Renal inflammation and fibrosis are the common pathways leading to progressive chronic kidney disease (CKD). We previously identified hematopoietic cell kinase (HCK) as upregulated in human chronic ...allograft injury promoting kidney fibrosis; however, the cellular source and molecular mechanisms are unclear. Here, using immunostaining and single cell sequencing data, we show that HCK expression is highly enriched in pro-inflammatory macrophages in diseased kidneys. HCK-knockout (KO) or HCK-inhibitor decreases macrophage M1-like pro-inflammatory polarization, proliferation, and migration in RAW264.7 cells and bone marrow-derived macrophages (BMDM). We identify an interaction between HCK and ATG2A and CBL, two autophagy-related proteins, inhibiting autophagy flux in macrophages. In vivo, both global or myeloid cell specific HCK-KO attenuates renal inflammation and fibrosis with reduces macrophage numbers, pro-inflammatory polarization and migration into unilateral ureteral obstruction (UUO) kidneys and unilateral ischemia reperfusion injury (IRI) models. Finally, we developed a selective boron containing HCK inhibitor which can reduce macrophage pro-inflammatory activity, proliferation, and migration in vitro, and attenuate kidney fibrosis in the UUO mice. The current study elucidates mechanisms downstream of HCK regulating macrophage activation and polarization via autophagy in CKD and identifies that selective HCK inhibitors could be potentially developed as a new therapy for renal fibrosis.
The glomerulus is a compact cluster of capillaries responsible for blood filtration and initiating urine production in the renal nephrons. A trilaminar structure in the capillary wall forms the ...glomerular filtration barrier (GFB), composed of glycocalyx-enriched and fenestrated endothelial cells adhering to the glomerular basement membrane and specialized visceral epithelial cells, podocytes, forming the outermost layer with a molecular slit diaphragm between their interdigitating foot processes. The unique dynamic and selective nature of blood filtration to produce urine requires the functionality of each of the GFB components, and hence, mimicking the glomerular filter
in vitro
has been challenging, though critical for various research applications and drug screening. Research efforts in the past few years have transformed our understanding of the structure and multifaceted roles of the cells and their intricate crosstalk in development and disease pathogenesis. In this review, we present a new wave of technologies that include glomerulus-on-a-chip, three-dimensional microfluidic models, and organoids all promising to improve our understanding of glomerular biology and to enable the development of GFB-targeted therapies. Here, we also outline the challenges and the opportunities of these emerging biomimetic systems that aim to recapitulate the complex glomerular filter, and the evolving perspectives on the sophisticated repertoire of cellular signaling that comprise the glomerular milieu.
Tissue architecture is a prerequisite for its biological functions. Recapitulating the three‐dimensional (3D) tissue structure represents one of the biggest challenges in tissue engineering. ...Two‐dimensional (2D) tissue fabrication methods are currently in the main stage for tissue engineering and disease modeling. However, due to their planar nature, the created models only represent very limited out‐of‐plane tissue structure. Here compressive buckling principle is harnessed to create 3D biomimetic cell‐laden microstructures from microfabricated planar patterns. This method allows out‐of‐plane delivery of cells and extracellular matrix patterns with high spatial precision. As a proof of principle, a variety of polymeric 3D miniature structures including a box, an octopus, a pyramid, and continuous waves are fabricated. A mineralized bone tissue model with spatially distributed cell‐laden lacunae structures is fabricated to demonstrate the fabrication power of the method. It is expected that this novel approach will help to significantly expand the utility of the established 2D fabrication techniques for 3D tissue fabrication. Given the widespread of 2D fabrication methods in biomedical research and the high demand for biomimetic 3D structures, this method is expected to bridge the gap between 2D and 3D tissue fabrication and open up new possibilities in tissue engineering and regenerative medicine.
This work represents the first study to explore the possibility of using compressive buckling‐based fabrication technology to build 3D cell‐laden microstructures from originally 2D planar structures. It is expected that this novel approach will help to significantly expand the utility of the well‐established 2D fabrication techniques and open up new possibilities in tissue engineering and regenerative medicine.
Maintenance of the intricate interdigitating morphology of podocytes is crucial for glomerular filtration. One of the key aspects of specialized podocyte morphology is the segregation and ...organization of distinct cytoskeletal filaments into different subcellular components, for which the exact mechanisms remain poorly understood.
Cells from rats, mice, and humans were used to describe the cytoskeletal configuration underlying podocyte structure. Screening the time-dependent proteomic changes in the rat puromycin aminonucleoside-induced nephropathy model correlated the actin-binding protein LIM-nebulette strongly with glomerular function. Single-cell RNA sequencing and immunogold labeling were used to determine
expression specificity in podocytes. Automated high-content imaging, super-resolution microscopy, atomic force microscopy (AFM), live-cell imaging of calcium, and measurement of motility and adhesion dynamics characterized the physiologic role of LIM-nebulette in podocytes.
knockout mice have increased susceptibility to adriamycin-induced nephropathy and display morphologic, cytoskeletal, and focal adhesion abnormalities with altered calcium dynamics, motility, and Rho GTPase activity. LIM-nebulette expression is decreased in diabetic nephropathy and FSGS patients at both the transcript and protein level. In mice, rats, and humans, LIM-nebulette expression is localized to primary, secondary, and tertiary processes of podocytes, where it colocalizes with focal adhesions as well as with vimentin fibers. LIM-nebulette shRNA knockdown in immortalized human podocytes leads to dysregulation of vimentin filament organization and reduced cellular elasticity as measured by AFM indentation.
LIM-nebulette is a multifunctional cytoskeletal protein that is critical in the maintenance of podocyte structural integrity through active reorganization of focal adhesions, the actin cytoskeleton, and intermediate filaments.
Abstract
Background and Aims
Diabetic kidney disease (DKD) is a leading cause of end-stage kidney disease (ESKD), however therapies targeting causal pathways have been limited by disease ...heterogeneity. Integrating electronic health record (EHR) data and genomics may uncover hidden subphenotypes in DKD. In this study, we use deep learning to identify a novel genetic variant of ARHGEF18 associated with significantly higher risk of DKD and ESKD (Figure 1A). We further employed quantitative microscopy techniques and biochemical assays to elucidate the mechanistic role of ARHGEF18 and its variant in podocytes.
Method
DKD patients from the Mount Sinai BioMe Biobank were used in this study. Unsupervised clustering and accounting for population structure in a deep learning framework identified two clusters: Cluster M (mild) and S (severe). We then performed a genome wide association study (GWAS) of patients within each cluster compared with healthy controls. For mechanistic studies of the novel variant, cytoplasmic, focal adhesion, cytoskeletal morphometrics as well as live-cell motility, Rho GTPase activity, and protein degradation experiments were performed using confocal and total internal reflection fluorescence (TIRF) microscopy as well as cell-free biochemical assays using immortalized human podocytes expressing ARHGEF18 wild-type (WT) and mutant transcripts.
Results
We employed autoencoders and unsupervised clustering of EHR data on 1,372 DKD patients to establish two clusters with differential prevalence of ESKD. There was a greater prevalence of proteinuria in Cluster S compared to Cluster M (Figure 1B). Further exome sequencing study in these patients identified a novel variant in ARHGEF18, a Rho guanine exchange factor highly enriched in podocytes (Figure 1A). Nephroseq database showed an increased ARHGEF18 expression in chronic kidney disease (CKD) kidney biopsy samples compared to healthy controls (Figure 1C). Overexpression of ARHGEF18 mutant transcripts in human immortalized podocytes led to impairments in cell adhesion, focal adhesion architecture, and cell motility. Live TIRF microscopy experiments showed preferential subcellular localization of GEF18 mutant to the periphery of migrating podocytes whereas GEF18 WT localized at the perinuclear/cytoplasmic region (Figure 2A, B). GEF18 mutant cells also displayed an increased RhoA activation (Figure 2C). Upon inhibition of protein synthesis using cycloheximide (CHX), we observed a significantly slower degradation of GEF18 mutant protein over a 12h period indicating increased protein stability (Figure 2D). GEF18 mutant also showed resistance to ubiquitin mediated degradation leading to pathologically increased protein levels.
Conclusions
We report a novel gain of function variant of ARHGEF18 that drives podocyte dysfunction through impaired protein localization and degradation. Targeting this pathway could help regulate RhoA activation and cytoskeletal rearrangements preventing podocyte effacement in DKD.
Abstract
Background and Aims
COVID-19 has been a significant public health concern for the last three years; however, not much is known about the mechanisms that lead to severe kidney outcomes in ...patients hospitalized with COVID-19. In this multicenter study, we combine isobaric TMT-tagged urinary proteomics and machine learning to predict severe kidney outcomes in hospitalized COVID-19 patients.
Method
Urine samples from hospitalized COVID-19 patients in two medical centers (Mount Sinai Hospital and University of Michigan) were used in this study in adherence with proper consenting protocols. Urine samples were prepared for LC-MS/MS analysis as previously reported 1. The obtained spectra were analyzed using Proteome discoverer software and matched against Uniprot human database. For constructing the ML algorithm, the samples were randomly divided into discovery and validation set at a 2:1 ratio. Severe outcomes were defined as ICU admission, mechanical ventilation, acute kidney injury (AKI), death, or length of stay more than 21 days. Limma test was used on the discovery set to identify differentially expressed proteins and then features were selected using Boruta feature selection method. 10-fold cross validation on a random forest model was then applied to obtain receiver operating characteristic (ROC) curves.
Results
Urine samples from 120 PCR-positive COVID-19 patients from two different medical centers were collected within one week of hospitalization. More than 3,000 unique urinary proteins were identified using TMT-tagged mass spectrometry. For constructing a predictive algorithm, patients were stratified into severe and mild outcomes. Using Limma test on the discovery set, we identified differentially expressed proteins (DEPs) in severe outcome cohort vs the mild outcome cohort (Figure 1A). A set of 12 top features were identified using Boruta feature selection method and used for random forest model construction within the discovery set with 10-fold cross validation (Figure 1B). The generated ROC curves show that the algorithm demonstrated good predictive power for both discovery and validation set with 87% and 79% accuracy, respectively and close to 90% specificity (Figure 1C, D). On average, major adverse kidney events were observed in patients within 5-13 days after hospitalization. Enrichment analysis of DEP in COVID-19 patients compared to healthy patients showed significant upregulation of immune related processes and downregulation of proteolytic and metabolic processes. Enrichment analysis of DEPs in severe COVID-19 patients compared to mild COVID-19 patients showed significant upregulation of exocytosis and some immune related processes and downregulation of cell adhesion and extracellular matrix organization related processes (Figure 2A, B). Upregulated proteins were associated with kidney proximal tubular cells in addition to pulmonary alveolar cells (Figure 2C). Downregulated proteins were associated strongly with kidney cells such as podocytes and mesangial cells in addition to endothelial cells (Figure 2D).
Conclusion
Here, we developed an algorithm for prediction of severity in COVID-19 patients within 5-13 days after hospitalization. We further delineate potential mechanisms that drive severe outcomes in COVID-19 patients. Learnings from this study can be used for developing therapeutic options for long COVID, in addition to better preparedness in the event of other respiratory illnesses in the future.
The integrity of the barrier between blood and the selective filtrate of solutes is important for homeostasis and its disruption contributes to many diseases. Microphysiological systems that ...incorporate synthetic or natural membranes with human cells can mimic biological filtration barriers, such as the glomerular filtration barrier in the kidney, and they can readily be used to study cellular filtration processes as well as drug effects and interactions. We present an affordable, open-source platform for the real-time monitoring of functional filtration status in engineered microphysiological systems. Using readily available components, our assay can linearly detect real-time concentrations of two target molecules, FITC-labeled inulin and Texas Red-labeled human-serum albumin, within clinically relevant ranges, and it can be easily modified for different target molecules of varying sizes and tags. We demonstrate the platform’s ability to determine the concentration of our target molecules automatically and consistently. We show through an acellular context that the platform enables real-time tracking of size-dependent diffusion with minimal fluid volume loss and without manual extraction of media, making it suitable for continuous operational monitoring of filtration status in microphysiological system applications. The platform’s affordability and integrability with microphysiological systems make it ideal for many precision medicine applications, including evaluation of drug nephrotoxicity and other forms of drug discovery.
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