The American Journal of Pathology

Cytomegalovirus (CMV) is a major opportunistic infection after liver transplantation and often mimics acute T cell-mediated rejection (TCMR), creating management uncertainty. We conducted a retrospective study to identify practical histologic features that distinguish CMV hepatitis from TCMR in routine practice. We included 10 recipients with CMV hepatitis and 5 with moderate to severe TCMR. Portal inflammation, bile duct injury, venous endotheliitis, lobular microgranulomas, neutrophilic microabscesses, and CMV inclusions were assessed. Clinical data were abstracted from the medical record. CMV hepatitis was diagnosed earlier after transplantation than TCMR (272 {+/-} 211 vs 549 {+/-} 522 days). Slides were available for 7 CMV and all 5 TCMR biopsies. Histologic findings and their diagnostic performance estimates were as follows: microgranulomas present in 7/7 CMV and 0/5 TCMR biopsies, sensitivity 100% and specificity 100%; bile duct injury minimal to absent in 6/7 CMV and 0/5 TCMR biopsies, sensitivity 86% and specificity 100%; neutrophilic microabscesses in 3/7 CMV and 0/5 TCMR biopsies, sensitivity 42.9% and specificity 100%. Antiviral therapy was administered in 9/10 CMV patients (90%); recurrent CMV viremia occurred in 4/10 (40%) and late chronic rejection in 2/10 (20%), while no CMV viremia occurred in the TCMR group. In routine practice, a pattern of portal lymphohistiocytic inflammation with lobular microgranulomas and minimal to absent bile duct injury supports CMV hepatitis over TCMR and can guide targeted search for inclusions and CMV PCR, which may help avoid unnecessary intensification of immunosuppression and enable timely antiviral therapy.

IntroductionAccurate intraoperative assessment of macrovesicular steatosis in donor liver biopsies is critical for transplantation decisions but is often limited by inter-observer variability and freezing artifacts that can obscure histological details. Artificial intelligence (AI) offers a potential solution for standardized and reproducible evaluation. To evaluate the diagnostic performance of two self-supervised learning (SSL)-based foundation models, Prov-GigaPath and UNI, for classifying macrovesicular steatosis in frozen liver biopsy sections, compared with assessments by surgical pathologists. MethodsWe retrospectively analyzed 131 frozen liver biopsy specimens from 68 donors collected between November 2022 and September 2024. Slides were digitized into whole-slide images, tiled into patches, and used to extract embeddings with Prov-GigaPath and UNI; slide-level classifiers were then trained and tested. Intraoperative diagnoses by on-call surgical pathologists were compared with ground truth determined from independent reviews of permanent sections by two liver pathologists. Accuracy was evaluated for both five-category classification and a clinically significant binary threshold (<30% vs. [&ge;]30%). ResultsFor binary classification, Prov-GigaPath achieved 96.4% accuracy, UNI 85.7%, and surgical pathologists 84.0% (P = .22). In five-category classification, accuracies were lower: Prov-GigaPath 57.1%, UNI 50.0%, and pathologists 58.7% (P = .70). Misclassification primarily occurred in intermediate categories (5%-<30% steatosis). ConclusionsSSL-based foundation models performed comparably to surgical pathologists in classifying macrovesicular steatosis, at the clinically relevant <30% vs. [&ge;]30% threshold. These findings support the potential role of AI in standardizing intraoperative evaluation of donor liver biopsies; however, the small sample size limits generalizability and requires validation in larger, balanced cohorts.

Hepatic stellate cells (HSCs) play a central role in liver fibrosis, shifting from quiescent vitamin A-storing cells to activated, myofibroblast-like cells that secrete collagen and other profibrotic factors1. HSCs have thus become a major focus in liver fibrosis research, and several Cre driver lines have been created to target HSCs in mice. However, early Cre lines had significant limitations. Glial fibrillary acidic protein (Gfap)-Cre labels only a subset of HSCs and also induces recombination in cholangiocytes2. Collagen type I alpha 1 (Col1a1)-Cre and alpha-smooth muscle actin (SMA)-Cre/CreERT2 primarily label activated myofibroblasts and broadly mark portal fibroblasts and vascular smooth muscle cells3,4. Platelet-derived growth factor receptor beta (Pdgfr{beta})-Cre reliably labels HSCs but also recombines pericytes and smooth muscle cells, limiting its specificity5. The introduction of lecithin-retinol acyltransferase (Lrat)-Cre marked a major advance, offering highly specific labeling of quiescent and activated HSCs and rapidly becoming the most widely used driver for HSC tracing and genetic perturbation2. However, the extrahepatic expression of Lrat-Cre remains incompletely understood. This is a critical limitation, given that liver biology is closely coordinated with other organs to maintain systemic metabolism. Addressing these gaps is essential for the accurate interpretation of HSC-specific genetic models in liver biology.

Background and AimsIn severe cases of acetaminophen (APAP) overdose, acute liver injury rapidly progresses to acute liver failure (ALF), producing life-threatening complications including, hepatic encephalopathy (HE) and multi-organ failure (MOF). Systemic levels of interleukin-6 (IL-6) and IL-10 are highest in ALF patients with the most severe complications and the poorest prognosis. The mechanistic basis for dysregulation of these cytokines, and their association with outcome in ALF, remain poorly defined. MethodsTo investigate the impact of IL-6 and IL-10 in ALF, we used an experimental setting of failed liver repair after APAP overdose in which a high dose of APAP is administered (i.e., 500-600 mg/kg). Mice were treated with neutralizing antibodies to block IL-6 and IL-10. ResultsIn mice with APAP-induced ALF, high levels of IL-10 reduced monocyte recruitment and trafficking in the liver resulting in impaired clearance of dead cell debris. Kupffer cells in these mice, displayed features of myeloid-derived suppressor cells, including high level expression of IL-10 and PD-L1, which were increased in an IL-6-dependent manner. Similar to ALF patients with HE, cerebral blood flow was reduced in mice with APAP-induced ALF. Remarkably, although IL-6 is hepatoprotective in mice treated with low doses of APAP (i.e., 300 mg/kg), IL-6 neutralization in mice with APAP-induced ALF fully restored cerebral blood flow and reduced mortality. ConclusionCollectively, these studies demonstrate that exaggerated production of IL-6 in APAP-induced ALF triggers immune suppression (i.e., high levels of IL-10 and PD-L1), reduces cerebral blood flow (a feature of hepatic encephalopathy), disrupts liver repair (i.e., failed clearance of dead cells), and increases mortality.

BackgroundTrauma and shock often severely affect the kidneys. This can lead to trauma-related acute kidney injury (TRAKI), which significantly increases the risk of adverse outcomes. MethodsTo study the pathophysiology of TRAKI, we developed a murine model of combined blunt thoracic trauma and pressure-controlled hemorrhage that induces mild transient TRAKI. ResultsThe mice showed early and transient increased plasma creatinine, urea, NGAL, and urine albumin, resolving 5 days after TRAKI induction. Despite normal kidney morphology, significant damage to proximal tubular cells and a loss of the brush border was observed. This included kidney stress responses, e.g., with induced heme oxygenase-1 expression in tubules. The upregulation of inflammatory mediators and kidney injury markers was followed by elevated leukocyte numbers, mainly consisting of monocytes/macrophages. Proteomic analyses revealed a distinct time course of intrarenal processes after trauma. 3D x-ray-based whole-organ histology by contrast-enhanced microcomputed tomography showed significant impairment of capillary blood flow, especially during the first day post THS, which was partly resolved by day 5. ConclusionsOur novel model of murine TRAKI has revealed previously unknown aspects of the complex temporal pathophysiological response of the kidney along the nephron after trauma and hemorrhage, which may provide mechanistic starting points for future therapeutic approaches.

Autologous pancreatic islet transplantation is an established therapy for patients with chronic pancreatitis. However, the long-term transplant outcomes are modest. Identifying indicators of graft function will aid the preservation of transplanted islets and glycemic control. To this end, we analyzed beta cell prohormone peptide levels in a retrospective cohort of total pancreatectomy autologous islet transplant patients (n=28). Proinsulin-to-C-peptide (PI/C) and proIAPP-to-total IAPP (proIAPP/IAPP) ratios measured at 3 months post-transplant were significantly higher in patients who remained insulin dependent at 1 year follow-up. In a mouse model of human islet transplantation, recipient mice that later became hyperglycemic displayed significantly higher PI/C ratios than mice that remained normoglycemic. Histological analysis of islet grafts showed reduced insulin- and proinsulin-positive area, but elevated glucagon-positive area in grafts that experienced greater secretory demand. Increased prohormone convertase 1/3 was detected in glucagon-positive cells, and glucagon-like peptide 1 (GLP-1) area was elevated in grafts from mice that displayed hyperglycemia or elevated plasma PI/C ratios, demonstrating intra-islet incretin production in metabolically challenged human islet grafts. These data indicate that in failing grafts, alpha cell prohormone processing is likely altered, and incomplete beta cell prohormone processing may be an early indicator of insulin dependency.

Snakebite claims 138,000 lives a year with an additional 400,000 patients left permanently disabled or disfigured1. Morbidity following envenoming includes the development of chronic wounds around the bite site. The understanding of the underlying pathophysiology of chronic snakebite wounds has been severely limited by the historical reliance on a preclinical model that only captures acute local envenoming pathology. Through the application of three medically important snake venoms (Echis ocellatus, Bothrops atrox and Naja nigricollis) to a recently developed preclinical model of chronic wounds, we have been able to characterise key features of venom wounds. We have been able to show that venom wounds share consistencies with non-venom induced preclinical wounds, and also display unique characteristics such as extracellular matrix degradation and eosinophilic infiltrate. This model will not only serve to increase our understanding the underlying pathophysiology of venom wounds, but will also provide a platform for exploring therapeutic interventions to reduce or resolve snakebite wounds.

PurposeProliferative vitreoretinopathy (PVR) is the most common cause of failure of surgically repaired rhegmatogenous retinal detachment (RRD). Chemically-induced and cell-injection PVR models do not fully simulate the clinical characteristics of PVR in the post-RRD context. There is an unmet need for translational models in which to study mechanisms and treatments specific to RRD-PVR. MethodsRRD-PVR was induced in adult Dutch Belted rabbits. Posterior segments of enucleated globes were fixed or processed for RNA-Seq at 6 hours and 2, 7, 14, and 35 days post-induction. Histochemical staining and immunolabeling for glial fibrillary acidic protein (GFAP), alpha smooth muscle actin (SMA), vascular endothelial growth factor receptor 2 (VEGFR2), CD68, and retinal pigment epithelium 65 kDa protein (RPE65) were performed, and labeling intensity was scored. Single cell RNA sequencing was performed. ResultsAcute histopathologic changes included intravitreal and intraretinal hemorrhage, leukocytic vitritis, chorioretinitis, and retinal rarefaction. Chronic lesions showed retinal atrophy, gliosis, fibrotic subretinal membranes, and epiretinal fibrovascular proliferation. Fibrillar collagen was present in the fibrocellular and fibrovascular membranes in chronic lesions. Moderate to strong labeling of glia and vasculature was detected in chronic lesions. At day 14, most cells profiled by single cell sequencing were identified as Muller glia and microglia, consistent with immunolabeling. Expression of several fibrillar collagen genes were upregulated in chronic lesions. ConclusionsHistologic and transcriptional features of this rabbit model simulate important features of human RRD-PVR, including the transition to chronic intra and periretinal fibrosis. This high-fidelity in vivo model of RRD-PVR will enable further research on targeted treatment interventions.

Capillary regression destabilizes tissue homeostasis and contributes to chronic organ dysfunction, yet the inflammatory pathways that drive pathological vessel loss remain incompletely defined. We previously identified the inflammatory cytokines TNF and IL-1 as conserved mediators of physiological vessel regression in the neonatal mouse eye, but whether these cytokines contribute to pathological capillary regression in adult mice is unknown. In this study, we investigated the capillary regression that occurs along with inflammation in murine kidneys following irreversible unilateral ureteral obstruction (UUO) surgical challenge. Mice lacking genes encoding global TNF, the endothelial IL-1 receptor IL-1R1, or both (double knockout, DKO) were examined at 10 days after UUO surgery. While loss of the individual genes did not affect peritubular capillary (PTC) regression, PTC regression was significantly reduced in DKO mice. This reduction in PTC regression correlated with less expression of the tubular epithelial injury marker KIM-1. DKO kidneys also displayed less fibrosis by Picrosirius Red and Massons trichrome staining. These findings demonstrate that TNF and endothelial IL-1R1 cooperatively drive pathological capillary regression in the irreversible UUO model of chronic kidney injury and that preservation of PTCs correlates with less renal tubular injury and fibrosis at 10 days after injury. NEW & NOTEWORTHYPathological PTC regression drives progressive kidney injury, but its inflammatory triggers are unclear. Using the UUO model, this study identifies cooperative TNF and endothelial IL-1R1 signaling as key drivers of capillary loss. While deletion of either pathway alone was insufficient, combined loss preserved PTCs, reduced tubular injury, and attenuated fibrosis. These findings highlight synergistic inflammatory signaling in microvascular loss and suggest dual targeting may help preserve PTC integrity in chronic kidney disease.

AimsDetermine if pathologic assessment of disease activity in steatohepatitis, performed using Whole Slide Images (WSIs) on the AISight Clinical Trials platform, yields results that are comparable to those obtained from the analysis performed using glass slides. Methods and ResultsThe accuracy of scoring for steatohepatitis (NAS [&ge;]4 with [&ge;]1 for each feature and absence of atypical features suggestive of other liver disease) performed on the WSI viewing platform was evaluated against scoring conducted on glass slides. Both methods were assessed for overall percent agreement (OPA) with a consensus ground truth (GT) score, defined as the median score of a panel of 3 expert pathologists on glass slides. Each case was also read by 3 different pathologists, once on glass and once using WSIs with a minimum 2-week washout period between glass and WSI reads. It was demonstrated that the average OPA across 3 pathologists of WSI scoring with GT was non-inferior to the average OPA of glass scoring with GT (non-inferiority margin of -0.05, difference of -0.001, 95% CI of (-0.027,0.026), and p<0.0001). For each pathologist, there was a similar average OPA of WSI and glass reads with glass GT (pathologist A 0.843 and 0.849, pathologist B 0.633 and 0.605 and pathologist C 0.755 and 0.780), with intra-reader, inter-modality agreements per histologic feature being greater than published intra-reader agreements. ConclusionAccuracy of digital reads for steatohepatitis using WSIs is equivalent to glass reads in the context of a clinical trial for scoring using the Clinical Research Network scoring system.

Endomucin (EMCN), an endothelial-specific glycocalyx component highly expressed in capillary and venous endothelium, plays a critical role in regulating VEGF receptor 2 (VEGFR2) endocytosis and downstream VEGF signaling. Using the first global EMCN knockout mouse model, we investigated the effects of EMCN deficiency on retinal vascularization during development and pathological angiogenesis. We found relatively high expression of EMCN in choroidal capillaries and retinal vasculature. Emcn-/- mice exhibited delayed retinal vascularization at postnatal day 5, with fewer tip cells and reduced vessel density. Ultrastructural examination revealed disrupted and reduced fenestrations in choroidal capillary endothelium. In an oxygen-induced retinopathy model, while Emcn-/- mice showed no significant difference in avascular area compared to Emcn+/+ mice at postnatal day 12, there was a significant reduction in neovascular tufts in Emcn-/- mice at postnatal day 17. Similarly, in a laser-induced choroidal neovascularization model, Emcn-/- mice showed a significant reduction in vascular leakage and lesion size. These findings suggest that EMCN plays a critical role in both vascular development and pathological neovascularization, highlighting its potential as a target for anti-angiogenic therapies.

Fuchs endothelial corneal dystrophy (FECD) impacts over 300 million individuals worldwide with corneal transplantation as the primary treatment. There is a dire need to establish non-surgical alternatives which are dependent on mouse models. Transcriptional co-activator with PDZ-binding motif (TAZ, encoded by Wwtr1) is a mechanotransducer implicated in maintaining homeostasis of corneal endothelial cells (CEnC). Wwtr1-/- (TAZ KO) mice serve as an animal model for late-onset FECD. We combined single-cell transcriptomics, transmission electron microscopy, and immunofluorescence staining to elucidate the mechanisms driving pathogenesis in young (2-month-old) and geriatric (11-month-old) mice. A progressive stress response was observed in TAZ KOs defined by endoplasmic reticulum (ER) stress, mitochondrial structural and functional abnormalities, and impaired Na+/K+ ATPase localization. These changes were accompanied by an altered expression of genes involved in extracellular matrix (ECM) organization, oxidative phosphorylation, macroautophagy and response to oxidative stress. Additionally, we noted age-related differences in cellular response with young TAZ KO CEnCs upregulating macroautophagy and downregulating ECM organization while geriatric TAZ KO CEnCs downregulated macroautophagy, and ECM organization. Both TAZ KO groups downregulated response to oxidative stress and cell-substrate adhesion. Together, these findings establish a mechanistic link between disrupted mechanotransduction and organelle stress in CEnC degeneration, further elaborating on potential mechanisms driving FECD pathogenesis. This positions TAZ KO mice as a translational platform for evaluating non-surgical therapeutic strategies targeting FECD. Significance statementFuchs endothelial corneal dystrophy (FECD) is a common, age-related cause of vision loss involving a depletion of corneal endothelial cells (CEnC) that necessitates corneal transplantation. Understanding why corneal endothelial cells progressively fail in this disease is essential for developing non-surgical therapies. Using transcriptomics, electron microscopy and immunofluorescence staining, we demonstrate that loss of the mechanotransducer TAZ disrupts cellular homeostasis by inducing endoplasmic reticulum stress, mitochondrial dysfunction and improper extracellular matrix and functional protein organization in CEnCs. By linking altered mechanotransduction to organelle stress and endothelial cell loss, these findings provide insight into fundamental disease mechanisms and identify pathways that may be targeted to preserve corneal endothelial function in FECD.

BackgroundThe novel coronavirus, SARS-CoV2 that causes COVID-19 has resulted in the death of more than 2.31 million people within the last year and yet no cure exists. Whereas passive immunization with COVID-19 convalescent plasma (CCP) provides a safe and viable option, selection of optimal units for therapy and lack of clear therapeutic benefit from transfusion remain as barriers to the use of CCP. Study design and methodsTo identify plasma that is expected to benefit recipients, we measured anti-SARS-CoV2 antibody levels using clinically available serological assays and correlated with the neutralizing activity of CCP from donors. Neutralizing titer of plasma samples was measured by assaying infectivity of SARS-CoV-2 spike protein pseudotyped retrovirus particles in the presence of dilutions of plasma samples. We also used this assay to identify evidence of passive transfusion of neutralizing activity in CCP recipients. ResultsViral neutralization and anti-spike protein antibodies in 109 samples from 87 plasma donors were highly varied but modestly correlated with each other. Recipients who died of COVID-19 were found to have been transfused with units with lower anti-spike antibody levels and neutralizing activity. Passive transfer of neutralization activity was documented in 62% of antibody naive plasma recipients. ConclusionsSince viral neutralization is the goal of CCP transfusion, our observations not only support the use of anti-spike SARS-CoV2 serology tests to identify beneficial CCP units, but also support the therapeutic value of convalescent plasma with high titers of anti-spike antibodies.

RationaleLung injury after hematopoietic stem cell transplantation (HCT) occurs due to infection, chemotherapy toxicity, and alloreactive inflammation. Analyses of bronchoalveolar lavage (BAL) fluid have revealed dominant pathobiologic signatures, but minimally-invasive diagnostics are needed. ObjectivesTo determine whether microbiome and gene expression perturbations are shared along the respiratory tract or isolated to the alveoli in pediatric HCT patients with lung injury. MethodsWe performed bulk RNA sequencing on 189 paired nasal and BAL samples from 160 patients enrolled at 28 childrens hospitals (2016-2021). Microbial and human transcripts were compared using multivariable models accounting for age, sex, and paired sampling. Measurements and Main ResultsBAL and nasal transcriptomes differed across 13,698 genes, 48 cellular components, and network interactions linking inflammation, reactive oxygen species, and immunometabolism. Minimal BAL-nasal correlation was observed in gene expression levels (median {rho}=0.03, IQR -0.03 to 0.08) or fractional abundance of key cells such as neutrophils and CD8+ T-cells. BAL microbiomes harbored fewer commensal bacteria and more fungi and DNA viruses. BAL bacterial RNA was associated with diminished immune signaling whereas nasal bacterial RNA aligned with inflammatory gene expression. Further, only BAL microbial RNA was linked to transcriptional shifts in epithelial injury response, keratinization, and collagen deposition. Finally, BAL commensal microbiome depletion, epithelial injury, and immune dysregulation signatures were associated with death or [&ge;]7 days of mechanical ventilation in 30% of patients, whereas nasal samples provided minimal prognostic information. ConclusionsThese data support alveolar compartmentalization in pediatric HCT and emphasize the ongoing need for minimally-invasive but informative diagnostics.

BackgroundAn emerging clinical phenomenon in patients with end stage liver disease is progressive skeletal muscle atrophy. This loss in lean mass predicts poor survival outcomes for liver disease patients and highlights an underappreciated crosstalk between injured liver and muscle that lacks defined mediators. The purpose of our study was to identify potential liver-muscle mediator(s) in pre-clinical in vivo models of liver injury which may contribute to the muscle loss observed in liver disease. MethodsUtilizing a mouse model of carbon tetrachloride CCl4-induced liver injury in the presence or absence of cardiotoxin-induced muscle injury, we evaluated whether neutralizing Activin type IIB receptor (ActRIIB) ligands, or specifically growth differentiation factor 8 (Gdf8), could preserve or reverse muscle atrophy associated with liver disease. ResultsWe found that hepatic injury via CCl4 or bile duct ligation (BDL) similarly caused significant muscle atrophy along with decreased gene expression in key myogenesis markers. This adverse effect of injured liver on muscle were completely prevented and reversed by the intervention of Activin type IIB receptor (ActRIIB)-Fc fusion protein, which neutralizes the ActRIIB ligands, including Activins and growth differentiation factor 8 (Gdf8 or myostatin). The results indicate that ActRIIB ligands promoted muscle atrophy which was manifested in response to hepatic injury/disease and conferred the negative communication of injured liver with muscle. Indeed, direct injection of exogenous Gdf8 protein into muscle along with acute focal muscle injury recapitulated similar dysregulated muscle regeneration as observed with liver injury. Furthermore, we found that hepatocytes produced Gdf8 in response to liver injury in rodents and in patients with end stage liver disease. A neutralizing antibody to Gdf8 attenuated muscle atrophy and unexpectedly ameliorated liver fibrosis in both CCl4 and BDL models. Following this observation, we demonstrated Gdf8s ability to induce fibrogenesis in stellate cells, potentially identifying a novel hepatic role for this protein. Moreover, hepatic Gdf8 promoted muscle wasting in response to liver damage and hindered skeletal muscle regeneration. ConclusionOur findings identified Gdf8 as a novel hepatomyokine contributing to injured liver-muscle negative crosstalk and liver injury progression. Moreover, we demonstrated a promising therapeutic strategy for muscle atrophy accompanying liver diseases.

Acute pancreatitis is a common inflammatory condition of the pancreas that can lead to severe complications such as chronic pancreatitis and pancreatic ductal adenocarcinoma. While vascular remodeling is a hallmark of many inflammatory conditions, the molecular mechanisms underlying vascular changes during pancreatitis remain largely unexplored. This study aimed to investigate the vascular changes associated with acute pancreatitis and to identify the molecular mechanisms underlying these changes. Acute pancreatitis was induced in wild-type mice through caerulein injections and resulted in progressive and substantial vascular changes, characterized by morphological remodeling, increased vessel density, and elevated vascular permeability. These structural changes were accompanied by molecular alterations, including increased expression of endothelial genes and decreased surface expression of vascular endothelial VE-cadherin. Injured acinar cells exhibited a significant increase in VEGF-A expression during pancreatitis. Acinar-specific VEGF-A inactivation led to marked impairments in vascular remodeling, with reduced vessel density and diminished number of vessels, without affecting immune cell infiltration, fibrosis, or acinar-to-ductal metaplasia. Together, our work identifies a mechanism by which increased expression of VEGF-A by acinar cells during pancreatitis is essential for maintaining vascular integrity and for driving vascular remodeling in the inflamed pancreas.

Background and aimsHepatocyte apoptosis is a key feature of non-alcoholic steatohepatitis (NASH), but the fate of apoptotic hepatocytes in NASH is poorly understood. Herein we explore the hypothesis that impaired TIM4-mediated clearance of dead hepatocytes by liver macrophages (efferocytosis) is impaired in NASH and drives the progression to liver fibrosis. MethodsKupffer cell (KC)-TIM4 expression and efferocytosis were assayed in normal and NASH liver from humans and diet-induced NASH mice. The engulfment of human and mouse apoptotic hepatocytes by primary human and mouse liver KCs was assayed ex vivo. Causation was assessed in NASH mice using anti-TIM4 antibodies, KC-TIM4-knockout, or inducible KC-TIM4 expression, with analyses focused on efferocytosis of apoptotic hepatocytes by liver macrophages and liver fibrosis. ResultsIn human and mouse NASH liver, apoptotic hepatocytes accumulated and was associated with the loss of the KC efferocytosis receptor TIM4. Anti-TIM4 inhibited the engulfment of apoptotic hepatocytes by primary human and mouse liver KCs ex vivo, and anti-TIM4 administration to early NASH mice worsened liver macrophage efferocytosis and accelerated the progression to fibrotic NASH. A similar result was obtained by genetically deleting TIM4 in KCs in NASH mice. Most importantly, genetic restoration of macrophage TIM4 in NASH mice enhanced the clearance of apoptotic hepatocytes by liver macrophages and decreased liver fibrosis. ConclusionsThe loss of macrophage TIM4 that occurs during NASH progression impairs the clearance of apoptotic hepatocytes by liver macrophages, which subsequently promotes the progression to fibrotic NASH. This pathogenic sequence of events can be prevented by restoring macrophage TIM4, suggesting that future therapeutic approaches designed to boost TIM4 expression in liver macrophages could represent a novel strategy to prevent fibrotic NASH progression. Lay summaryNonalcoholic steatohepatitis (NASH) is emerging as the leading cause of liver disease, but the processes leading to liver fibrosis in NASH, which determines clinical outcome, are incompletely understood. Our study provides evidence impaired clearance of dead liver cells by liver macrophages in NASH, which is due to loss of a macrophage receptor called TIM4, contributes to liver fibrosis. Knowledge of this process may suggest new ways to bolster the clearance of dead liver cells in NASH and thereby prevent the progression to liver fibrosis and subsequent liver disease.

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 pathologists 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.

Cell-associated kidney injury molecule-1 (KIM-1) exerts an anti-inflammatory role following kidney injury by mediating efferocytosis and downregulating the NF-{kappa}B pathway. KIM-1 cleavage blunts its anti-inflammatory activities. We reported that Mucin 1 (MUC1) is protective in a mouse model of ischemia-reperfusion injury (IRI). As both KIM-1 and MUC1 are induced in the proximal tubule (PT) during IRI and are ADAM17 substrates, we tested the hypothesis that MUC1 protects KIM-1 activity. Muc1 KO mice and wild-type (WT) littermates were subjected to IRI. KIM-1, MUC1 and ADAM17 levels (and signaling pathways) were assessed by immunoblotting. PT localization was assessed by confocal microscopy and in situ proximity ligation assay. Findings were extended using human kidneys and urine, and KIM-1-mediated efferocytosis assays in mouse PT cultures. In response to tubular injury in mouse and human kidneys, we observed induction and co-expression of KIM-1 and MUC1 in the PT. Compared to WT, Muc1 KO mice had higher urinary KIM-1 and lower kidney KIM-1. KIM-1 was apical in PT of WT kidneys, but predominately with luminal debris in Muc1 KO mice. Efferocytosis was reduced in Muc1 KO PT cultures when compared to WT cells, while inflammation was increased in Muc1 KO kidneys when compared to WT mice. MUC1 was cleaved by ADAM17 in PT cultures, and blocked KIM-1 shedding in MDCK cells. We conclude that KIM-1-mediated efferocytosis and thus anti-inflammatory activity during IRI is preserved in the injured kidney by MUC1 inhibition of KIM-1 shedding. NEW & NOTEWORTHYKIM-1 plays a key role in the recovery of the tubule epithelium during renal IRI by mediating efferocytosis and associated signaling that suppresses inflammation. Excessive cleavage of KIM-1 by ADAM17 provides decoy receptor that aggravates efferocytosis and subsequent signaling. Our data from studies in mice, patients and cultured cells show that MUC1 is also induced during IRI and competes with KIM-1 for cleavage by ADAM17. Consequently, MUC1 protects KIM-1 anti-inflammatory activity in the damaged kidney.

Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD) prevalent in Type-2 Diabetes (T2D) and contributes to progression of Non-Alcoholic Steatohepatitis (NASH). Acetyl-High Mobility Group Box 1 (HMGB1,) a proinflammatory isoform of HMGB1, is released as a DAMP in T2D associated hepatic inflammatory condition. Chromosomal Maintenance 1 (CRM1), a nuclear transporter protein, maintains the nuclear-cytoplasm translocation of hepatic HMGB1 in T2D. We hypothesize that inhibition of CRM1/HMGB1 nuclear shuttling is a therapeutic target for MASLD in T2D. Methods: We performed immunohistochemical analysis of acetyl-HMGB1 and CRM1 in human liver biopsies from control, T2D, and T2D-NASH (n:4 per group). H&E staining to evaluate inflammation and disease stratification. In vitro, studies involved targeted inhibition of CRM1 using Leptomycin-B and HMGB1 with Glycyrrhizin in T2D Huh7 human hepatocytes. Results: T2D subjects with NASH exhibit an increase in acetyl-HMGB1 nuclear and cytoplasmic translocation compared to DM and controls. Acetyl-HMGB1 increased 2-fold in the nucleus and 4-fold in the cytoplasm; CRM1 increased 6-fold in the nucleus and 8-fold in the cytoplasm of T2D/NASH subjects compared to controls. Targeted inhibition of CRM1 and HMGB1 prevented acetyl-HMGB1 hepatocyte release with the most prominent effect in T2D-NASH conditions. Conclusions: Hepatic CRM1/HMGB1 inhibition could be a potential therapeutic target for T2D-driven MASLD.