Project description:we followed the course of anti-Thy1 nephritis from days 1 to 5, establishing gene expression profiles at each timepoint using microarrays to explore the development of immunity. Overall design: We established anti-Thy1 model by injection of anti-Thy1 antibody. The models were killed from day 1 to 5. The normal control group was killed at day 0. Three glomeruli of each of six rats per timepoint were subjected to microarray analysis.
Project description:Glomerular damage mediated by glomerulus-infiltrating myeloid-derived cells is a key pathogenic event in lupus nephritis (LN), but the process is poorly understood. Confocal microscopy of kidney sections and flow cytometry analysis of glomerular cells from magnetic bead-purified glomeruli have identified glomerulus-infiltrating leukocyte populations in NZM2328 (NZM) lupus-prone mice with spontaneous chronic glomerulonephritis (GN) and anti-glomerular basement membrane-induced nephritis. The occurrence of a major glomerulus-infiltrating CD11b+F4/80-I-A- macrophage population exhibiting the markers programmed death ligand-1 (PD-L1), Mac-2, and macrophage mannose receptor (CD206) and producing Klf4, Il10, Retnla, Tnf, and Il6 mRNA, which are known to be expressed by alternatively activated (M2b) macrophages, correlated with proteinuria status. In NZM mice with spontaneous LN, glomerular macrophage infiltration is predominant. CD11b+F4/80-I-A- intraglomerular macrophages and polymorphonuclear neutrophils (PMN) are important in inducing GN, as anti-CD11b and -ICAM-1 mAb inhibited both proteinuria and macrophage and PMN infiltration. The predominant and high expression of PD-L1 by CD11b+F4/80-I-A- glomerular macrophages in kidneys of mice with GN and the inhibition of proteinuria by anti-PD-L1 mAb supported the pathogenic role of these macrophages but not the PD-L1- PMN in GN development and in inducing podocyte damage. In NZM mice with spontaneous chronic GN and severe proteinuria, few glomerulus-infiltrating PMN were found, leaving macrophages and, to a less extent, dendritic cells as the major infiltrating leukocytes. Taken together, these data support the important pathogenic effect of CD11b+F4/80-I-A- M2b-like glomerulus-infiltrating macrophages in LN and reinforce macrophages as a promising target for GN treatment.
Project description:Anti-Thy1 glomerulonephritis is a rat nephritis model closely simulating human mesangial proliferative glomerulonephritis. It affects primarily the mesangium, yet displays substantial proteinuria during the course. This study investigated the molecular signals underlying proteinuria in this disease and the modulation of which by the known antiproteinuric agent, pentoxifylline. Male Wistar rats were randomly divided into a control group and nephritic groups with or without treatment with IMD-0354 (an I?B kinase inhibitor), SB431542 (an activin receptor-like kinase inhibitor) or pentoxifylline. Kidney sections were prepared for histological examinations. Glomeruli were isolated for mRNA and protein analysis. Urine samples were collected for protein and nephrin quantitation. One day after nephritis induction, proteinuria developed together with ultrastructural changes of the podocyte and downregulation of podocyte mRNA and protein expression. These were associated with upregulation of tumor necrosis factor (TNF)-? and transforming growth factor (TGF)-?/activins mRNAs and activation of nuclear factor (NF)-?B p65 and Smad2/3. IMD-0354 attenuated proteinuria on d 1, whereas SB431542 decreased proteinuria on d 3 and 5, in association with partial restoration of downregulated podocyte mRNA and protein expression. Pentoxifylline attenuated proteinuria and nephrinuria through the course, plus inhibition of p-NF-?B p65 (d 1) and p-Smad2/3 (d 5) and partial reversal of downregulated podocyte mRNA and protein. Our data show that the pathogenesis of proteinuria in anti-Thy1 glomerulonephritis involves TNF-? and TGF-?/activin pathways, and the evolution of this process can be attenuated by pentoxifylline via downregulation of NF-?B and Smad signals and restoration of the podocyte component of the glomerular filtration barrier.
Project description:MicroRNAs contribute to the pathogenesis of certain diseases and may serve as biomarkers. We analyzed glomerular microRNA expression in B6.MRLc1, which serve as a mouse model of autoimmune glomerulonephritis. We found that miR-26a was the most abundantly expressed microRNA in the glomerulus of normal C57BL/6 and that its glomerular expression in B6.MRLc1 was significantly lower than that in C57BL/6. In mouse kidneys, podocytes mainly expressed miR-26a, and glomerular miR-26a expression in B6.MRLc1 mice correlated negatively with the urinary albumin levels and podocyte-specific gene expression. Puromycin-induced injury of immortalized mouse podocytes decreased miR-26a expression, perturbed the actin cytoskeleton, and increased the release of exosomes containing miR-26a. Although miR-26a expression increased with differentiation of immortalized mouse podocytes, silencing miR-26a decreased the expression of genes associated with the podocyte differentiation and formation of the cytoskeleton. In particular, the levels of vimentin and actin significantly decreased. In patients with lupus nephritis and IgA nephropathy, glomerular miR-26a levels were significantly lower than those of healthy controls. In B6.MRLc1 and patients with lupus nephritis, miR-26a levels in urinary exosomes were significantly higher compared with those for the respective healthy control. These data indicate that miR-26a regulates podocyte differentiation and cytoskeletal integrity, and its altered levels in glomerulus and urine may serve as a marker of injured podocytes in autoimmune glomerulonephritis.
Project description:BACKGROUND:Inflammatory processes play an important role in the pathogenesis of glomerulopathies. Finding novel ways to suppress glomerular inflammation may offer a new way to stop disease progression. However, the molecular mechanisms that initiate and drive inflammation in the glomerulus are still poorly understood. METHODS:We performed large-scale gene expression profiling of glomerulus-associated G protein-coupled receptors (GPCRs) to identify new potential therapeutic targets for glomerulopathies. The expression of Gprc5b in disease was analyzed using quantitative PCR and immunofluorescence, and by analyzing published microarray data sets. In vivo studies were carried out in a podocyte-specific Gprc5b knockout mouse line. Mechanistic studies were performed in cultured human podocytes. RESULTS:We identified an orphan GPCR, Gprc5b, as a novel gene highly enriched in podocytes that was significantly upregulated in common human glomerulopathies, including diabetic nephropathy, IgA nephropathy, and lupus nephritis. Similar upregulation of Gprc5b was detected in LPS-induced nephropathy in mice. Studies in podocyte-specific Gprc5b knockout mice showed that Gprc5b was not essential for normal development of the glomerular filtration barrier. However, knockout mice were partially protected from LPS-induced proteinuria and recruitment of inflammatory cells. Mechanistically, RNA sequencing in Gprc5b knockouts mice and experiments in cultured human podocytes showed that Gpr5cb regulated inflammatory response in podocytes via NF-?B signaling. CONCLUSIONS:GPRC5b is a novel podocyte-specific receptor that regulates inflammatory response in the glomerulus by modulating the NF-?B signaling pathway. Upregulation of Gprc5b in human glomerulopathies suggests that it may play a role in their pathogenesis.
Project description:<h4>Background</h4>The glomerulus is a specialized capillary bed that is involved in urine production and BP control. Glomerular injury is a major cause of CKD, which is epidemic and without therapeutic options. Single-cell transcriptomics has radically improved our ability to characterize complex organs, such as the kidney. Cells of the glomerulus, however, have been largely underrepresented in previous single-cell kidney studies due to their paucity and intractability.<h4>Methods</h4>Single-cell RNA sequencing comprehensively characterized the types of cells in the glomerulus from healthy mice and from four different disease models (nephrotoxic serum nephritis, diabetes, doxorubicin toxicity, and CD2AP deficiency).<h4>Results</h4>All cell types in the glomerulus were identified using unsupervised clustering analysis. Novel marker genes and gene signatures of mesangial cells, vascular smooth muscle cells of the afferent and efferent arterioles, parietal epithelial cells, and three types of endothelial cells were identified. Analysis of the disease models revealed cell type-specific and injury type-specific responses in the glomerulus, including acute activation of the Hippo pathway in podocytes after nephrotoxic immune injury. Conditional deletion of YAP or TAZ resulted in more severe and prolonged proteinuria in response to injury, as well as worse glomerulosclerosis.<h4>Conclusions</h4>Generation of comprehensive high-resolution, single-cell transcriptomic profiles of the glomerulus from healthy and injured mice provides resources to identify novel disease-related genes and pathways.
Project description:The obesity epidemic is developing into the most costly health problem facing the world. Obesity, characterized by excessive adipogenesis and enlarged adipocytes, promotes morbidities, such as diabetes, cardiovascular disease, and cancer. Regulation of adipogenesis is critical to our understanding of how fat cell formation causes obesity and associated health problems. Thy1 (also called CD90), a widely used stem cell marker, blocks adipogenesis and reduces lipid accumulation. Thy1-knockout mice are prone to diet-induced obesity. Although the importance of Thy1 in adipogenesis and obesity is now evident, how its expression is regulated is not. We hypothesized that DNA methylation has a role in promoting adipogenesis and affects Thy1 expression. Using the methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-dC), we investigated whether DNA methylation alters Thy1 expression during adipogenesis in both mouse 3T3-L1 preadipocytes and mouse mesenchymal stem cells. Thy1 protein and mRNA levels were decreased dramatically during adipogenesis. However, 5-aza-dC treatment prevented that phenomenon. Methylation-sensitive pyrosequencing analysis showed that CpG sites at the Thy1 locus have increased methylation during adipogenesis, as well as increased methylation in adipose tissue from diet-induced obese mice. These new findings highlight the potential role of Thy1 and DNA methylation in adipogenesis and obesity.-Flores, E. M., Woeller, C. F., Falsetta, M. L., Susiarjo, M., Phipps, R. P. Thy1 (CD90) expression is regulated by DNA methylation during adipogenesis.
Project description:The mechanism by which anti-DNA antibodies mediate lupus nephritis has yet to be conclusively determined. Previously, we found that treatment of mesangial cells with anti-DNA antibodies induced high expression of neutrophil gelatinase-associated lipocalin (NGAL), an iron-binding protein up-regulated in response to kidney injury. We undertook this study to determine whether NGAL is instrumental in the pathogenesis of nephritis, is induced as part of repair, or is irrelevant to damage/repair pathways.To investigate the role of NGAL in antibody-mediated nephritis, we induced nephrotoxic nephritis by passive antibody transfer to 129/SyJ and C57BL/6 mice. To determine if NGAL up-regulation is instrumental, we compared the severity of renal damage in NGAL wild-type mice and NGAL-knockout mice following induction of nephrotoxic nephritis.We found that kidney NGAL expression, as well as urine NGAL levels, were significantly increased in mice with nephrotoxic nephritis as compared to control-injected mice. Tight correlations were observed between NGAL expression, renal histopathology, and urine NGAL excretion. NGAL-knockout mice had attenuated proteinuria and improved renal histopathology compared to wild-type mice. Similarly, following nephritis induction, NGAL injection significantly exacerbated nephritis and decreased survival. NGAL induced apoptosis via caspase 3 activation and up-regulated inflammatory gene expression in kidney cells in vitro and when injected in vivo.We conclude that kidney binding of pathogenic antibodies stimulates local expression of NGAL, which plays a crucial role in the pathogenesis of nephritis via promotion of inflammation and apoptosis. NGAL blockade may be a novel therapeutic approach for the treatment of nephritis mediated by pathogenic antibodies, including anti-glomerular basement membrane disease and lupus nephritis.
Project description:Lupus nephritis (LN) is a severe manifestation of systemic lupus erythematosus (SLE), which often progresses to end-stage renal disease (ESRD) and ultimately leads to death. At present, there are no definitive therapies towards LN, so that illuminating the molecular mechanism behind the disease has become an urgent task for researchers. Bioinformatics has become a widely utilized method for exploring genes related to disease. This study set out to conduct weighted gene co-expression network analysis (WGCNA) and screen the hub gene of LN. We performed WGCNA on the microarray expression profile dataset of GSE104948 from the Gene Expression Omnibus (GEO) database with 18 normal and 21 LN samples of glomerulus. A total of 5,942 genes were divided into 5 co-expression modules, one of which was significantly correlated to LN. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted on the LN-related module, and the module was proved to be associated mainly with the activation of inflammation, immune response, cytokines, and immune cells. Genes in the most significant GO terms were extracted for sub-networks of WGNCA. We evaluated the centrality of genes in the sub-networks by Maximal Clique Centrality (MCC) method and CD36 was ultimately screened out as a hub candidate gene of the pathogenesis of LN. The result was verified by its differentially expressed level between normal and LN in GSE104948 and the other three multi-microarray datasets of GEO. Moreover, we further demonstrated that the expression level of CD36 is related to the WHO Lupus Nephritis Class of LN patients with the help of Nephroseq database. The current study proposed CD36 as a vital candidate gene in LN for the first time and CD36 may perform as a brand-new biomarker or therapeutic target of LN in the future.
Project description:<b>Background:</b> Both membranous nephropathy (MN) and lupus nephritis (LN) are autoimmune kidney disease. In recent years, with the deepening of research, some similarities have been found in the pathogenesis of these two diseases. However, the mechanism of their interrelationship is not clear. The purpose of this study was to investigate the differences in molecular mechanisms and key biomarkers between MN and LN. <b>Method:</b> The expression profiles of GSE99325, GSE99339, GSE104948 and GSE104954 were downloaded from GEO database, and the differentially expressed genes (DEGs) of MN and LN samples were obtained. We used Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) for enrichment analysis of DEGs. A protein-protein interaction (PPI) network of DEGs was constructed using Metascape. We filtered DEGs with NetworkAnalyst. Finally, we used receiver operating characteristic (ROC) analysis to identify the most significant DEGs for MN and LN. <b>Result:</b> Compared with LN in the glomerulus, 14 DEGs were up-regulated and 77 DEGs were down-regulated in MN. Compared with LN in renal tubules, 21 DEGs were down-regulated, but no up-regulated genes were found in MN. According to the result of GO and KEGG enrichment, PPI network and Networkanalyst, we screened out six genes (IFI6, MX1, XAF1, HERC6, IFI44L, IFI44). Interestingly, among PLA2R, THSD7A and NELL1, which are the target antigens of podocyte in MN, the expression level of NELL1 in MN glomerulus is significantly higher than that of LN, while there is no significant difference in the expression level of PLA2R and THSD7A. <b>Conclusion:</b> Our study provides new insights into the pathogenesis of MN and LN by analyzing the differences in gene expression levels between MN and LN kidney samples, and is expected to be used to prepare an animal model of MN that is more similar to human.