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Background The impact of artificial intelligence combined with advanced techniques is ever-increasing in the biomedical field appearing promising, among others, in chronic kidney disease (CKD) diagnosis. However, existing models are often single-aetiology specific. Proposed here is a pipeline for the development of single models able to distinguish and spatially visualize multiple CKD aetiologies. Methods Acquired were from the Human Urinary Proteome Database the urinary peptide data of 1850 healthy control (HC) and CKD (diabetic kidney disease-DKD, IgA nephropathy-IgAN, vasculitis) participants. The uniform manifold approximation and projection (UMAP) method was coupled to a support vector machine (SVM) algorithm. Binary (DKD, HC) and multiclass (DKD, HC, IgAN, vasculitis) classifications were performed, including or skipping the UMAP step. Last, the pipeline was compared to the current state-of-the-art single-aetiology CKD urinary models. Findings In an independent test set, the developed models (including the UMAP step) achieved 90.35% and 70.13% overall predictive accuracies, respectively, for the binary and the multiclass classifications (96.14% and 85.06%, skipping the UMAP step). Overall, the HC class was distinguished with the highest accuracy. The different classes displayed a tendency to form distinct clusters in the 3D-space based on their disease state. Interpretation Urinary peptide data appear to potentially be an effective basis for CKD aetiology differentiation. The UMAP step may provide a unique visualization advantage capturing the relevant molecular (patho)physiology. Further studies are warranted to validate the pipelines clinical potential in the presented as well as other CKD aetiologies or even other diseases.

Thrombospondin type-1 domain-containing 7A (THSD7A) is a large extracellular protein that is found in podocyte foot processes of the kidney glomerulus. It has been established as a causative autoantigen in membranous nephropathy. Amongst the predicted 21 thrombospondin repeat domains of its extracellular segment, the highest frequency of autoimmune response has been associated with the two N-terminal domains. Here, we show that antibodies against this THSD7A segment in mice induce typical clinical and morphological signs of membranous nephropathy. The high-resolution structure of these two domains reveals a non-canonical thrombospondin repeat fold that is distinct from the established type 1 thrombospondin repeat. As it shares a conserved disulfide pattern with the canonical fold, we refer to these domains d1 and d2 as type 1A thrombospondin repeats. Both domains comprise a seven layered CC-W-PP-R-W-QQ-CC pattern, which is only partly shared by other THSD7A thrombospondin repeat domains. The two domains form a well-defined V-shaped tandem arrangement. Our findings provide crucial insight into specific structural features of these two domains that are distinct from other regions of THSD7A and hence could cause the high level of antigenicity found for these two domains.

Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), has been associated mainly with a range of neurological symptoms, including brain fog and brain tissue loss, raising concerns about the virus’s acute and potential chronic impact on the central nervous system. In this study, we utilized mouse models and human post-mortem tissues to investigate the presence and distribution of the SARS-CoV-2 spike protein in the skull-meninges-brain axis. Our results revealed the accumulation of the spike protein in the skull marrow, brain meninges, and brain parenchyma. The injection of the spike protein alone caused cell death in the brain, highlighting a direct effect on brain tissue. Furthermore, we observed the presence of spike protein in the skull of deceased long after their COVID-19 infection, suggesting that the spike’s persistence may contribute to long-term neurological symptoms. The spike protein was associated with neutrophil-related pathways and dysregulation of the proteins involved in the PI3K-AKT as well as complement and coagulation pathway. Overall, our findings suggest that SARS-CoV-2 spike protein trafficking from CNS borders into the brain parenchyma and identified differentially regulated pathways may present insights into mechanisms underlying immediate and long-term consequences of SARS-CoV-2 and present diagnostic and therapeutic opportunities.Graphical SummaryShort SummaryThe accumulation of SARS-CoV-2 spike protein in the skull-meninges-brain axis presents potential molecular mechanisms and therapeutic targets for neurological complications in long-COVID-19 patients.

SummaryT regulatory type 1 (Tr1) cells, which are defined by their regulatory function, lack of Foxp3, high expression of IL-10, CD49b, and LAG3, are known to be able to suppress Th1 and Th17 in the intestine. Th1 and Th17 cells are also the main drivers of crescentic glomerulonephritis, the most severe form of renal autoimmune disease. However, whether Tr1 cells emerge in renal inflammation and moreover, whether they exhibit regulatory function during glomerulonephritis has not been thoroughly investigated yet. To address these questions, we used a mouse model of experimental crescentic glomerulonephritis and double Foxp3mRFPIL-10eGFPreporter mice. We found that Foxp3negIL-10-producing CD4+T cells infiltrate the kidneys during glomerulonephritis progression. Using single-cell RNA- sequencing, we could show that these cells express the core transcriptional factors characteristic of Tr1 cells. In line with this, Tr1 cells showed a strong suppressive activityex vivoand were protective in experimental crescentic glomerulonephritisin vivo. Finally, we could also identify Tr1 cells in the kidneys of patients with anti-neutrophil cytoplasmic autoantibody (ANCA)-associated glomerulonephritis and define their transcriptional profile. Tr1 cells are currently used in several immune-mediated inflammatory diseases, e.g. as T- cell therapy. Thus, our study provides proof of concept for Tr1 cell-based therapies in experimental glomerulonephritis.

Crescentic glomerulonephritis (cGN), most often caused by anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis, is an aggressive form of immune-mediated kidney disease and represents an important cause of end-stage renal failure. Although it is known that T cells infiltrate the kidney in cGN, their precise role in autoimmune kidney disease remains to be fully elucidated. By performing single-cell analysis, we identified activated, clonally expanded CD8+ T cells with a cytotoxic gene expression profile in the kidneys of patients with ANCA-associated cGN. Using an experimental model of cGN, we demonstrated that clonally expanded murine CD8+ T cells highly expressed the cytotoxic molecule granzyme B. Moreover, lack of CD8+ T cells or granzyme B resulted in an ameliorated course of cGN. This was associated with reduced cleaved caspase-3 induction in renal tissue cells. Our data indicate that clonally expanded cytotoxic CD8+ T cells have a previously unrecognized pathogenic function in aggravating immune-mediated kidney disease.

Mechanotransduction is an important process of sensing physical forces in the environment of organisms, tissues and cells and transducing them into a biochemical response. Due to their position on the glomerular capillaries, podocytes are exposed to near-constant biomechanical force, which can fluctuate widely. These include shear stress and hydrostatic pressure. A pathological increase in these forces can induce morphological change to podocytes, their detachment from the glomerular basement membrane and subsequent loss into the primary urine. The ability to sense and respond to variations in mechanical force would be beneficial to a cell exposed to these conditions. It is likely podocytes have such mechanisms, however their identity are unknown. Here we investigated the hypothesis that the mechanotransducer Piezo is involved in a mechanotransduction pathway in Drosophila nephrocytes, the podocyte homologue in the fly. We find Piezo is expressed in nephrocytes and localizes to the nephrocyte diaphragm. The Piezo agonist YODA, which stimulates channel opening in the absence of mechanical force, leads to a significant increase in intracellular Ca++ upon shear stress in the nephrocyte. This leads to activation of Rho1, delineating a putative Piezo mechanotransductive pathway in these cells. Loss of function analysis revealed minor defects in nephrocyte filtration function. In contrast, we show that elevated Piezo levels resulted in constantly oscillating Ca++ signals even in the absence of shear stress, increased active Rho1 and accumulation of actin stress fibers, culminating in a severe nephrocyte filtration phenotype, suggesting that pathway hyperactivity is detrimental. We asked if this phenotype could be reversed by blocking Piezo activity pharmacologically using the tarantula toxin GsMTx4. Treatment with GsMTx4 brought levels of activated Rho1 into the normal range. This work delineates a mechanotransductive pathway in nephrocytes involving Piezo, Ca++, Rho1 and the actin-cytoskeleton, and suggest this is part of a mechanism by which nephrocytes sense and adapt to changes in mechanical force.

1AbstractBackgroundAnalyses in hospitalized patients and small autopsy series suggest that severe SARS-CoV-2 infection may affect the heart. We investigated heart tissue by in situ hybridization, immunohistochemistry and RNA sequencing in consecutive autopsy cases to quantify virus load and characterize cardiac involvement in COVID-19.MethodsLeft ventricular tissue from 95 deceased with diagnosed SARS-CoV-2 infection undergoing autopsy was analyzed and clinical data were collected. RNA was isolated to examine virus load of SARS- CoV-2 and its replication in the heart. A virus load >1000 copies per µg RNA was defined as relevant. Viral RNA and inflammatory cells were assessed using histology. RNA sequencing and gene ontology (GO) enrichment were performed in 10 cases with high cardiac virus load and 10 age-matched cases without cardiac infection.ResultsA relevant SARS-CoV-2 virus load was detected in 41 out of 95 deceased (43%). The median cardiac virus load was 7952 copies per µg RNA (IQR 2507, 32 005). In situ hybridization revealed SARS- CoV-2 RNA primarily in the interstitium or interstitial cells. Virus detection was not associated with increased inflammatory cells. Relevant cardiac infection was associated with increased expression of the entry factor TMPRSS2. Cardiac virus replication was found in 14/95 hearts (15%). Remarkably, cardiac virus replication was associated with shorter time between diagnosis and death. RNA sequencing revealed clear activation of immune response pathways to virus infection and destruction of cardiomyocytes. Hearts with high virus load showed activation of the GO term “extracellular exosomes”.ConclusionSARS-CoV-2 infection including virus replication and distinct transcriptomic alterations without signs of myocarditis demonstrate a cardiac involvement. In this autopsy series, cardiac replication of SARS-CoV-2 was associated with early death.

Stroke and central nervous system dysfunction are cardinal symptoms in critically ill corona virus disease 19 (COVID-19) patients. In an autopsy series of 32 COVID-19 patients, we investigated whether carotid arteries were infected with SARS-CoV-2 by employing genomic, virologic, histochemical and transcriptomic analyses. We show that SARS-CoV-2 productively infects and modulates vascular responses in carotid arteries. This finding has far reaching implications for the understanding and clinical treatment of COVID-19.

Morphologic examination of tissue biopsies is essential for histopathological diagnosis. However, accurate and scalable cellular quantification in human samples remains challenging. Here, we present a deep learning-based approach for antigen-specific cellular morphometrics in human kidney biopsies, which combines indirect immunofluorescence imaging with U-Net-based architectures for image-to-image translation and dual segmentation tasks, achieving human-level accuracy. In the kidney, podocyte loss represents a hallmark of glomerular injury and can be estimated in diagnostic biopsies. Thus, we profiled over 27,000 podocytes from 110 human samples, including patients with anti-neutrophil cytoplasmic antibody-associated glomerulonephritis (ANCA-GN), an immune-mediated disease with aggressive glomerular damage and irreversible loss of kidney function. Previously unknown morphometric signatures of podocyte depletion were identified in patients with ANCA-GN, which allowed patient classification and showed potential for risk stratification in combination with routine clinical tools. Together, our approach enables robust and scalable molecular morphometric analysis of human tissues, yielding deeper biological insights into the human kidney pathophysiology.SummaryDeep learning enables robust and scalable molecular morphometric analysis of human tissues, yielding deeper biological insights into the human kidney pathophysiology.

PurposeThe kidney glomerulus comprises a syncytium of podocytes, mesangial and endothelial cells, which jointly determine glomerular filtration barrier function, and thereby kidney and cardiovascular health. The understanding of this intricate functional unit and its intracellular communication beyond the transcriptome requires bulk isolation of these cell-types from glomeruli for subsequent biochemical investigations. Therefore, we developed a globally applicable tripartite isolation method for murine mesangial and endothelial cells and podocytes (timMEP).MethodsGlomerular cells were separated via a novel FACS-sort depending on a cell-specific antibody labeling in wildtype mice or based on a combination of transgenic fluorescent protein expression and antibody labeling in mT/mG mice. The purity of isolated cell-types was validated by qPCR and immunoblot. The proteome of podocytes, mesangial and endothelial cells was determined and compared between species, ages and gender of wildtype and mT/mG mice. The method was also applied to the podocyte-targeting immunologic injury model of THSD7A-associated membranous glomerulonephritis.ResultsTimMEP enabled protein-biochemical analyses of podocytes, mesangial and endothelial cells derived from a single reporter free mouse. Proteomic analyses allowed the first characterization of podocyte, endothelial and mesangial proteomes of individual mice. Marker proteins for mesangial and endothelial proteins were determined, and protein-based interaction and intraglomerular cell communication networks were elucidated. Interestingly, analyses revealed significant cell-type specific proteome differences between mouse strains, artefacts induced by reporters, and alterations depending on gender and age. Within the glomerulus, timMEP resolved a fine-tuned initial stress response exclusively in podocytes after exposure to anti-THSD7A antibodies, which was not detectable using conventional analyses in whole glomeruli.ConclusionGlobally applicable timMEP abolishes the need for costly, time- and animal-consuming breeding of mice to glomerular cell-type reporters. TimMEP enables glomerular cell-type resolved investigations at the transcriptional and protein biochemical level in health and disease, while avoiding reporter-based artefacts, paving the way towards the comprehensive and systematic characterization of glomerular cell-type biology.Key messagesA tripartite isolation method for mesangial, endothelial and podocyte cell-types from reporter-free mice.Generation of bulk cell-type samples and primary co-cultures for biochemical and protein-based analyses.Strain and transgene-dependent expression of proteins among glomerular cell-types, including protein profiles, intra-glomerular communication machineries, and reporter-dependent artefacts.Disease specific time-resolved resolution of glomerular cell-type’s response to injury.

Kidney transplantation is the preferred renal replacement therapy available. Yet, the biological processes during and after kidney transplantation and how they translate into the overall functional graft outcome are insufficiently understood. Recent developments in the field of extracellular vesicle research allow the deeper exploitation of this non-invasive source. We separated small urinary extracellular vesicles (suEVs) throughout the course of living donor kidney transplantation. SuEVs were collected longitudinally from both the donor and the recipient in 22 living donor kidney transplantations. Unbiased proteomic analysis revealed specific temporal patterns of suEV proteins indicative of the cellular processes involved in the allograft’s response after transplantation with proteins playing a role in complement activation being among the most dynamically regulated components. Using a leave-one-out cross validation model, we identified potential prognostic markers of kidney function at 1 year after transplantation. One of the proteins identified – phosphoenol pyruvate carboxykinase (PCK2) – could be confirmed in an independent validation cohort of another 22 donor-recipient pairs using targeted mass spectrometry. This study sheds the light on early molecular processes during the course of kidney transplantation and shows the future potential of suEVs as a source of biomarkers in this setting. The data set is provided as a unique resource directly accessible through an online tool that allows dynamic interrogation of this first comprising suEV proteome atlas after kidney transplantation.One Sentence SummaryThis study represents the first atlas of the proteomic changes in small urinary extracellular vesicles throughout living donor kidney transplantation identifying PCK2 abundance as a biomarker for renal function 12 months after transplantation

In diseases of many parenchymatous organs, heterogenous detoriation of individual functional units determines the clinical prognosis. However, the molecular characterization of these subunits remains a technological challenge that needs to be addressed in order to better understand pathological mechanisms. Sclerotic and proteinuric glomerular kidney disease is a frequent and heterogeneous disease which affects a fraction of nephrons, glomeruli and draining tubules, to variable extents, and for which no treatment exists. Here, we developed and applied an antibody-independent methodology to investigate heterogeneity of individual nephron segment proteomes from mice with proteinuric kidney disease. This “one-segment-one-proteome-approach” defines mechanistic connections between upstream (glomerular) and downstream (tubular) nephron segment populations. In single glomeruli from two different mouse models of sclerotic glomerular disease, we identified a coherent protein expression module consisting of extracellular matrix protein deposition (reflecting glomerular sclerosis), glomerular albumin (reflecting proteinuria) and LAMP1, a lysosomal protein. This module was associated with a loss of podocyte marker proteins. In an attempt to target this protein co-expression module, genetic ablation of LAMP1-correlated lysosomal proteases in mice could ameliorate glomerular damage. Furthermore, individual glomeruli from patients with genetic sclerotic and non-sclerotic proteinuric diseases demonstrated increased abundance of lysosomal proteins, in combination with a decreased abundance of the mutated gene products. Therefore, increased glomerular lysosomal load is a conserved key mechanism in proteinuric kidney diseases, and the technology applied here can be implemented to address heterogeneous pathophysiology in a variety of diseases at a sub-biopsy scale