Histopathological biomarkers for predicting the tumour accumulation of nanomedicines.

The clinical prospects of cancer nanomedicines depend on effective patient stratification. Here we report the identification of predictive biomarkers of the accumulation of nanomedicines in tumour tissue. By using supervised machine learning on data of the accumulation of nanomedicines in tumour models in mice, we identified the densities of blood vessels and of tumour-associated macrophages as key predictive features. On the basis of these two features, we derived a biomarker score correlating with the concentration of liposomal doxorubicin in tumours and validated it in three syngeneic tumour models in immunocompetent mice and in four cell-line-derived and six patient-derived tumour xenografts in mice. The score effectively discriminated tumours according to the accumulation of nanomedicines (high versus low), with an area under the receiver operating characteristic curve of 0.91. Histopathological assessment of 30 tumour specimens from patients and of 28 corresponding primary tumour biopsies confirmed the score's effectiveness in predicting the tumour accumulation of liposomal doxorubicin. Biomarkers of the tumour accumulation of nanomedicines may aid the stratification of patients in clinical trials of cancer nanomedicines.

tRigon: an R package and Shiny App for integrative (path-)omics data analysis.

BACKGROUND: Pathomics facilitates automated, reproducible and precise histopathology analysis and morphological phenotyping. Similar to molecular omics, pathomics datasets are high-dimensional, but also face large outlier variability and inherent data missingness, making quick and comprehensible data analysis challenging. To facilitate pathomics data analysis and interpretation as well as support a broad implementation we developed tRigon (Toolbox foR InteGrative (path-)Omics data aNalysis), a Shiny application for fast, comprehensive and reproducible pathomics analysis. RESULTS: tRigon is available via the CRAN repository ( https://cran.r-project.org/web/packages/tRigon ) with its source code available on GitLab ( https://git-ce.rwth-aachen.de/labooratory-ai/trigon ). The tRigon package can be installed locally and its application can be executed from the R console via the command 'tRigon::run_tRigon()'. Alternatively, the application is hosted online and can be accessed at https://labooratory.shinyapps.io/tRigon . We show fast computation of small, medium and large datasets in a low- and high-performance hardware setting, indicating broad applicability of tRigon. CONCLUSIONS: tRigon allows researchers without coding abilities to perform exploratory feature analyses of pathomics and non-pathomics datasets on their own using a variety of hardware.

[Advances in computational quantitative nephropathology]. / Fortschritte in der computergestützten quantitativen Nephropathologie.

BACKGROUND: Semiquantitative histological scoring systems are frequently used in nephropathology. In computational nephropathology, the focus is on generating quantitative data from histology (so-called pathomics). Several recent studies have collected such data using next-generation morphometry (NGM) based on segmentations by artificial neural networks and investigated their usability for various clinical or diagnostic purposes. AIM: To present an overview of the current state of studies regarding renal pathomics and to identify current challenges and potential solutions. MATERIALS AND METHODS: Due to the literature restriction (maximum of 30 references), studies were selected based on a database search that processed as much data as possible, used innovative methodologies, and/or were ideally multicentric in design. RESULTS AND DISCUSSION: Pathomics studies in the kidney have impressively demonstrated that morphometric data are useful clinically (for example, for prognosis assessment) and translationally. Further development of NGM requires overcoming some challenges, including better standardization and generation of prospective evidence.

Detection of PatIent-Level distances from single cell genomics and pathomics data with Optimal Transport (PILOT).

Although clinical applications represent the next challenge in single-cell genomics and digital pathology, we still lack computational methods to analyze single-cell or pathomics data to find sample-level trajectories or clusters associated with diseases. This remains challenging as single-cell/pathomics data are multi-scale, i.e., a sample is represented by clusters of cells/structures, and samples cannot be easily compared with each other. Here we propose PatIent Level analysis with Optimal Transport (PILOT). PILOT uses optimal transport to compute the Wasserstein distance between two individual single-cell samples. This allows us to perform unsupervised analysis at the sample level and uncover trajectories or cellular clusters associated with disease progression. We evaluate PILOT and competing approaches in single-cell genomics or pathomics studies involving various human diseases with up to 600 samples/patients and millions of cells or tissue structures. Our results demonstrate that PILOT detects disease-associated samples from large and complex single-cell or pathomics data. Moreover, PILOT provides a statistical approach to find changes in cell populations, gene expression, and tissue structures related to the trajectories or clusters supporting interpretation of predictions.

Banff Digital Pathology Working Group: Image Bank, Artificial Intelligence Algorithm, and Challenge Trial Developments.

The Banff Digital Pathology Working Group (DPWG) was established with the goal to establish a digital pathology repository; develop, validate, and share models for image analysis; and foster collaborations using regular videoconferencing. During the calls, a variety of artificial intelligence (AI)-based support systems for transplantation pathology were presented. Potential collaborations in a competition/trial on AI applied to kidney transplant specimens, including the DIAGGRAFT challenge (staining of biopsies at multiple institutions, pathologists' visual assessment, and development and validation of new and pre-existing Banff scoring algorithms), were also discussed. To determine the next steps, a survey was conducted, primarily focusing on the feasibility of establishing a digital pathology repository and identifying potential hosts. Sixteen of the 35 respondents (46%) had access to a server hosting a digital pathology repository, with 2 respondents that could serve as a potential host at no cost to the DPWG. The 16 digital pathology repositories collected specimens from various organs, with the largest constituent being kidney (n = 12,870 specimens). A DPWG pilot digital pathology repository was established, and there are plans for a competition/trial with the DIAGGRAFT project. Utilizing existing resources and previously established models, the Banff DPWG is establishing new resources for the Banff community.

Finerenone Added to RAS/SGLT2 Blockade for CKD in Alport Syndrome. Results of a Randomized Controlled Trial with Col4a3-/- Mice.

SIGNIFICANCE STATEMENT: We hypothesized that triple therapy with inhibitors of the renin-angiotensin system (RAS), sodium-glucose transporter (SGLT)-2, and the mineralocorticoid receptor (MR) would be superior to dual RAS/SGLT2 blockade in attenuating CKD progression in Col4a3 -deficient mice, a model of Alport syndrome. Late-onset ramipril monotherapy or dual ramipril/empagliflozin therapy attenuated CKD and prolonged overall survival by 2 weeks. Adding the nonsteroidal MR antagonist finerenone extended survival by 4 weeks. Pathomics and RNA sequencing revealed significant protective effects on the tubulointerstitium when adding finerenone to RAS/SGLT2 inhibition. Thus, triple RAS/SGLT2/MR blockade has synergistic effects and might attenuate CKD progression in patients with Alport syndrome and possibly other progressive chronic kidney disorders. BACKGROUND: Dual inhibition of the renin-angiotensin system (RAS) plus sodium-glucose transporter (SGLT)-2 or the mineralocorticoid receptor (MR) demonstrated additive renoprotective effects in large clinical trials. We hypothesized that triple therapy with RAS/SGLT2/MR inhibitors would be superior to dual RAS/SGLT2 blockade in attenuating CKD progression. METHODS: We performed a preclinical randomized controlled trial (PCTE0000266) in Col4a3 -deficient mice with established Alport nephropathy. Treatment was initiated late (age 6 weeks) in mice with elevated serum creatinine and albuminuria and with glomerulosclerosis, interstitial fibrosis, and tubular atrophy. We block-randomized 40 male and 40 female mice to either nil (vehicle) or late-onset food admixes of ramipril monotherapy (10 mg/kg), ramipril plus empagliflozin (30 mg/kg), or ramipril plus empagliflozin plus finerenone (10 mg/kg). Primary end point was mean survival. RESULTS: Mean survival was 63.7±10.0 days (vehicle), 77.3±5.3 days (ramipril), 80.3±11.0 days (dual), and 103.1±20.3 days (triple). Sex did not affect outcome. Histopathology, pathomics, and RNA sequencing revealed that finerenone mainly suppressed the residual interstitial inflammation and fibrosis despite dual RAS/SGLT2 inhibition. CONCLUSION: Experiments in mice suggest that triple RAS/SGLT2/MR blockade may substantially improve renal outcomes in Alport syndrome and possibly other progressive CKDs because of synergistic effects on the glomerular and tubulointerstitial compartments.

Next-Generation Morphometry for pathomics-data mining in histopathology.

Pathology diagnostics relies on the assessment of morphology by trained experts, which remains subjective and qualitative. Here we developed a framework for large-scale histomorphometry (FLASH) performing deep learning-based semantic segmentation and subsequent large-scale extraction of interpretable, quantitative, morphometric features in non-tumour kidney histology. We use two internal and three external, multi-centre cohorts to analyse over 1000 kidney biopsies and nephrectomies. By associating morphometric features with clinical parameters, we confirm previous concepts and reveal unexpected relations. We show that the extracted features are independent predictors of long-term clinical outcomes in IgA-nephropathy. We introduce single-structure morphometric analysis by applying techniques from single-cell transcriptomics, identifying distinct glomerular populations and morphometric phenotypes along a trajectory of disease progression. Our study provides a concept for Next-generation Morphometry (NGM), enabling comprehensive quantitative pathology data mining, i.e., pathomics.

Tackling stain variability using CycleGAN-based stain augmentation.

Background: Considerable inter- and intra-laboratory stain variability exists in pathology, representing a challenge in development and application of deep learning (DL) approaches. Since tackling all sources of stain variability with manual annotation is not feasible, we here investigated and compared unsupervised DL approaches to reduce the consequences of stain variability in kidney pathology. Methods: We aimed to improve the applicability of a pretrained DL segmentation model to 3 external multi-centric cohorts with large stain variability. In contrast to the traditional approach of training generative adversarial networks (GAN) for stain normalization, we here propose to tackle stain variability by data augmentation. We augment the training data of the pretrained model by the stain variability using CycleGANs and then retrain the model on the stain-augmented dataset. We compared the performance of i/ the unmodified pretrained segmentation model with ii/ CycleGAN-based stain normalization, iii/ a feature-preserving modification to ii/ for improved normalization, and iv/ the proposed stain-augmented model. Results: The proposed stain-augmented model showed highest mean segmentation accuracy in all external cohorts and maintained comparable performance on the training cohort. However, the increase in performance was only marginal compared to the pretrained model. CycleGAN-based stain normalization suffered from encoded imperceptible information into the normalizations that confused the pretrained model and thus resulted in slightly worse performance. Conclusions: Our findings suggest that stain variability can be tackled more effectively by augmenting data by it than by following the commonly used approach of normalizing the stain. However, the applicability of this approach providing only a rather slight performance increase has to be weighted against an additional carbon footprint.

On the Acceptance of "Fake" Histopathology: A Study on Frozen Sections Optimized with Deep Learning.

BACKGROUND: The fast acquisition process of frozen sections allows surgeons to wait for histological findings during the interventions to base intrasurgical decisions on the outcome of the histology. Compared with paraffin sections, however, the quality of frozen sections is often strongly reduced, leading to a lower diagnostic accuracy. Deep neural networks are capable of modifying specific characteristics of digital histological images. Particularly, generative adversarial networks proved to be effective tools to learn about translation between two modalities, based on two unconnected data sets only. The positive effects of such deep learning-based image optimization on computer-aided diagnosis have already been shown. However, since fully automated diagnosis is controversial, the application of enhanced images for visual clinical assessment is currently probably of even higher relevance. METHODS: Three different deep learning-based generative adversarial networks were investigated. The methods were used to translate frozen sections into virtual paraffin sections. Overall, 40 frozen sections were processed. For training, 40 further paraffin sections were available. We investigated how pathologists assess the quality of the different image translation approaches and whether experts are able to distinguish between virtual and real digital pathology. RESULTS: Pathologists' detection accuracy of virtual paraffin sections (from pairs consisting of a frozen and a paraffin section) was between 0.62 and 0.97. Overall, in 59% of images, the virtual section was assessed as more appropriate for a diagnosis. In 53% of images, the deep learning approach was preferred to conventional stain normalization (SN). CONCLUSION: Overall, expert assessment indicated slightly improved visual properties of converted images and a high similarity to real paraffin sections. The observed high variability showed clear differences in personal preferences.

Complement activation induces excessive T cell cytotoxicity in severe COVID-19.

Severe COVID-19 is linked to both dysfunctional immune response and unrestrained immunopathology, and it remains unclear whether T cells contribute to disease pathology. Here, we combined single-cell transcriptomics and single-cell proteomics with mechanistic studies to assess pathogenic T cell functions and inducing signals. We identified highly activated CD16+ T cells with increased cytotoxic functions in severe COVID-19. CD16 expression enabled immune-complex-mediated, T cell receptor-independent degranulation and cytotoxicity not found in other diseases. CD16+ T cells from COVID-19 patients promoted microvascular endothelial cell injury and release of neutrophil and monocyte chemoattractants. CD16+ T cell clones persisted beyond acute disease maintaining their cytotoxic phenotype. Increased generation of C3a in severe COVID-19 induced activated CD16+ cytotoxic T cells. Proportions of activated CD16+ T cells and plasma levels of complement proteins upstream of C3a were associated with fatal outcome of COVID-19, supporting a pathological role of exacerbated cytotoxicity and complement activation in COVID-19.

Activation of Notch3 in Renal Tubular Cells Leads to Progressive Cystic Kidney Disease.

BACKGROUND: Polycystic kidney disease (PKD) is a genetic disorder affecting millions of people worldwide that is characterized by fluid-filled cysts and leads to end-stage renal disease (ESRD). The hallmarks of PKD are proliferation and dedifferentiation of tubular epithelial cells, cellular processes known to be regulated by Notch signaling. METHODS: We found increased Notch3 expression in human PKD and renal cell carcinoma biopsies. To obtain insight into the underlying mechanisms and the functional consequences of this abnormal expression, we developed a transgenic mouse model with conditional overexpression of the intracellular Notch3 (ICN3) domain specifically in renal tubules. We evaluated the alterations in renal function (creatininemia, BUN) and structure (cysts, fibrosis, inflammation) and measured the expression of several genes involved in Notch signaling and the mechanisms of inflammation, proliferation, dedifferentiation, fibrosis, injury, apoptosis and regeneration. RESULTS: After one month of ICN3 overexpression, kidneys were larger with tubules grossly enlarged in diameter, with cell hypertrophy and hyperplasia, exclusively in the outer stripe of the outer medulla. After three months, mice developed numerous cysts in proximal and distal tubules. The cysts had variable sizes and were lined with a single- or multilayered, flattened, cuboid or columnar epithelium. This resulted in epithelial hyperplasia, which was observed as protrusions into the cystic lumen in some of the renal cysts. The pre-cystic and cystic epithelium showed increased expression of cytoskeletal filaments and markers of epithelial injury and dedifferentiation. Additionally, the epithelium showed increased proliferation with an aberrant orientation of the mitotic spindle. These phenotypic tubular alterations led to progressive interstitial inflammation and fibrosis. CONCLUSIONS: In summary, Notch3 signaling promoted tubular cell proliferation, the alignment of cell division, dedifferentiation and hyperplasia, leading to cystic kidney diseases and pre-neoplastic lesions.

Deep learning-based classification of kidney transplant pathology: a retrospective, multicentre, proof-of-concept study.

BACKGROUND: Histopathological assessment of transplant biopsies is currently the standard method to diagnose allograft rejection and can help guide patient management, but it is one of the most challenging areas of pathology, requiring considerable expertise, time, and effort. We aimed to analyse the utility of deep learning to preclassify histology of kidney allograft biopsies into three main broad categories (ie, normal, rejection, and other diseases) as a potential biopsy triage system focusing on transplant rejection. METHODS: We performed a retrospective, multicentre, proof-of-concept study using 5844 digital whole slide images of kidney allograft biopsies from 1948 patients. Kidney allograft biopsy samples were identified by a database search in the Departments of Pathology of the Amsterdam UMC, Amsterdam, Netherlands (1130 patients) and the University Medical Center Utrecht, Utrecht, Netherlands (717 patients). 101 consecutive kidney transplant biopsies were identified in the archive of the Institute of Pathology, RWTH Aachen University Hospital, Aachen, Germany. Convolutional neural networks (CNNs) were trained to classify allograft biopsies as normal, rejection, or other diseases. Three times cross-validation (1847 patients) and deployment on an external real-world cohort (101 patients) were used for validation. Area under the receiver operating characteristic curve (AUROC) was used as the main performance metric (the primary endpoint to assess CNN performance). FINDINGS: Serial CNNs, first classifying kidney allograft biopsies as normal (AUROC 0·87 [ten times bootstrapped CI 0·85-0·88]) and disease (0·87 [0·86-0·88]), followed by a second CNN classifying biopsies classified as disease into rejection (0·75 [0·73-0·76]) and other diseases (0·75 [0·72-0·77]), showed similar AUROC in cross-validation and deployment on independent real-world data (first CNN normal AUROC 0·83 [0·80-0·85], disease 0·83 [0·73-0·91]; second CNN rejection 0·61 [0·51-0·70], other diseases 0·61 [0·50-0·74]). A single CNN classifying biopsies as normal, rejection, or other diseases showed similar performance in cross-validation (normal AUROC 0·80 [0·73-0·84], rejection 0·76 [0·66-0·80], other diseases 0·50 [0·36-0·57]) and generalised well for normal and rejection classes in the real-world data. Visualisation techniques highlighted rejection-relevant areas of biopsies in the tubulointerstitium. INTERPRETATION: This study showed that deep learning-based classification of transplant biopsies could support pathological diagnostics of kidney allograft rejection. FUNDING: European Research Council; German Research Foundation; German Federal Ministries of Education and Research, Health, and Economic Affairs and Energy; Dutch Kidney Foundation; Human(e) AI Research Priority Area of the University of Amsterdam; and Max-Eder Programme of German Cancer Aid.

Multisystemic Cellular Tropism of SARS-CoV-2 in Autopsies of COVID-19 Patients.

Multiorgan tropism of SARS-CoV-2 has previously been shown for several major organs. We have comprehensively analyzed 25 different formalin-fixed paraffin-embedded (FFPE) tissues/organs from autopsies of fatal COVID-19 cases (n = 8), using histopathological assessment, detection of SARS-CoV-2 RNA using polymerase chain reaction and RNA in situ hybridization, viral protein using immunohistochemistry, and virus particles using transmission electron microscopy. SARS-CoV-2 RNA was mainly localized in epithelial cells across all organs. Next to lung, trachea, kidney, heart, or liver, viral RNA was also found in tonsils, salivary glands, oropharynx, thyroid, adrenal gland, testicles, prostate, ovaries, small bowel, lymph nodes, skin and skeletal muscle. Viral RNA was predominantly found in cells expressing ACE2, TMPRSS2, or both. The SARS-CoV-2 replicating RNA was also detected in these organs. Immunohistochemistry and electron microscopy were not suitable for reliable and specific SARS-CoV-2 detection in autopsies. These findings were validated using in situ hybridization on external COVID-19 autopsy samples (n = 9). Apart from the lung, correlation of viral detection and histopathological assessment did not reveal any specific alterations that could be attributed to SARS-CoV-2. In summary, SARS-CoV-2 and its replication could be observed across all organ systems, which co-localizes with ACE2 and TMPRSS2 mainly in epithelial but also in mesenchymal and endothelial cells. Apart from the respiratory tract, no specific (histo-)morphologic alterations could be assigned to the SARS-CoV-2 infection.

SARS-CoV-2 RNA screening in routine pathology specimens.

Virus detection methods are important to cope with the SARS-CoV-2 pandemics. Apart from the lung, SARS-CoV-2 was detected in multiple organs in severe cases. Less is known on organ tropism in patients developing mild or no symptoms, and some of such patients might be missed in symptom-indicated swab testing. Here, we tested and validated several approaches and selected the most reliable RT-PCR protocol for the detection of SARS-CoV-2 RNA in patients' routine diagnostic formalin-fixed and paraffin-embedded (FFPE) specimens available in pathology, to assess (i) organ tropism in samples from COVID-19-positive patients, (ii) unrecognized cases in selected tissues from negative or not-tested patients during a pandemic peak, and (iii) retrospectively, pre-pandemic lung samples. We identified SARS-CoV-2 RNA in seven samples from confirmed COVID-19 patients, in two gastric biopsies, one small bowel and one colon resection, one lung biopsy, one pleural resection and one pleural effusion specimen, while all other specimens were negative. In the pandemic peak cohort, we identified one previously unrecognized COVID-19 case in tonsillectomy samples. All pre-pandemic lung samples were negative. In conclusion, SARS-CoV-2 RNA detection in FFPE pathology specimens can potentially improve surveillance of COVID-19, allow retrospective studies, and advance our understanding of SARS-CoV-2 organ tropism and effects.

A multicenter blinded preclinical randomized controlled trial on Jak1/2 inhibition in MRL/MpJ-Faslpr mice with proliferative lupus nephritis predicts low effect size.

Data reproducibility and single-center bias are concerns in preclinical research and compromise translation from animal to human. Multicenter preclinical randomized controlled trials (pRCT) may reduce the gap between experimental studies and RCT and improve the predictability of results, for example Jak1/2 inhibition in lupus nephritis. To evaluate this, we conducted the first pRCT in the kidney domain at two Spanish and two German academic sites. Eligible MRL/MpJ-Faslpr mice (female, age13-14 weeks, stress scores of less than two and no visible tumor or signs of infection) were equally randomized to either oral treatment with the Jak1/2 inhibitor baricitinib or vehicle for four weeks. Central blinded histology analysis was performed at an independent fifth site. The primary endpoint was the urinary protein/creatinine ratio. Baricitinib treatment did not significantly affect proteinuria, histological markers of activity and chronicity, or the glomerular filtration rate but significantly improved plasma autoantibody levels and lymphadenopathy. Data heterogeneity was noted across the different centers referring in part to phenotype differences between MRL/MpJ-Faslpr mice bred at different sites, mimicking well patient phenotype diversity in lupus trials. Multicenter pRCT can overcome single-center bias at the cost of increasing variability and reducing effect size. Thus, our pRCT predicts a low effect size of baricitinib treatment on human lupus nephritis in heterogeneous study populations.

Multistain segmentation of renal histology: first steps toward artificial intelligence-augmented digital nephropathology.

Artificial intelligence (AI), and particularly deep learning (DL), are showing great potential in improving pathology diagnostics in many aspects, 1 of which is the segmentation of histology into (diagnostically) relevant compartments. Although most current studies focus on AI and DL in oncologic pathology, an increasing number of studies explore their application to nephropathology, including the study published in this issue of Kidney International by Jayapandian et al.

Vascular neutrophilic inflammation and immunothrombosis distinguish severe COVID-19 from influenza pneumonia.

OBJECTIVE: Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can lead to severe pneumonia, but also thrombotic complications and non-pulmonary organ failure. Recent studies suggest intravascular neutrophil activation and subsequent immune cell-triggered immunothrombosis as a central pathomechanism linking the heterogenous clinical picture of coronavirus disease 2019 (COVID-19). We sought to study whether immunothrombosis is a pathognomonic factor in COVID-19 or a general feature of (viral) pneumonia, as well as to better understand its upstream regulation. APPROACH AND RESULTS: By comparing histopathological specimens of SARS-CoV-2 with influenza-affected lungs, we show that vascular neutrophil recruitment, NETosis, and subsequent immunothrombosis are typical features of severe COVID-19, but less prominent in influenza pneumonia. Activated neutrophils were typically found in physical association with monocytes. To explore this further, we combined clinical data of COVID-19 cases with comprehensive immune cell phenotyping and bronchoalveolar lavage fluid scRNA-seq data. We show that a HLADRlow CD9low monocyte population expands in severe COVID-19, which releases neutrophil chemokines in the lungs, and might in turn explain neutrophil expansion and pulmonary recruitment in the late stages of severe COVID-19. CONCLUSIONS: Our data underline an innate immune cell axis causing vascular inflammation and immunothrombosis in severe SARS-CoV-2 infection.

Deep Learning-Based Segmentation and Quantification in Experimental Kidney Histopathology.

BACKGROUND: Nephropathologic analyses provide important outcomes-related data in experiments with the animal models that are essential for understanding kidney disease pathophysiology. Precision medicine increases the demand for quantitative, unbiased, reproducible, and efficient histopathologic analyses, which will require novel high-throughput tools. A deep learning technique, the convolutional neural network, is increasingly applied in pathology because of its high performance in tasks like histology segmentation. METHODS: We investigated use of a convolutional neural network architecture for accurate segmentation of periodic acid-Schiff-stained kidney tissue from healthy mice and five murine disease models and from other species used in preclinical research. We trained the convolutional neural network to segment six major renal structures: glomerular tuft, glomerulus including Bowman's capsule, tubules, arteries, arterial lumina, and veins. To achieve high accuracy, we performed a large number of expert-based annotations, 72,722 in total. RESULTS: Multiclass segmentation performance was very high in all disease models. The convolutional neural network allowed high-throughput and large-scale, quantitative and comparative analyses of various models. In disease models, computational feature extraction revealed interstitial expansion, tubular dilation and atrophy, and glomerular size variability. Validation showed a high correlation of findings with current standard morphometric analysis. The convolutional neural network also showed high performance in other species used in research-including rats, pigs, bears, and marmosets-as well as in humans, providing a translational bridge between preclinical and clinical studies. CONCLUSIONS: We developed a deep learning algorithm for accurate multiclass segmentation of digital whole-slide images of periodic acid-Schiff-stained kidneys from various species and renal disease models. This enables reproducible quantitative histopathologic analyses in preclinical models that also might be applicable to clinical studies.

Keratins are novel markers of renal epithelial cell injury.

Keratins, the intermediate filaments of the epithelial cell cytoskeleton, are up-regulated and post-translationally modified in stress situations. Renal tubular epithelial cell stress is a common finding in progressive kidney diseases, but little is known about keratin expression and phosphorylation. Here, we comprehensively describe keratin expression in healthy and diseased kidneys. In healthy mice, the major renal keratins, K7, K8, K18, and K19, were expressed in the collecting ducts and K8, K18 in the glomerular parietal epithelial cells. Tubular expression of all 4 keratins increased by 20- to 40-fold in 5 different models of renal tubular injury as assessed by immunohistochemistry, Western blot, and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). The up-regulation became significant early after disease induction, increased with disease progression, was found de novo in distal tubules and was accompanied by altered subcellular localization. Phosphorylation of K8 and K18 increased under stress. In humans, injured tubules also exhibited increased keratin expression. Urinary K18 was only detected in mice and patients with tubular cell injury. Keratins labeled glomerular parietal epithelial cells forming crescents in patients and animals. Thus, all 4 major renal keratins are significantly, early, and progressively up-regulated upon tubular injury regardless of the underlying disease and may be novel sensitive markers of renal tubular cell stress.