Bone marrow (BM) niches are often inaccessible for controlled experimentation due to their difficult accessibility, biological complexity, and three-dimensional (3D) geometry.

Recent developments in optical tissue clearing have been difficult to apply for the morphometric analysis of organs with high cellular content and small functional structures, such as the kidney. Here, we establish combinations of genetic and immuno-labelling for single cell identification, tissue clearing and subsequent de-clarification for histoimmunopathology and transmission electron microscopy. Using advanced light microscopy and computational analyses, we investigated a murine model of crescentic nephritis, an inflammatory kidney disease typified by immune-mediated damage to glomeruli leading to the formation of hypercellular lesions and the rapid loss of kidney function induced by nephrotoxic serum. Results show a graded susceptibility of the glomeruli, significant podocyte loss and capillary injury. These effects are associated with activation of parietal epithelial cells and formation of glomerular lesions that may evolve and obstruct the kidney tubule, thereby explaining the loss of kidney function. Thus, our work provides new high-throughput endpoints for the analysis of complex tissues with single-cell resolution.

Microvascular homeostasis is strictly regulated, requiring close interaction between endothelial cells and pericytes. Here, we aimed to improve our understanding of how microvascular crosstalk affects pericytes. Human-derived pericytes, cultured in absence, or presence of human endothelial cells, were studied by RNA sequencing. Compared with mono-cultured pericytes, a total of 6704 genes were differentially expressed in co-cultured pericytes. Direct endothelial contact induced transcriptome profiles associated with pericyte maturation, suppression of extracellular matrix production, proliferation, and morphological adaptation. In vitro studies confirmed enhanced pericyte proliferation mediated by endothelial-derived PDGFB and pericyte-derived HB-EGF and FGF2. Endothelial-induced PLXNA2 and ACTR3 upregulation also triggered pericyte morphological adaptation. Pathway analysis predicted a key role for TGFβ signaling in endothelial-induced pericyte differentiation, whereas the effect of signaling via gap- and adherens junctions was limited. We demonstrate that endothelial cells have a major impact on the transcriptional profile of pericytes, regulating endothelial-induced maturation, proliferation, and suppression of ECM production.

Kidney failure is frequently observed during and after COVID-19, but it remains elusive whether this is a direct effect of the virus. Here, we report that SARS-CoV-2 directly infects kidney cells and is associated with increased tubule-interstitial kidney fibrosis in patient autopsy samples. To study direct effects of the virus on the kidney independent of systemic effects of COVID-19, we infected human-induced pluripotent stem-cell-derived kidney organoids with SARS-CoV-2. Single-cell RNA sequencing indicated injury and dedifferentiation of infected cells with activation of profibrotic signaling pathways. Importantly, SARS-CoV-2 infection also led to increased collagen 1 protein expression in organoids. A SARS-CoV-2 protease inhibitor was able to ameliorate the infection of kidney cells by SARS-CoV-2. Our results suggest that SARS-CoV-2 can directly infect kidney cells and induce cell injury with subsequent fibrosis. These data could explain both acute kidney injury in COVID-19 patients and the development of chronic kidney disease in long COVID.

Dexamethasone is widely used as an immunosuppressive therapy and recently as COVID-19 treatment. Here, we demonstrate that dexamethasone sensitizes to ferroptosis, a form of iron-catalyzed necrosis, previously suggested to contribute to diseases such as acute kidney injury, myocardial infarction, and stroke, all of which are triggered by glutathione (GSH) depletion. GSH levels were significantly decreased by dexamethasone. Mechanistically, we identified that dexamethasone up-regulated the GSH metabolism regulating protein dipeptidase-1 (DPEP1) in a glucocorticoid receptor (GR)-dependent manner. DPEP1 knockdown reversed the phenotype of dexamethasone-induced ferroptosis sensitization. Ferroptosis inhibitors, the DPEP1 inhibitor cilastatin, or genetic DPEP1 inactivation reversed the dexamethasone-induced increase in tubular necrosis in freshly isolated renal tubules. Our data indicate that dexamethasone sensitizes to ferroptosis by a GR-mediated increase in DPEP1 expression and GSH depletion. Together, we identified a previously unknown mechanism of glucocorticoid-mediated sensitization to ferroptosis bearing clinical and therapeutic implications.

Calciphylaxis is a rare disease with high mortality mainly involving patients with chronic kidney disease (CKD). Sodium thiosulphate (STS) has been used as an off-label therapeutic in calciphylaxis, but there is a lack of clinical trials and studies that demonstrate its effect compared with those without STS treatment.

Mesenchymal stem cells (MSCs) reside in the perivascular niche of many organs, including kidney, lung, liver, and heart, although their roles in these tissues are poorly understood. Here, we demonstrate that Gli1 marks perivascular MSC-like cells that substantially contribute to organ fibrosis. In vitro, Gli1(+) cells express typical MSC markers, exhibit trilineage differentiation capacity, and possess colony-forming activity, despite constituting a small fraction of the platelet-derived growth factor-β (PDGFRβ)(+) cell population. Genetic lineage tracing analysis demonstrates that tissue-resident, but not circulating, Gli1(+) cells proliferate after kidney, lung, liver, or heart injury to generate myofibroblasts. Genetic ablation of these cells substantially ameliorates kidney and heart fibrosis and preserves ejection fraction in a model of induced heart failure. These findings implicate perivascular Gli1(+) MSC-like cells as a major cellular origin of organ fibrosis and demonstrate that these cells may be a relevant therapeutic target to prevent solid organ dysfunction after injury.

The casein kinase 1A1 gene (CSNK1A1) is a putative tumor suppressor gene located in the common deleted region for del(5q) myelodysplastic syndrome (MDS). We generated a murine model with conditional inactivation of Csnk1a1 and found that Csnk1a1 haploinsufficiency induces hematopoietic stem cell expansion and a competitive repopulation advantage, whereas homozygous deletion induces hematopoietic stem cell failure. Based on this finding, we found that heterozygous inactivation of Csnk1a1 sensitizes cells to a CSNK1 inhibitor relative to cells with two intact alleles. In addition, we identified recurrent somatic mutations in CSNK1A1 on the nondeleted allele of patients with del(5q) MDS. These studies demonstrate that CSNK1A1 plays a central role in the biology of del(5q) MDS and is a promising therapeutic target.

Bone marrow fibrosis (BMF) develops in various hematological and non-hematological conditions and is a central pathological feature of myelofibrosis. Effective cell-targeted therapeutics are needed, but the cellular origin of BMF remains elusive. Here, we show using genetic fate tracing in two murine models of BMF that Gli1+ mesenchymal stromal cells (MSCs) are recruited from the endosteal and perivascular niche to become fibrosis-driving myofibroblasts in the bone marrow. Genetic ablation of Gli1+ cells abolished BMF and rescued bone marrow failure. Pharmacological targeting of Gli proteins with GANT61 inhibited Gli1+ cell expansion and myofibroblast differentiation and attenuated fibrosis severity. The same pathway is also active in human BMF, and Gli1 expression in BMF significantly correlates with the severity of the disease. In addition, GANT61 treatment reduced the myofibroblastic phenotype of human MSCs isolated from patients with BMF, suggesting that targeting of Gli proteins could be a relevant therapeutic strategy.

A major drawback of single-cell ATAC-seq (scATAC-seq) is its sparsity, i.e., open chromatin regions with no reads due to loss of DNA material during the scATAC-seq protocol. Here, we propose scOpen, a computational method based on regularized non-negative matrix factorization for imputing and quantifying the open chromatin status of regulatory regions from sparse scATAC-seq experiments. We show that scOpen improves crucial downstream analysis steps of scATAC-seq data as clustering, visualization, cis-regulatory DNA interactions, and delineation of regulatory features. We demonstrate the power of scOpen to dissect regulatory changes in the development of fibrosis in the kidney. This identifies a role of Runx1 and target genes by promoting fibroblast to myofibroblast differentiation driving kidney fibrosis.

Nephrotic syndrome (NS) is characterized by severe proteinuria as a consequence of kidney glomerular injury due to podocyte damage. In vitro models mimicking in vivo podocyte characteristics are a prerequisite to resolve NS pathogenesis. The detailed characterization of organoid podocytes resulting from a hybrid culture protocol showed a podocyte population that resembles adult podocytes and was superior compared with 2D counterparts, based on single-cell RNA sequencing, super-resolution imaging and electron microscopy. In this study, these next-generation podocytes in kidney organoids enabled personalized idiopathic nephrotic syndrome modeling, as shown by activated slit diaphragm signaling and podocyte injury following protamine sulfate, puromycin aminonucleoside treatment and exposure to NS plasma containing pathogenic permeability factors. Organoids cultured from cells of a patient with heterozygous NPHS2 mutations showed poor NPHS2 expression and aberrant NPHS1 localization, which was reversible after genetic correction. Repaired organoids displayed increased VEGFA pathway activity and transcription factor activity known to be essential for podocyte physiology, as shown by RNA sequencing. This study shows that organoids are the preferred model of choice to study idiopathic and congenital podocytopathies.

Scattered tubular cells (STCs) are a phenotypically distinct cell population in the proximal tubule that increase in number after acute kidney injury. We aimed to characterize the human STC population. Three-dimensional human tissue analysis revealed that STCs are preferentially located within inner bends of the tubule and are barely present in young kidney tissue (<2 years), and their number increases with age. Increased STC numbers were associated with acute tubular injury (kidney injury molecule 1) and interstitial fibrosis (alpha smooth muscle actin). Isolated CD13+ CD24- CD133- proximal tubule epithelial cells (PTECs) and CD13+ CD24+ and CD13+ CD133+ STCs were analyzed using RNA sequencing. Transcriptome analysis revealed an upregulation of nuclear factor κB, tumor necrosis factor alpha, and inflammatory pathways in STCs, whereas metabolism, especially the tricarboxylic acid cycle and oxidative phosphorylation, was downregulated, without showing signs of cellular senescence. Using immunostaining and a publicly available single-cell sequencing database of human kidneys, we demonstrate that STCs represent a heterogeneous population in a transient state. In conclusion, STCs are dedifferentiated PTECs showing a metabolic shift toward glycolysis, which could facilitate cellular survival after kidney injury. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Vascular calcification (VC) is a major risk factor for cardiovascular morbidity and mortality. Depending on the location of mineral deposition within the arterial wall, VC is classified as intimal and medial calcification. Using in vitro mineralization assays, we developed protocols triggering both types of calcification in vascular smooth muscle cells (SMCs) following diverging molecular pathways.

Plasma IL-6 is elevated after myocardial infarction (MI) and is associated with increased morbidity and mortality. Which cardiac cell type preferentially contributes to IL-6 expression and how its production is regulated are largely unknown. Here, we studied the cellular source and purinergic regulation of IL-6 formation in a murine MI model. We found that IL-6, measured in various cell types in post-MI hearts at the protein level and by quantitative PCR and RNAscope, was preferentially formed by cardiac fibroblasts (CFs). Single-cell RNA-Seq (scRNA-Seq) in infarcted mouse and human hearts confirmed this finding. We found that adenosine stimulated fibroblast IL-6 formation via the adenosine receptor A2bR in a Gq-dependent manner. CFs highly expressed Adora2b and rapidly degraded extracellular ATP to AMP but lacked CD73. In mice and humans, scRNA-Seq revealed that Adora2B was also mainly expressed by fibroblasts. We assessed global IL-6 production in isolated hearts from mice lacking CD73 on T cells (CD4-CD73-/-), a condition known to be associated with adverse cardiac remodeling. The ischemia-induced release of IL-6 was strongly attenuated in CD4-CD73-/- mice, suggesting adenosine-mediated modulation. Together, these findings demonstrate that post-MI IL-6 was mainly derived from activated CFs and was controlled by T cell-derived adenosine. We show that purinergic metabolic cooperation between CFs and T cells is a mechanism that modulates IL-6 formation by the heart and has therapeutic potential.

Acute kidney injury (AKI) is morphologically characterized by a synchronized plasma membrane rupture of cells in a specific section of a nephron, referred to as acute tubular necrosis (ATN). Whereas the involvement of necroptosis is well characterized, genetic evidence supporting the contribution of ferroptosis is lacking. Here, we demonstrate that the loss of ferroptosis suppressor protein 1 (Fsp1) or the targeted manipulation of the active center of the selenoprotein glutathione peroxidase 4 (Gpx4cys/-) sensitize kidneys to tubular ferroptosis, resulting in a unique morphological pattern of tubular necrosis. Given the unmet medical need to clinically inhibit AKI, we generated a combined small molecule inhibitor (Nec-1f) that simultaneously targets receptor interacting protein kinase 1 (RIPK1) and ferroptosis in cell lines, in freshly isolated primary kidney tubules and in mouse models of cardiac transplantation and of AKI and improved survival in models of ischemia-reperfusion injury. Based on genetic and pharmacological evidence, we conclude that GPX4 dysfunction hypersensitizes mice to ATN during AKI. Additionally, we introduce Nec-1f, a solid inhibitor of RIPK1 and weak inhibitor of ferroptosis.

Vascular calcification is an active process that increases cardiovascular disease (CVD) risk. There is still no consensus on an appropriate biomarker for vascular calcification. We reasoned that the biomarker for vascular calcification is the collection of all blood components that can be sensed and integrated into a calcification response by human vascular smooth muscle cells (hVSMCs).

Tubular injury is the hallmark of acute kidney injury (AKI) with a tremendous impact on patients and health-care systems. During injury, any differentiated proximal tubular cell (PT) may transition into a specific injured phenotype, so-called "scattered tubular cell" (STC)-phenotype. To understand the fate of this specific phenotype, we generated transgenic mice allowing inducible, reversible, and irreversible tagging of these cells in a murine AKI model, the unilateral ischemia-reperfusion injury (IRI). For lineage tracing, we analyzed the kidneys using single-cell profiling during disease development at various time points. Labeled cells, which we defined by established endogenous markers, already appeared 8 h after injury and showed a distinct expression set of genes. We show that STCs re-differentiate back into fully differentiated PTs upon the resolution of the injury. In summary, we show the dynamics of the phenotypic transition of PTs during injury, revealing a reversible transcriptional program as an adaptive response during disease.

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 antineutrophil cytoplasmic antibody-associated glomerulonephritis (ANCA-GN), an immune-mediated disease with aggressive glomerular damage and irreversible loss of kidney function. We identified previously unknown morphometric signatures of podocyte depletion in patients with ANCA-GN, which allowed patient classification and, in combination with routine clinical tools, showed potential for risk stratification. Our approach enables robust and scalable molecular morphometric analysis of human tissues, yielding deeper biological insights into the human kidney pathophysiology.

Cardiovascular disease is the major cause of death in end-stage renal disease (ESRD). To develop better means to assess cardiovascular risk in these patients, we compared conventional echocardiography-derived left ventricular ejection fraction (EF) with the novel method of 2D speckle-tracking echocardiography to determine cardiac strain.

Desmosomes are intercellular junctions connecting keratin intermediate filaments of neighboring cells. The cadherins desmoglein 2 (Dsg2) and desmocollin 2 mediate cell-cell adhesion, whereas desmoplakin (Dsp) provides the attachment of desmosomes to keratins. Although the importance of the desmosome-keratin network is well established in mechanically challenged tissues, we aimed to assess the currently understudied function of desmosomal proteins in intestinal epithelia.