Publications by authors named "Rafael Kramann"

90 Publications

Genetically determined NLRP3 inflammasome activation associates with systemic inflammation and cardiovascular mortality.

Eur Heart J 2021 Mar 22. Epub 2021 Mar 22.

Department of Internal Medicine IV, Nephrology and Hypertension, Saarland University Hospital, Kirrberger Strasse, Building 41, 66424 Homburg/Saar, Germany.

Aims: Inflammation plays an important role in cardiovascular disease (CVD) development. The NOD-like receptor protein-3 (NLRP3) inflammasome contributes to the development of atherosclerosis in animal models. Components of the NLRP3 inflammasome pathway such as interleukin-1β can therapeutically be targeted. Associations of genetically determined inflammasome-mediated systemic inflammation with CVD and mortality in humans are unknown.

Methods And Results: We explored the association of genetic NLRP3 variants with prevalent CVD and cardiovascular mortality in 538 167 subjects on the individual participant level in an explorative gene-centric approach without performing multiple testing. Functional relevance of single-nucleotide polymorphisms on NLRP3 inflammasome activation has been evaluated in monocyte-enriched peripheral blood mononuclear cells (PBMCs). Genetic analyses identified the highly prevalent (minor allele frequency 39.9%) intronic NLRP3 variant rs10754555 to affect NLRP3 gene expression. rs10754555 carriers showed significantly higher C-reactive protein and serum amyloid A plasma levels. Carriers of the G allele showed higher NLRP3 inflammasome activation in isolated human PBMCs. In carriers of the rs10754555 variant, the prevalence of coronary artery disease was significantly higher as compared to non-carriers with a significant interaction between rs10754555 and age. Importantly, rs10754555 carriers had significantly higher risk for cardiovascular mortality during follow-up. Inflammasome inducers (e.g. urate, triglycerides, apolipoprotein C3) modulated the association between rs10754555 and mortality.

Conclusion: The NLRP3 intronic variant rs10754555 is associated with increased systemic inflammation, inflammasome activation, prevalent coronary artery disease, and mortality. This study provides evidence for a substantial role of genetically driven systemic inflammation in CVD and highlights the NLRP3 inflammasome as a therapeutic target.
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http://dx.doi.org/10.1093/eurheartj/ehab107DOI Listing
March 2021

Deep learning-based molecular morphometrics for kidney biopsies.

JCI Insight 2021 Apr 8;6(7). Epub 2021 Apr 8.

III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

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.
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http://dx.doi.org/10.1172/jci.insight.144779DOI Listing
April 2021

Causal integration of multi-omics data with prior knowledge to generate mechanistic hypotheses.

Mol Syst Biol 2021 01;17(1):e9730

Faculty of Medicine, and Heidelberg University Hospital, Institute for Computational Biomedicine, Heidelberg University, Heidelberg, Germany.

Multi-omics datasets can provide molecular insights beyond the sum of individual omics. Various tools have been recently developed to integrate such datasets, but there are limited strategies to systematically extract mechanistic hypotheses from them. Here, we present COSMOS (Causal Oriented Search of Multi-Omics Space), a method that integrates phosphoproteomics, transcriptomics, and metabolomics datasets. COSMOS combines extensive prior knowledge of signaling, metabolic, and gene regulatory networks with computational methods to estimate activities of transcription factors and kinases as well as network-level causal reasoning. COSMOS provides mechanistic hypotheses for experimental observations across multi-omics datasets. We applied COSMOS to a dataset comprising transcriptomics, phosphoproteomics, and metabolomics data from healthy and cancerous tissue from eleven clear cell renal cell carcinoma (ccRCC) patients. COSMOS was able to capture relevant crosstalks within and between multiple omics layers, such as known ccRCC drug targets. We expect that our freely available method will be broadly useful to extract mechanistic insights from multi-omics studies.
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http://dx.doi.org/10.15252/msb.20209730DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7838823PMC
January 2021

Experimental and computational technologies to dissect the kidney at the single-cell level.

Nephrol Dial Transplant 2020 Dec 17. Epub 2020 Dec 17.

Division of Nephrology, RWTH Aachen University, Aachen, Germany.

The field of single-cell technologies, in particular single-cell genomics with transcriptomics and epigenomics, and most recently single-cell proteomics, is rapidly growing and holds promise to advance our understanding of organ homoeostasis and disease, and facilitate the identification of novel therapeutic targets and biomarkers. This review offers an introduction to these technologies. In addition, as the size and complexity of the data require sophisticated computational methods for analysis and interpretation, we will also provide an overview of these methods and summarize the single-cell literature specifically pertaining to the kidney.
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http://dx.doi.org/10.1093/ndt/gfaa233DOI Listing
December 2020

Heterogeneous bone-marrow stromal progenitors drive myelofibrosis via a druggable alarmin axis.

Cell Stem Cell 2021 Apr 9;28(4):637-652.e8. Epub 2020 Dec 9.

Department of Hematology, Erasmus Medical Center, Rotterdam 3015GD, the Netherlands; Department of Cell Biology, Institute for Biomedical Engineering, Faculty of Medicine, RWTH Aachen University, Pauwelsstrasse 30, 52074 Aachen, Germany; Oncode Institute, Erasmus Medical Center, Rotterdam 3015GD, the Netherlands. Electronic address:

Functional contributions of individual cellular components of the bone-marrow microenvironment to myelofibrosis (MF) in patients with myeloproliferative neoplasms (MPNs) are incompletely understood. We aimed to generate a comprehensive map of the stroma in MPNs/MFs on a single-cell level in murine models and patient samples. Our analysis revealed two distinct mesenchymal stromal cell (MSC) subsets as pro-fibrotic cells. MSCs were functionally reprogrammed in a stage-dependent manner with loss of their progenitor status and initiation of differentiation in the pre-fibrotic and acquisition of a pro-fibrotic and inflammatory phenotype in the fibrotic stage. The expression of the alarmin complex S100A8/S100A9 in MSC marked disease progression toward the fibrotic phase in murine models and in patient stroma and plasma. Tasquinimod, a small-molecule inhibiting S100A8/S100A9 signaling, significantly ameliorated the MPN phenotype and fibrosis in JAK2V617F-mutated murine models, highlighting that S100A8/S100A9 is an attractive therapeutic target in MPNs.
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http://dx.doi.org/10.1016/j.stem.2020.11.004DOI Listing
April 2021

Implementation of Pericytes in Vascular Regeneration Strategies.

Tissue Eng Part B Rev 2021 Jan 20. Epub 2021 Jan 20.

Department of Nephrology and Hypertension, University Medical Center Utrecht, Utrecht, The Netherlands.

For the survival and integration of complex large-sized tissue-engineered (TE) organ constructs that exceed the maximal nutrients and oxygen diffusion distance required for cell survival, graft (pre)vascularization to ensure medium or blood supply is crucial. To achieve this, the morphology and functionality of the microcapillary bed should be mimicked by incorporating vascular cell populations, including endothelium and mural cells. Pericytes play a crucial role in microvascular function, blood vessel stability, angiogenesis, and blood pressure regulation. In addition, tissue-specific pericytes are important in maintaining specific functions in different organs, including vitamin A storage in the liver, renin production in the kidneys and maintenance of the blood-brain-barrier. Together with their multipotential differentiation capacity, this makes pericytes the preferred cell type for application in TE grafts. The use of a tissue-specific pericyte cell population that matches the TE organ may benefit organ function. In this review, we provide an overview of the literature for graft (pre)-vascularization strategies and highlight the possible advantages of using tissue-specific pericytes for specific TE organ grafts. Impact statement The use of a tissue-specific pericyte cell population that matches the tissue-engineered (TE) organ may benefit organ function. In this review, we provide an overview of the literature for graft (pre)vascularization strategies and highlight the possible advantages of using tissue-specific pericytes for specific TE organ grafts.
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http://dx.doi.org/10.1089/ten.TEB.2020.0229DOI Listing
January 2021

Decoding myofibroblast origins in human kidney fibrosis.

Nature 2021 01 11;589(7841):281-286. Epub 2020 Nov 11.

Division of Nephrology and Clinical Immunology, RWTH Aachen University, Aachen, Germany.

Kidney fibrosis is the hallmark of chronic kidney disease progression; however, at present no antifibrotic therapies exist. The origin, functional heterogeneity and regulation of scar-forming cells that occur during human kidney fibrosis remain poorly understood. Here, using single-cell RNA sequencing, we profiled the transcriptomes of cells from the proximal and non-proximal tubules of healthy and fibrotic human kidneys to map the entire human kidney. This analysis enabled us to map all matrix-producing cells at high resolution, and to identify distinct subpopulations of pericytes and fibroblasts as the main cellular sources of scar-forming myofibroblasts during human kidney fibrosis. We used genetic fate-tracing, time-course single-cell RNA sequencing and ATAC-seq (assay for transposase-accessible chromatin using sequencing) experiments in mice, and spatial transcriptomics in human kidney fibrosis, to shed light on the cellular origins and differentiation of human kidney myofibroblasts and their precursors at high resolution. Finally, we used this strategy to detect potential therapeutic targets, and identified NKD2 as a myofibroblast-specific target in human kidney fibrosis.
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http://dx.doi.org/10.1038/s41586-020-2941-1DOI Listing
January 2021

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

J Am Soc Nephrol 2021 Jan 5;32(1):52-68. Epub 2020 Nov 5.

Institute of Imaging and Computer Vision, RWTH Aachen University, Aachen, Germany.

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.
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http://dx.doi.org/10.1681/ASN.2020050597DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7894663PMC
January 2021

Only Hyperuricemia with Crystalluria, but not Asymptomatic Hyperuricemia, Drives Progression of Chronic Kidney Disease.

J Am Soc Nephrol 2020 12 16;31(12):2773-2792. Epub 2020 Sep 16.

Division of Nephrology, Department of Medicine IV, Ludwig-Maximilian's-University Hospital, Munich, Germany

Background: The roles of asymptomatic hyperuricemia or uric acid (UA) crystals in CKD progression are unknown. Hypotheses to explain links between UA deposition and progression of CKD include that () asymptomatic hyperuricemia does not promote CKD progression unless UA crystallizes in the kidney; () UA crystal granulomas may form due to pre-existing CKD; and () proinflammatory granuloma-related M1-like macrophages may drive UA crystal-induced CKD progression.

Methods: MALDI-FTICR mass spectrometry, immunohistochemistry, 3D confocal microscopy, and flow cytometry were used to characterize a novel mouse model of hyperuricemia and chronic UA crystal nephropathy with granulomatous nephritis. Interventional studies probed the role of crystal-induced inflammation and macrophages in the pathology of progressive CKD.

Results: Asymptomatic hyperuricemia alone did not cause CKD or drive the progression of aristolochic acid I-induced CKD. Only hyperuricemia with UA crystalluria due to urinary acidification caused tubular obstruction, inflammation, and interstitial fibrosis. UA crystal granulomas surrounded by proinflammatory M1-like macrophages developed late in this process of chronic UA crystal nephropathy and contributed to the progression of pre-existing CKD. Suppressing M1-like macrophages with adenosine attenuated granulomatous nephritis and the progressive decline in GFR. In contrast, inhibiting the JAK/STAT inflammatory pathway with tofacitinib was not renoprotective.

Conclusions: Asymptomatic hyperuricemia does not affect CKD progression unless UA crystallizes in the kidney. UA crystal granulomas develop late in chronic UA crystal nephropathy and contribute to CKD progression because UA crystals trigger M1-like macrophage-related interstitial inflammation and fibrosis. Targeting proinflammatory macrophages, but not JAK/STAT signaling, can attenuate granulomatous interstitial nephritis.
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http://dx.doi.org/10.1681/ASN.2020040523DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7790211PMC
December 2020

Fibrosis and Immune Cell Infiltration Are Separate Events Regulated by Cell-Specific Receptor Notch3 Expression.

J Am Soc Nephrol 2020 11 28;31(11):2589-2608. Epub 2020 Aug 28.

Clinic of Nephrology and Hypertension, Diabetes and Endocrinology, Otto-von-Guericke University Magdeburg, Magdeburg, Germany

Background: Kidney injuries that result in chronic inflammation initiate crosstalk between stressed resident cells and infiltrating immune cells. In animal models, whole-body receptor deficiency protects from leukocyte infiltration and organ fibrosis. However, the relative contribution of expression in tissue versus infiltrating immune cells is unknown.

Methods: Chimeric mice deficient for in hematopoietic cells and/or resident tissue cells were generated, and kidney fibrosis and inflammation after unilateral ureteral obstruction (UUO) were analyzed. Adoptive transfer of labeled bone marrow-derived cells validated the results in a murine ear infection model. adhesion assays, integrin activation, and extracellular matrix production were analyzed.

Results: Fibrosis follows UUO, but inflammatory cell infiltration mostly depends upon Notch3 expression in hematopoietic cells, which coincides with an enhanced proinflammatory milieu (., CCL2 and CCL5 upregulation). Notch3 expression on CD45 leukocytes plays a prominent role in efficient cell transmigration. Functionally, leukocyte adhesion and integrin activation are abrogated in the absence of receptor Notch3. Chimeric animal models also reveal that tubulointerstitial fibrosis develops, even in the absence of prominent leukocyte infiltrates after ureteral obstruction. Deleting Notch3 receptors on resident cells blunts kidney fibrosis, ablates NF-B signaling, and lessens matrix deposition.

Conclusions: Cell-specific receptor Notch3 signaling independently orchestrates leukocyte infiltration and organ fibrosis. Interference with Notch3 signaling may present a novel therapeutic approach in inflammatory as well as fibrotic diseases.
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http://dx.doi.org/10.1681/ASN.2019121289DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7608975PMC
November 2020

Big Data Approaches in Heart Failure Research.

Curr Heart Fail Rep 2020 10;17(5):213-224

Institute for Computational Biomedicine, Bioquant, Heidelberg University, Faculty of Medicine, and Heidelberg University Hospital, Heidelberg, Germany.

Purpose Of Review: The goal of this review is to summarize the state of big data analyses in the study of heart failure (HF). We discuss the use of big data in the HF space, focusing on "omics" and clinical data. We address some limitations of this data, as well as their future potential.

Recent Findings: Omics are providing insight into plasmal and myocardial molecular profiles in HF patients. The introduction of single cell and spatial technologies is a major advance that will reshape our understanding of cell heterogeneity and function as well as tissue architecture. Clinical data analysis focuses on HF phenotyping and prognostic modeling. Big data approaches are increasingly common in HF research. The use of methods designed for big data, such as machine learning, may help elucidate the biology underlying HF. However, important challenges remain in the translation of this knowledge into improvements in clinical care.
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http://dx.doi.org/10.1007/s11897-020-00469-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7496059PMC
October 2020

Increased CXCL4 expression in hematopoietic cells links inflammation and progression of bone marrow fibrosis in MPN.

Blood 2020 10;136(18):2051-2064

Department of Hematology, Erasmus Medical Center Cancer Institute, Rotterdam, The Netherlands.

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm (MPN) that leads to progressive bone marrow (BM) fibrosis. Although the cellular mutations involved in the pathogenesis of PMF have been extensively investigated, the sequential events that drive stromal activation and fibrosis by hematopoietic-stromal cross-talk remain elusive. Using an unbiased approach and validation in patients with MPN, we determined that the differential spatial expression of the chemokine CXCL4/platelet factor-4 marks the progression of fibrosis. We show that the absence of hematopoietic CXCL4 ameliorates the MPN phenotype, reduces stromal cell activation and BM fibrosis, and decreases the activation of profibrotic pathways in megakaryocytes, inflammation in fibrosis-driving cells, and JAK/STAT activation in both megakaryocytes and stromal cells in 3 murine PMF models. Our data indicate that higher CXCL4 expression in MPN has profibrotic effects and is a mediator of the characteristic inflammation. Therefore, targeting CXCL4 might be a promising strategy to reduce inflammation in PMF.
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http://dx.doi.org/10.1182/blood.2019004095DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7678060PMC
October 2020

Vitamin K2 Needs an RDI Separate from Vitamin K1.

Nutrients 2020 Jun 21;12(6). Epub 2020 Jun 21.

Department of Biochemistry, Cardiovascular Research Institute Maastricht, 6200MD Maastricht, The Netherlands.

Vitamin K and its essential role in coagulation (vitamin K [Koagulation]) have been well established and accepted the world over. Many countries have a Recommended Daily Intake (RDI) for vitamin K based on early research, and its necessary role in the activation of vitamin K-dependent coagulation proteins is known. In the past few decades, the role of vitamin K-dependent proteins in processes beyond coagulation has been discovered. Various isoforms of vitamin K have been identified, and vitamin K2 specifically has been highlighted for its long half-life and extrahepatic activity, whereas the dietary form vitamin K1 has a shorter half-life. In this review, we highlight the specific activity of vitamin K2 based upon proposed frameworks necessary for a bioactive substance to be recommended for an RDI. Vitamin K2 meets all these criteria and should be considered for a specific dietary recommendation intake.
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http://dx.doi.org/10.3390/nu12061852DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7353270PMC
June 2020

Different subpopulations of kidney interstitial cells produce erythropoietin and factors supporting tissue oxygenation in response to hypoxia in vivo.

Kidney Int 2020 10 23;98(4):918-931. Epub 2020 May 23.

Institute of Physiology, University of Regensburg, Regensburg, Germany.

Genetic induction of hypoxia signaling by deletion of the von Hippel-Lindau (Vhl) protein in mesenchymal PDGFR-β cells leads to abundant HIF-2 dependent erythropoietin (EPO) expression in the cortex and outer medulla of the kidney. This rather unique feature of kidney PDGFR-β cells promote questions about their special characteristics and general functional response to hypoxia. To address these issues, we characterized kidney PDGFR-β EPO expressing cells based on additional cell markers and their gene expression profile in response to hypoxia signaling induced by targeted deletion of Vhl or exposure to low oxygen and carbon monoxide respectively, and after unilateral ureteral obstruction. CD73, Gli1, tenascin C and interstitial SMMHC cells were identified as zonally distributed subpopulations of PDGFR-β cells. EPO expression could be induced by Vhl deletion in all PDGFR-β subpopulations. Under hypoxemic conditions, recruited EPO cells were mostly part of the CD73 subpopulation. Besides EPO production, expression of adrenomedullin and regulator of G-protein signaling 4 was upregulated in PDGFR-β subpopulations in response to the different hypoxic stimuli. Thus, different kidney interstitial PDGFR-β subpopulations exist, capable of producing EPO in response to different stimuli. Activation of hypoxia signaling in these cells also induces factors likely contributing to improved kidney interstitial tissue oxygenation.
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http://dx.doi.org/10.1016/j.kint.2020.04.040DOI Listing
October 2020

Cardiac Remodeling in Chronic Kidney Disease.

Toxins (Basel) 2020 03 5;12(3). Epub 2020 Mar 5.

Clinic for Renal and Hypertensive Disorders, Rheumatological and Immunological Disease, University Hospital of the RWTH Aachen, 52074 Aachen, Germany.

Cardiac remodeling occurs frequently in chronic kidney disease patients and affects quality of life and survival. Current treatment options are highly inadequate. As kidney function declines, numerous metabolic pathways are disturbed. Kidney and heart functions are highly connected by organ crosstalk. Among others, altered volume and pressure status, ischemia, accelerated atherosclerosis and arteriosclerosis, disturbed mineral metabolism, renal anemia, activation of the renin-angiotensin system, uremic toxins, oxidative stress and upregulation of cytokines stress the sensitive interplay between different cardiac cell types. The fatal consequences are left-ventricular hypertrophy, fibrosis and capillary rarefaction, which lead to systolic and/or diastolic left-ventricular failure. Furthermore, fibrosis triggers electric instability and sudden cardiac death. This review focuses on established and potential pathophysiological cardiorenal crosstalk mechanisms that drive uremia-induced senescence and disease progression, including potential known targets and animal models that might help us to better understand the disease and to identify novel therapeutics.
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http://dx.doi.org/10.3390/toxins12030161DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7150902PMC
March 2020

Lumenal calcification and microvasculopathy in fetuin-A-deficient mice lead to multiple organ morbidity.

PLoS One 2020 19;15(2):e0228503. Epub 2020 Feb 19.

Helmholtz Institute for Biomedical Engineering, Biointerface Lab, RWTH Aachen University Hospital, Aachen, Germany.

The plasma protein fetuin-A mediates the formation of protein-mineral colloids known as calciprotein particles (CPP)-rapid clearance of these CPP by the reticuloendothelial system prevents errant mineral precipitation and therefore pathological mineralization (calcification). The mutant mouse strain D2,Ahsg-/- combines fetuin-A deficiency with the calcification-prone DBA/2 genetic background, having a particularly severe compound phenotype of microvascular and soft tissue calcification. Here we studied mechanisms leading to soft tissue calcification, organ damage and death in these mice. We analyzed mice longitudinally by echocardiography, X-ray-computed tomography, analytical electron microscopy, histology, mass spectrometry proteomics, and genome-wide microarray-based expression analyses of D2 wildtype and Ahsg-/- mice. Fetuin-A-deficient mice had calcified lesions in myocardium, lung, brown adipose tissue, reproductive organs, spleen, pancreas, kidney and the skin, associated with reduced growth, cardiac output and premature death. Importantly, early-stage calcified lesions presented in the lumen of the microvasculature suggesting precipitation of mineral containing complexes from the fluid phase of blood. Genome-wide expression analysis of calcified lesions and surrounding (not calcified) tissue, together with morphological observations, indicated that the calcification was not associated with osteochondrogenic cell differentiation, but rather with thrombosis and fibrosis. Collectively, these results demonstrate that soft tissue calcification can start by intravascular mineral deposition causing microvasculopathy, which impacts on growth, organ function and survival. Our study underscores the importance of fetuin-A and related systemic regulators of calcified matrix metabolism to prevent cardiovascular disease, especially in dysregulated mineral homeostasis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0228503PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7029858PMC
May 2020

A Functional Landscape of CKD Entities From Public Transcriptomic Data.

Kidney Int Rep 2020 Feb 13;5(2):211-224. Epub 2019 Nov 13.

Faculty of Medicine, RWTH Aachen University, Joint Research Centre for Computational Biomedicine (JRC-COMBINE), Aachen, Germany.

Introduction: To develop effective therapies and identify novel early biomarkers for chronic kidney disease, an understanding of the molecular mechanisms orchestrating it is essential. We here set out to understand how differences in chronic kidney disease (CKD) origin are reflected in gene expression. To this end, we integrated publicly available human glomerular microarray gene expression data for 9 kidney disease entities that account for most of CKD worldwide. Our primary goal was to demonstrate the possibilities and potential on data analysis and integration to the nephrology community.

Methods: We integrated data from 5 publicly available studies and compared glomerular gene expression profiles of disease with that of controls from nontumor parts of kidney cancer nephrectomy tissues. A major challenge was the integration of the data from different sources, platforms, and conditions that we mitigated with a bespoke stringent procedure.

Results: We performed a global transcriptome-based delineation of different kidney disease entities, obtaining a transcriptomic diffusion map of their similarities and differences based on the genes that acquire a consistent differential expression between each kidney disease entity and nephrectomy tissue. We derived functional insights by inferring the activity of signaling pathways and transcription factors from the collected gene expression data and identified potential drug candidates based on expression signature matching. We validated representative findings by immunostaining in human kidney biopsies indicating, for example, that the transcription factor FOXM1 is significantly and specifically expressed in parietal epithelial cells in rapidly progressive glomerulonephritis (RPGN) whereas not expressed in control kidney tissue. Furthermore, we found drug candidates by matching the signature on expression of drugs to that of the CKD entities, in particular, the Food and Drug Administration-approved drug nilotinib.

Conclusion: These results provide a foundation to comprehend the specific molecular mechanisms underlying different kidney disease entities that can pave the way to identify biomarkers and potential therapeutic targets. To facilitate further use, we provide our results as a free interactive Web application: https://saezlab.shinyapps.io/ckd_landscape/. However, because of the limitations of the data and the difficulties in its integration, any specific result should be considered with caution. Indeed, we consider this study rather an illustration of the value of functional genomics and integration of existing data.
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http://dx.doi.org/10.1016/j.ekir.2019.11.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7000845PMC
February 2020

Valvular Calcification in Chronic Kidney Disease.

Adv Chronic Kidney Dis 2019 11;26(6):464-471

Department of Nephrology, University Hospital RWTH Aachen, Aachen, Germany; Department of Internal Medicine, Nephrology and Transplantation, Erasmus University Medical Center, Rotterdam, the Netherlands.

Accelerated and premature cardiovascular calcification is a hallmark of patients with chronic kidney disease (CKD) or end-stage renal disease (ESRD). The presence and the amount of cardiovascular calcification are among the driving forces of increased morbidity and mortality in renal patients. Cardiovascular calcification occurs at different sites, including the cardiac valves-a location that is of particular importance for both the patient and the treating physician. The correlation between degree of calcification and functional impairment is particularly close at the aortic valve, that is, the amount of calcification predicts the degree of stenosis. Calcific aortic stenosis (CAS) is the most prevalent valvular heart disease in Western societies. CAS is particularly prevalent in patients with underlying CKD or ESRD. CAS increases afterload and hence contributes to the widespread finding of left ventricular hypertrophy in CKD/ESRD patients. Medical treatment options to prevent the development and progression of CAS are limited. Hence, close surveillance and timely referral of patients for heart valve replacement therapy is a mainstay of current therapy. Novel treatment approaches, such as transcatheter aortic valve implantation, offer promising yet challenging options for elderly, comorbid, and often frail patients with CAS in combination with advanced CKD/ESRD.
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http://dx.doi.org/10.1053/j.ackd.2019.10.004DOI Listing
November 2019

Apolipoprotein C3 induces inflammation and organ damage by alternative inflammasome activation.

Nat Immunol 2020 01 9;21(1):30-41. Epub 2019 Dec 9.

Department of Internal Medicine IV (Nephrology and Hypertension), Saarland University, Homburg, Germany.

NLRP3-inflammasome-driven inflammation is involved in the pathogenesis of a variety of diseases. Identification of endogenous inflammasome activators is essential for the development of new anti-inflammatory treatment strategies. Here, we identified that apolipoprotein C3 (ApoC3) activates the NLRP3 inflammasome in human monocytes by inducing an alternative NLRP3 inflammasome via caspase-8 and dimerization of Toll-like receptors 2 and 4. Alternative inflammasome activation in human monocytes is mediated by the Toll-like receptor adapter protein SCIMP. This triggers Lyn/Syk-dependent calcium entry and the production of reactive oxygen species, leading to activation of caspase-8. In humanized mouse models, ApoC3 activated human monocytes in vivo to impede endothelial regeneration and promote kidney injury in an NLRP3- and caspase-8-dependent manner. These data provide new insights into the regulation of the NLRP3 inflammasome and the pathophysiological role of triglyceride-rich lipoproteins containing ApoC3. Targeting ApoC3 might prevent organ damage and provide an anti-inflammatory treatment for vascular and kidney diseases.
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http://dx.doi.org/10.1038/s41590-019-0548-1DOI Listing
January 2020

The authors reply.

Kidney Int 2019 12;96(6):1422-1423

RWTH Aachen University, Department of Nephrology and Clinical Immunology, Aachen, Germany; Department of Internal Medicine, Nephrology and Transplantation, Erasmus Medical Center, Rotterdam, The Netherlands. Electronic address:

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http://dx.doi.org/10.1016/j.kint.2019.09.011DOI Listing
December 2019

Transcriptome analysis reveals microvascular endothelial cell-dependent pericyte differentiation.

Sci Rep 2019 10 30;9(1):15586. Epub 2019 Oct 30.

Experimental Cardiology, Department of Cardiology, Thoraxcenter, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands.

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.
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http://dx.doi.org/10.1038/s41598-019-51838-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6821775PMC
October 2019

mTOR-mediated podocyte hypertrophy regulates glomerular integrity in mice and humans.

JCI Insight 2019 09 19;4(18). Epub 2019 Sep 19.

Development and Stem Cells Program, Monash Biomedicine Discovery Institute and Department of Anatomy and Developmental Biology, Monash University, Melbourne, Australia.

The cellular origins of glomerulosclerosis involve activation of parietal epithelial cells (PECs) and progressive podocyte depletion. While mammalian target of rapamycin-mediated (mTOR-mediated) podocyte hypertrophy is recognized as an important signaling pathway in the context of glomerular disease, the role of podocyte hypertrophy as a compensatory mechanism preventing PEC activation and glomerulosclerosis remains poorly understood. In this study, we show that glomerular mTOR and PEC activation-related genes were both upregulated and intercorrelated in biopsies from patients with focal segmental glomerulosclerosis (FSGS) and diabetic nephropathy, suggesting both compensatory and pathological roles. Advanced morphometric analyses in murine and human tissues identified podocyte hypertrophy as a compensatory mechanism aiming to regulate glomerular functional integrity in response to somatic growth, podocyte depletion, and even glomerulosclerosis - all of this in the absence of detectable podocyte regeneration. In mice, pharmacological inhibition of mTOR signaling during acute podocyte loss impaired hypertrophy of remaining podocytes, resulting in unexpected albuminuria, PEC activation, and glomerulosclerosis. Exacerbated and persistent podocyte hypertrophy enabled a vicious cycle of podocyte loss and PEC activation, suggesting a limit to its beneficial effects. In summary, our data highlight a critical protective role of mTOR-mediated podocyte hypertrophy following podocyte loss in order to preserve glomerular integrity, preventing PEC activation and glomerulosclerosis.
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http://dx.doi.org/10.1172/jci.insight.99271DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6795295PMC
September 2019

Speckle Tracking Echocardiography and All-Cause and Cardiovascular Mortality Risk in Chronic Kidney Disease Patients.

Kidney Blood Press Res 2019 15;44(4):690-703. Epub 2019 Aug 15.

Department of Nephrology and Clinical Immunology, Uniklinik RWTH Aachen, Aachen, Germany,

Background And Objectives: Patients with chronic kidney disease (CKD) exhibit a highly increased risk of cardiovascular (CV) morbidity and mortality. Subtle changes in left ventricular function can be detected by two-dimensional (2D) speckle tracking echocardiography (STE). This study investigated whether myocardial dysfunction detected by 2D STE may aid in CV and all-cause mortality risk assessment in patients with CKD stages 3 and 4.

Method: A study group of 285 patients (CKD 3: 193 patients; CKD 4: 92 patients) and a healthy control group (34 participants) were included in the retrospective study. 2D STE values as well as early and late diastolic strain rates were measured in ventricular longitudinal, circumferential and radial directions. Patients' CV and all-cause outcome was determined.

Results: In the CKD group all measured longitudinal STE values and radial strain were significantly reduced compared to the control group. Cox proportional hazards regression revealed global longitudinal strain to predict CV and all-cause mortality (hazard ratio [HR] 1.15, 95% CI 1.06-1.25; p = 0.0008 and HR 1.09, 95% CI 1.04-1.14; p = 0.0003). After adjustment for sex, age, diabetes, estimated glomerular filtration rate, and preexisting CV disease, this association was maintained for CV mortality and all-cause mortality (HR 1.16, 95% CI 1.06-1.27; p = 0.0019 and HR 1.08, 95% CI 1.03-1.14; p = 0.0026, respectively).

Conclusions: The present study shows that 2D STE detects reduced left ventricular myocardial function and allows the prediction of CV and all-cause mortality in patients at CKD stages 3 and 4.
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http://dx.doi.org/10.1159/000501225DOI Listing
January 2020

Optical Clearing and Imaging of Immunolabeled Kidney Tissue.

J Vis Exp 2019 07 22(149). Epub 2019 Jul 22.

Division of Nephrology and Clinical Immunology, University Hospital RWTH Aachen; Department of Internal Medicine, Nephrology and Transplantation, Erasmus Medical Center.

Optical clearing techniques render tissue transparent by equilibrating the refractive index throughout a sample for subsequent three-dimensional (3-D) imaging. They have received great attention in all research areas for the potential to analyze microscopic multicellular structures that extend over macroscopic distances. Given that kidney tubules, vasculature, nerves, and glomeruli extend in many directions, which have been only partially captured by traditional two-dimensional techniques so far, tissue clearing also opened up many new areas of kidney research. The list of optical clearing methods is rapidly growing, but it remains difficult for beginners in this field to choose the best method for a given research question. Provided here is a simple method that combines antibody labeling of thick mouse kidney slices; optical clearing with cheap, non-toxic and ready-to-use chemical ethyl cinnamate; and confocal imaging. This protocol describes how to perfuse kidneys and use an antigen-retrieval step to increase antibody- binding without requiring specialized equipment. Its application is presented in imaging different multicellular structures within the kidney, and how to troubleshoot poor antibody penetration into tissue is addressed. We also discuss the potential difficulties of imaging endogenous fluorophores and acquiring very large samples and how to overcome them. This simple protocol provides an easy-to-setup and comprehensive tool to study tissue in three dimensions.
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http://dx.doi.org/10.3791/60002DOI Listing
July 2019

Heterogeneity and plasticity in healthy and atherosclerotic vasculature explored by single-cell sequencing.

Cardiovasc Res 2019 Oct;115(12):1705-1715

Pathology Department, CARIM School for Cardiovascular Diseases, MUMC Maastricht, P. Debyelaan 25, Maastricht, the Netherlands.

Cellular characteristics and their adjustment to a state of disease have become more evident due to recent advances in imaging, fluorescent reporter mice, and whole genome RNA sequencing. The uncovered cellular heterogeneity and/or plasticity potentially complicates experimental studies and clinical applications, as markers derived from whole tissue 'bulk' sequencing is unable to yield a subtype transcriptome and specific markers. Here, we propose definitions on heterogeneity and plasticity, discuss current knowledge thereof in the vasculature and how this may be improved by single-cell sequencing (SCS). SCS is emerging as an emerging technique, enabling researchers to investigate different cell populations in more depth than ever before. Cell selection methods, e.g. flow assisted cell sorting, and the quantity of cells can influence the choice of SCS method. Smart-Seq2 offers sequencing of the complete mRNA molecule on a low quantity of cells, while Drop-seq is possible on large numbers of cells on a more superficial level. SCS has given more insight in heterogeneity in healthy vasculature, where it revealed that zonation is crucial in gene expression profiles among the anatomical axis. In diseased vasculature, this heterogeneity seems even more prominent with discovery of new immune subsets in atherosclerosis as proof. Vascular smooth muscle cells and mesenchymal cells also share these plastic characteristics with the ability to up-regulate markers linked to stem cells, such as Sca-1 or CD34. Current SCS studies show some limitations to the number of replicates, quantity of cells used, or the loss of spatial information. Bioinformatical tools could give some more insight in current datasets, making use of pseudo-time analysis or RNA velocity to investigate cell differentiation or polarization. In this review, we discuss the use of SCS in unravelling heterogeneity in the vasculature, its current limitations and promising future applications.
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http://dx.doi.org/10.1093/cvr/cvz185DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6873093PMC
October 2019

Novel 3D analysis using optical tissue clearing documents the evolution of murine rapidly progressive glomerulonephritis.

Kidney Int 2019 08 15;96(2):505-516. Epub 2019 Mar 15.

Department of Nephrology and Clinical Immunology, RWTH Aachen University Clinic, Aachen, Germany; Interdisciplinary Centre for Clinical Research (IZKF Aachen), RWTH Aachen University Hospital, Aachen, Germany; Heisenberg Chair for Preventive and Translational Nephrology, Division of Nephrology, RWTH Aachen University, Aachen, Germany. Electronic address:

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.
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http://dx.doi.org/10.1016/j.kint.2019.02.034DOI Listing
August 2019

Non-canonical HIF-1 stabilization contributes to intestinal tumorigenesis.

Oncogene 2019 07 1;38(28):5670-5685. Epub 2019 May 1.

Molecular Tumor Biology, Department of General Visceral and Transplantation Surgery, RWTH University Hospital, Pauwelsstraße 30, 52074, Aachen, Germany.

The hypoxia-inducible transcription factor HIF-1 is appreciated as a promising target for cancer therapy. However, conditional deletion of HIF-1 and HIF-1 target genes in cells of the tumor microenvironment can result in accelerated tumor growth, calling for a detailed characterization of the cellular context to fully comprehend HIF-1's role in tumorigenesis. We dissected cell type-specific functions of HIF-1 for intestinal tumorigenesis by lineage-restricted deletion of the Hif1a locus. Intestinal epithelial cell-specific Hif1a loss reduced activation of Wnt/β-catenin, tumor-specific metabolism and inflammation, significantly inhibiting tumor growth. Deletion of Hif1a in myeloid cells reduced the expression of fibroblast-activating factors in tumor-associated macrophages resulting in decreased abundance of tumor-associated fibroblasts (TAF) and robustly reduced tumor formation. Interestingly, hypoxia was detectable only sparsely and without spatial association with HIF-1α, arguing for an importance of hypoxia-independent, i.e., non-canonical, HIF-1 stabilization for intestinal tumorigenesis that has not been previously appreciated. This adds a further layer of complexity to the regulation of HIF-1 and suggests that hypoxia and HIF-1α stabilization can be uncoupled in cancer. Collectively, our data show that HIF-1 is a pivotal pro-tumorigenic factor for intestinal tumor formation, controlling key oncogenic programs in both the epithelial tumor compartment and the tumor microenvironment.
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http://dx.doi.org/10.1038/s41388-019-0816-4DOI Listing
July 2019

Big science and big data in nephrology.

Kidney Int 2019 06 5;95(6):1326-1337. Epub 2019 Mar 5.

RWTH Aachen, Department of Nephrology and Clinical Immunology, Aachen, Germany; Department of Internal Medicine, Nephrology and Transplantation, Erasmus Medical Center, Rotterdam, The Netherlands. Electronic address:

There have been tremendous advances during the last decade in methods for large-scale, high-throughput data generation and in novel computational approaches to analyze these datasets. These advances have had a profound impact on biomedical research and clinical medicine. The field of genomics is rapidly developing toward single-cell analysis, and major advances in proteomics and metabolomics have been made in recent years. The developments on wearables and electronic health records are poised to change clinical trial design. This rise of 'big data' holds the promise to transform not only research progress, but also clinical decision making towards precision medicine. To have a true impact, it requires integrative and multi-disciplinary approaches that blend experimental, clinical and computational expertise across multiple institutions. Cancer research has been at the forefront of the progress in such large-scale initiatives, so-called 'big science,' with an emphasis on precision medicine, and various other areas are quickly catching up. Nephrology is arguably lagging behind, and hence these are exciting times to start (or redirect) a research career to leverage these developments in nephrology. In this review, we summarize advances in big data generation, computational analysis, and big science initiatives, with a special focus on applications to nephrology.
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http://dx.doi.org/10.1016/j.kint.2018.11.048DOI Listing
June 2019

Elastin imaging enables noninvasive staging and treatment monitoring of kidney fibrosis.

Sci Transl Med 2019 04;11(486)

Institute of Pathology, RWTH Aachen University Hospital, 52074 Aachen, Germany.

Fibrosis is the common endpoint and currently the best predictor of progression of chronic kidney diseases (CKDs). Despite several drawbacks, biopsies remain the only available means to specifically assess the extent of renal fibrosis. Here, we show that molecular imaging of the extracellular matrix protein elastin allows for noninvasive staging and longitudinal monitoring of renal fibrosis. Elastin was hardly expressed in healthy mouse, rat, and human kidneys, whereas it was highly up-regulated in cortical, medullar, and perivascular regions in progressive CKD. Compared to a clinically relevant control contrast agent, the elastin-specific magnetic resonance imaging agent ESMA specifically detected elastin expression in multiple mouse models of renal fibrosis and also in fibrotic human kidneys. Elastin imaging allowed for repetitive and reproducible assessment of renal fibrosis, and it enabled longitudinal monitoring of therapeutic interventions, accurately capturing anti-fibrotic therapy effects. Last, in a model of reversible renal injury, elastin imaging detected ensuing fibrosis not identifiable via routine assessment of kidney function. Elastin imaging thus has the potential to become a noninvasive, specific imaging method to assess renal fibrosis.
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http://dx.doi.org/10.1126/scitranslmed.aat4865DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7115882PMC
April 2019

Disruption of CUL3-mediated ubiquitination causes proximal tubule injury and kidney fibrosis.

Sci Rep 2019 03 14;9(1):4596. Epub 2019 Mar 14.

Division of Nephrology and Hypertension, Oregon Health and Science University, Portland, Oregon, USA.

Cullin 3 (CUL3) is part of the ubiquitin proteasomal system and controls several cellular processes critical for normal organ function including the cell cycle, and Keap1/Nrf2 signaling. Kidney tubule-specific Cul3 disruption causes tubulointerstitial fibrosis, but little is known about the mechanisms. Therefore, we tested the hypothesis that dysregulation of the cell cycle and Keap1/Nrf2 pathway play a role in initiating the kidney injury upon Cul3 disruption. Cul3 deletion increased expression of cyclin E and p21, associated with uncontrolled proliferation, DNA damage, and apoptosis, all of which preceded proximal tubule injury. The cdk2-cyclin E inhibitor roscovitine did not prevent the effects of Cul3 deletion, but instead exacerbated the kidney injury. Injury occurred despite accumulation and activation of CUL3 substrate Keap1/Nrf2, proposed to be protective in kidney injury. Cul3 disruption led to progressive interstitial inflammation, functionally relevant renal fibrosis and death. Finally, we observed reduced CUL3 expression in several AKI and CKD mouse models and in fibrotic human kidney tissue. These data establish CUL3 knockout mice as a novel genetic CKD model in which dysregulation of the cell cycle may play a primary role in initiating tubule injury, and that CUL3 dysregulation could contribute to acute and fibrotic kidney disease.
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http://dx.doi.org/10.1038/s41598-019-40795-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6418206PMC
March 2019