerosis on 29 kidney biopsies, comparing their assessments using H&E images alone versus using both H&E and virtual PAS images. Among general pathologists' evaluations, ICC scores from the H&E to virtual PAS stain increased for IF/TA percentage, inflammation percentage, and arteriosclerosis number. For renal pathologists' evaluations, ICC scores from the H&E to virtual PAS stain increased for the percentage of sclerotic glomeruli, IF/TA percentage, inflammation percentage, and number of arteriosclerotic lesions. No improvement in ICC score was observed for arteriolar hyalinosis in either pathologist's group. We have demonstrated that the use of frozen virtual PAS stains enables higher consistency among pathologist evaluations when compared to the use of frozen section H&E alone for various metrics used to assess donor kidney viability. This approach has the potential to reduce diagnostic variability, improve transplant decision-making, and optimize donor kidney utilization.