Murine models are valuable instruments in defining the pathogenesis of diabetic nephropathy (DN), but they only partially recapitulate disease manifestations of human DN, limiting their utility. To define the molecular similarities and differences between human and murine DN, we performed a cross-species comparison of glomerular transcriptional networks. Glomerular gene expression was profiled in patients with early type 2 DN and in three mouse models (streptozotocin DBA/2, C57BLKS db/db, and eNOS-deficient C57BLKS db/db mice). Species-specific transcriptional networks were generated and compared with a novel network-matching algorithm. Three shared human–mouse cross-species glomerular transcriptional networks containing 143 (Human-DBA STZ), 97 (Human-BKS db/db), and 162 (Human-BKS eNOS−/−
db/db) gene nodes were generated. Shared nodes across all networks reflected established pathogenic mechanisms of diabetes complications, such as elements of Janus kinase (JAK)/signal transducer and activator of transcription (STAT) and vascular endothelial growth factor receptor (VEGFR) signaling pathways. In addition, novel pathways not previously associated with DN and cross-species gene nodes and pathways unique to each of the human–mouse networks were discovered. The human–mouse shared glomerular transcriptional networks will assist DN researchers in selecting mouse models most relevant to the human disease process of interest. Moreover, they will allow identification of new pathways shared between mice and humans.
Nephrotic syndrome (NS) gene discovery efforts are now occurring in small kindreds and cohorts of sporadic cases. Power to identify causal variants in these groups beyond a statistical significance threshold is challenging due to small sample size and/or lack of family information. There is a need to develop novel methods to identify NS-associated variants. One way to determine putative functional relevance of a gene is to measure its strength of negative selection, as variants in genes under strong negative selection are more likely to be deleterious. We created a gene-level, integrated metric of negative selection (GIMS) score for 20,079 genes by combining multiple comparative genomics and population genetics measures. To understand the utility of GIMS for NS gene discovery, we examined this score in a diverse set of NS-relevant gene sets. These included genes known to cause monogenic forms of NS in humans as well as genes expressed in the cells of the glomerulus and, particularly, the podocyte. We found strong negative selection in the following NS-relevant gene sets: (1) autosomal-dominant Mendelian focal segmental glomerulosclerosis (FSGS) genes (p= 0.03 compared to reference), (2) glomerular expressed genes (p = 4×10-23), and (3) predicted podocyte genes (p = 3×10-9). Eight genes causing autosomal dominant forms of FSGS had a stronger combined score of negative selection and podocyte enrichment as compared to all other genes (p=1 x 10-3). As a whole, recessive FSGS genes were not enriched for negative selection. Thus, we also created a transcript-level, integrated metric of negative selection (TIMS) to quantify negative selection on an isoform level. These revealed transcripts of known autosomal recessive disease-causing genes that were nonetheless under strong selection. We suggest that a filtering strategy that includes measuring negative selection on a gene or isoform level could aid in identifying NS-related genes. Our GIMS and TIMS scores are available at http://glom.sph.umich.edu/GIMS/.
Integrating large-scale functional genomic data has significantly accelerated our understanding of gene functions. However, no algorithm has been developed to differentiate functions for isoforms of the same gene using high-throughput genomic data. This is because standard supervised learning requires ‘ground-truth’ functional annotations, which are lacking at the isoform level. To address this challenge, we developed a generic framework that interrogates public RNA-seq data at the transcript level to differentiate functions for alternatively spliced isoforms. For a specific function, our algorithm identifies the ‘responsible’ isoform(s) of a gene and generates classifying models at the isoform level instead of at the gene level. Through cross-validation, we demonstrated that our algorithm is effective in assigning functions to genes, especially the ones with multiple isoforms, and robust to gene expression levels and removal of homologous gene pairs. We identified genes in the mouse whose isoforms are predicted to have disparate functionalities and experimentally validated the ‘responsible’ isoforms using data from mammary tissue. With protein structure modeling and experimental evidence, we further validated the predicted isoform functional differences for the genes Cdkn2a and Anxa6. Our generic framework is the first to predict and differentiate functions for alternatively spliced isoforms, instead of genes, using genomic data. It is extendable to any base machine learner and other species with alternatively spliced isoforms, and shifts the current gene-centered function prediction to isoform-level predictions.
In mammalian genomes, a single gene can be alternatively spliced into multiple isoforms which greatly increase the functional diversity of the genome. In the human, more than 95% of multi-exon genes undergo alternative splicing. It is hard to computationally differentiate the functions for the splice isoforms of the same gene, because they are almost always annotated with the same functions and share similar sequences. In this paper, we developed a generic framework to identify the ‘responsible’ isoform(s) for each function that the gene carries out, and therefore predict functional assignment on the isoform level instead of on the gene level. Within this generic framework, we implemented and evaluated several related algorithms for isoform function prediction. We tested these algorithms through both computational evaluation and experimental validation of the predicted ‘responsible’ isoform(s) and the predicted disparate functions of the isoforms of Cdkn2a and of Anxa6. Our algorithm represents the first effort to predict and differentiate isoforms through large-scale genomic data integration.
To define shared and unique features of SLE nephritis in mouse models of proliferative and glomerulosclerotic renal disease.
Perfused kidneys from NZB/W F1, NZW/BXSB and NZM2410 mice were harvested before and after nephritis onset. Affymetrix based gene expression profiles of kidney RNA were analyzed using Genomatix Pathway Systems and Ingenuity Pathway Analysis software. Gene expression patterns were confirmed using real-time PCR.
955, 1168 and 755 genes were regulated in the kidneys of nephritic NZB/W F1, NZM2410 and NZW/BXSB mice respectively. 263 genes were regulated concordantly in all three strains reflecting immune cell infiltration, endothelial cell activation, complement activation, cytokine signaling, tissue remodeling and hypoxia. STAT3 was the top associated transcription factor, having a binding site in the gene promoter of 60/263 regulated genes. The two strains with proliferative nephritis shared a macrophage/DC infiltration and activation signature. NZB/W and NZM2410 mice shared a mitochondrial dysfunction signature. Dominant T cell and plasma cell signatures in NZB/W mice reflected lymphoid aggregates; this was the only strain with regulatory T cell infiltrates. NZW/BXSB mice manifested tubular regeneration and NZM2410 mice had the most metabolic stress and manifested loss of nephrin, indicating podocyte loss.
These findings identify shared inflammatory mechanisms of SLE nephritis that can be therapeutically targeted. Nevertheless, the heterogeneity of effector mechanisms suggests that individualized therapy might need to be based on biopsy findings. Some common mechanisms are shared with non-immune–mediated renal diseases, suggesting that strategies to prevent tissue hypoxia and remodeling may be useful in SLE nephritis.
The Nephrotic Syndrome Study Network (NEPTUNE) is a North American multi-center collaborative consortium established to develop a translational research infrastructure for Nephrotic Syndrome. This includes a longitudinal observational cohort study, a pilot and ancillary studies program, a training program, and a patient contact registry. NEPTUNE will enroll 450 adults and children with minimal change disease, focal segmental glomerulosclerosis and membranous nephropathy for detailed clinical, histopathologic, and molecular phenotyping at the time of clinically-indicated renal biopsy. Initial visits will include an extensive clinical history, physical examination, collection of urine, blood and renal tissue samples, and assessments of quality of life and patient-reported outcomes. Follow-up history, physical measures, urine and blood samples, and questionnaires will be obtained every 4 months in the first year and bi-annually, thereafter. Molecular profiles and gene expression data will be linked to phenotypic, genetic, and digitalized histologic data for comprehensive analyses using systems biology approaches. Analytical strategies were designed to transform descriptive information to mechanistic disease classification for Nephrotic Syndrome and to identify clinical, histological, and genomic disease predictors. Thus, understanding the complexity of the disease pathogenesis will guide further investigation for targeted therapeutic strategies.
Diabetic kidney disease (DKD) is a microvascular complication of type 1 and 2 diabetes with a devastating impact on individuals with the disease, their families and society as a whole. DKD is the single most frequent cause of incident chronic kidney disease (CKD) cases and accounts for over 40% of the population with end stage renal disease (ESRD). Contributing factors for the high prevalence are the increase in obesity and subsequent diabetes combined with an improved long–term survival with diabetes. Environment and genetic variations contribute to DKD susceptibility and progressive loss of kidney function. How the molecular mechanisms of genetic and environmental exposures interact during DKD initiation and progression are the focus of ongoing research efforts. The development of standardized, unbiased high throughput profiling technologies of human DKD samples opens new avenues in capturing the multiple layers of DKD pathobiology. These techniques routinely interrogate analytes on a genome–wide scale generating comprehensive DKD associated fingerprints. Linking the molecular fingerprints to deep clinical phenotypes may ultimately elucidate the intricate molecular interplay in a disease stage and subtype specific manner. This insight will form the basis for accurate prognosis and facilitate targeted therapeutic interventions. In this review, we present ongoing efforts from large scale data integration translating “–omics” research efforts into improved and individualized health care in DKD.
diabetic nephropathy; systems biology; high throughput molecular profiling; transcriptional regulatory networks; proteomics; metabolomics
Lupus nephritis (LN) is a serious manifestation of SLE. Therapeutic studies in mouse LN models do not always predict outcomes of human therapeutic trials, raising concerns about the human relevance of these pre-clinical models. In this study we used an unbiased transcriptional network approach to define in molecular terms similarities and differences between three lupus models and human LN. Genome wide gene expression networks were generated using natural language processing and automated promoter analysis and compared across species via suboptimal graph matching.
The three murine models and human LN share both common and unique features. The 20 commonly shared network nodes reflect the key pathologic processes of immune cell infiltration/activation, endothelial cell activation/injury and tissue remodeling/fibrosis, with macrophage/dendritic cell activation as a dominant cross-species shared transcriptional pathway. The unique nodes reflect differences in numbers and types of infiltrating cells and degree of remodeling between the three mouse strains. To define mononuclear phagocyte derived pathways in human LN, gene sets activated in isolated NZB/W renal mononuclear cells were compared with human LN kidney profiles. A tissue compartment specific macrophage activation pattern was seen, with NFκB1 and PPARγ as major regulatory nodes in the tubulointerstitial and glomerular networks respectively.
Our study defines which pathologic processes in murine models of LN recapitulate the key transcriptional processes active in human LN and suggests that there are functional differences between mononuclear phagocytes infiltrating different renal microenvironments.
Lupus nephritis (LN) is one of the most severe complications of systemic lupus erythematosus (SLE). Current treatments for LN lack sufficient efficacy as they do not necessarily target the LN responsible pathways and therapeutic responses vary widely in the patient population. LN mouse models have been useful in delineating disease pathogenesis and for testing novel therapies, but they do not entirely represent the events happening in human LN. This review describes how recently developed systems biology technologies can help to integrate current knowledge with large scale experimental data to generate new hypotheses and insight into the regulatory events occurring in LN.
Systems biology; SLE; Nephritis; Mouse models; Human
Chronic kidney disease (CKD) remains a major challenge in nephrology and for public health care, affecting 14–15% of the adult U.S. population and consuming significant health care resources. In the next 20 years, the number of patients with end stage renal disease is projected to increase by 50%. Ideal biomarkers that allow early identification of CKD patients at high risk of progression are urgently needed for early and targeted treatment to improve patient care. Recent success of integrating molecular approaches for personalized management of neoplastic diseases, including diagnosis, staging, prognosis, treatment selection and monitoring, has strongly encouraged kidney researchers to pursue molecular definitions of patients with kidney disease. Challenges for molecular marker identification in CKD are a high degree of cellular heterogeneity of the kidney and the paucity of human tissue availability for molecular studies. Despite these limitations potential molecular biomarker candidates have been uncovered at multiple levels along the genome – phenome continuum. Here we will review the identification and validation of potential genomic biomarker candidates of CKD and CKD progression in clinical studies. The challenges in predicting CKD progression, as well as the promises and opportunities resulting from a molecular definition of CKD will be discussed.
genomic biomarkers; CKD progression; GFR slope; RNA marker panel
In patients with Type 1 Diabetes (T1D) who develop microalbuminuria, progressive decline in glomerular filtration rate (GFR) may be initiated by leakage into the urine of toxic proteins (txUPs). This study tested this hypothesis.
After archiving baseline urine, we followed T1D patients with microalbuminuria for 8–12 years to distinguish those in whom GFR declined (Decliners) and those in whom it remained stable (Non-decliners). Human proximal tubular cells (HK-2 cells) were grown in serum-free medium enriched with pooled urines from Decliners or Non-decliners. We determined genome-wide expression profiles in extracted mRNA.
The two pooled urines induced differential expression of 312 genes. In terms of gene ontology, molecular functions of the 119 up-regulated genes were enriched for protein binding and peptidase inhibitor activities. Their biologic processes were enriched for defense response, responses to other organisms, regulation of cellular processes, or response to stress or stimulus, and programmed cell death. The 195 down-regulated genes were disproportionately represented in molecular functions of cation binding, hydrolase activity, and DNA binding. They were disproportionately represented in biological processes for regulation of metabolic processes, nucleic acid metabolic processes, cellular response to stress and macromolecule biosynthesis. The set of up-regulated genes in HK-2 cells overlaps significantly with sets of over-expressed genes in tubular and interstitial compartments of kidney biopsies from patients with advanced DN (33 genes in one study and 25 in the other compared with 10.3 expected by chance, p<10−9 and p<10−4, respectively). The overlap included genes encoding chemokines and cytokines. Overlap of down-regulated genes was no more than expected by chance.
Molecular processes in tubules and interstitium seen in advanced diabetic nephropathy can be induced in vitro by exposure to urine from patients with minimal microalbuminuria who subsequently developed progressive renal function decline, presumably due to putative txUPs.
The National Center for Integrative and Biomedical Informatics (NCIBI) is one of the eight NCBCs. NCIBI supports information access and data analysis for biomedical researchers, enabling them to build computational and knowledge models of biological systems to address the Driving Biological Problems (DBPs). The NCIBI DBPs have included prostate cancer progression, organ-specific complications of type 1 and 2 diabetes, bipolar disorder, and metabolic analysis of obesity syndrome. Collaborating with these and other partners, NCIBI has developed a series of software tools for exploratory analysis, concept visualization, and literature searches, as well as core database and web services resources. Many of our training and outreach initiatives have been in collaboration with the Research Centers at Minority Institutions (RCMI), integrating NCIBI and RCMI faculty and students, culminating each year in an annual workshop. Our future directions include focusing on the TranSMART data sharing and analysis initiative.
Integrative; bioinformatics; computational biology; biomedical informatics; biomedical informatics; genomics; national center
This study investigated the mechanisms underlying tubular apoptosis in diabetes by identifying proapoptotic genes that are differentially upregulated by reactive oxygen species in renal proximal tubular cells (RPTCs) in models of diabetes. Total RNAs isolated from renal proximal tubules (RPTs) of 20-week-old heterozygous db/m+, db/db, and db/db catalase (CAT)-transgenic (Tg) mice were used for DNA chip microarray analysis. Real-time quantitative PCR assays, immunohistochemistry, and mice rendered diabetic with streptozotocin were used to validate the proapoptotic gene expression in RPTs. Cultured rat RPTCs were used to confirm the apoptotic activity and regulation of proapoptotic gene expression. Additionally, studies in kidney tissues from patients with and without diabetes were used to confirm enhanced proapoptotic gene expression in RPTs. Bcl-2–modifying factor (Bmf) was differentially upregulated (P < 0.01) in RPTs of db/db mice compared with db/m+ and db/db CAT-Tg mice and in RPTs of streptozotocin-induced diabetic mice in which insulin reversed this finding. In vitro, Bmf cDNA overexpression in rat RPTCs coimmunoprecipated with Bcl-2, enhanced caspase-3 activity, and promoted apoptosis. High glucose (25 mmol/L) induced Bmf mRNA expression in RPTCs, whereas rotenone, catalase, diphenylene iodinium, and apocynin decreased it. Knockdown of Bmf with small interfering RNA reduced high glucose–induced apoptosis in RPTCs. More important, enhanced Bmf expression was detected in RPTs of kidneys from patients with diabetes. These data demonstrate differential upregulation of Bmf in diabetic RPTs and suggest a potential role for Bmf in regulating RPTC apoptosis and tubular atrophy in diabetes.
Systemic lupus erythematosus (SLE) is an autoimmune disease with heterogeneous manifestations including severe organ damage and vascular dysfunction leading to premature atherosclerosis. Interferon-alpha (IFN-α) has been proposed to have an important role in the development of lupus and lupus-related cardiovascular disease, partly by repression of IL-1 pathways leading to impairments in vascular repair induced by endothelial progenitor cells (EPCs) and circulating angiogenic cells (CACs). Counterintuitively, SLE patients also display transcriptional upregulation of the IL-1β/IL-18 processing machinery, the inflammasome. To understand this dichotomy and its impact on SLE-related cardiovascular disease, we examined cultures of human and murine control or lupus EPC/CACs to determine the role of the inflammasome in endothelial differentiation. We show that caspase-1 inhibition improves dysfunctional SLE EPC/CAC differentiation into mature endothelial cells and blocks IFN-α-mediated repression of this differentiation, implicating inflammasome activation as a crucial downstream pathway leading to aberrant vasculogenesis. Furthermore, serum IL-18 levels are elevatedin SLE and correlate with EPC/CAC dysfunction. Exogenous IL-18 inhibits endothelial differentiation in control EPC/CACs and neutralization of IL-18 in SLE EPC/CAC cultures restores their capacity to differentiate into mature endothelial cells, supporting a deleterious effect of IL-18 on vascular repair in vivo. Upregulation of the inflammasome machinery was operational in vivo, as evidenced by gene array analysis of lupus nephritis biopsies. Thus, the effects of IFN-α are complex and contribute to an elevated risk of cardiovascular disease by suppression of IL-1β pathways and by upregulation of the inflammasome machinery and potentiation of IL-18 activation.
Diabetic neuropathy is a common complication of diabetes. While multiple pathways are implicated in the pathophysiology of diabetic neuropathy, there are no specific treatments and no means to predict diabetic neuropathy onset or progression. Here, we identify gene expression signatures related to diabetic neuropathy and develop computational classification models of diabetic neuropathy progression. Microarray experiments were performed on 50 samples of human sural nerves collected during a 52-week clinical trial. A series of bioinformatics analyses identified differentially expressed genes and their networks and biological pathways potentially responsible for the progression of diabetic neuropathy. We identified 532 differentially expressed genes between patient samples with progressing or non-progressing diabetic neuropathy, and found these were functionally enriched in pathways involving inflammatory responses and lipid metabolism. A literature-derived co-citation network of the differentially expressed genes revealed gene subnetworks centred on apolipoprotein E, jun, leptin, serpin peptidase inhibitor E type 1 and peroxisome proliferator-activated receptor gamma. The differentially expressed genes were used to classify a test set of patients with regard to diabetic neuropathy progression. Ridge regression models containing 14 differentially expressed genes correctly classified the progression status of 92% of patients (P < 0.001). To our knowledge, this is the first study to identify transcriptional changes associated with diabetic neuropathy progression in human sural nerve biopsies and describe their potential utility in classifying diabetic neuropathy. Our results identifying the unique gene signature of patients with progressive diabetic neuropathy will facilitate the development of new mechanism-based diagnostics and therapies.
biomarkers; diabetic neuropathy; classification model; sural nerve; gene expression
The genetic architecture responsible for chronic kidney disease (CKD) remains incompletely described. The Oligosyndactyly (Os) mouse models focal and segmental glomerulosclerosis (FSGS), which is associated with reduced nephron number caused by the Os mutation. The Os mutation leads to FSGS in multiple strains including the ROP-Os/+. However, on the C57Bl/6J background the mutation does not cause FSGS, although nephron number in these mice are equivalent to those in ROP-Os/+ mice. We exploited this phenotypic variation to identify genes that potentially contribute to glomerulosclerosis.
To identify such novel genes, which regulate susceptibility or resistance to renal disease progression, we generated and compared the renal transcriptomes using serial analysis of gene expression (SAGE) from the sclerosis-prone ROP-Os/+ and sclerosis resistant C57-Os/+ mouse kidneys. We confirmed the validity of the differential gene expression using multiple approaches. We also used an Ingenuity Pathway Analysis engine to assemble differentially regulated molecular networks. Cell culture techniques were employed to confirm functional relevance of selected genes.
A comparative analysis of the kidney transcriptomes revealed multiple genes, with expression levels that were statistically different. These novel, candidate, renal disease susceptibility/resistance genes included neuropilin2 (Nrp2), glutathione-S-transferase theta (Gstt1) and itchy (Itch). Of 34 genes with the most robust statistical difference in expression levels between ROP-Os/+ and C57-Os/+ mice, 13 and 3 transcripts localized to glomerular and tubulointerstitial compartments, respectively, from micro-dissected human FSGS biopsies. Network analysis of all significantly differentially expressed genes identified 13 connectivity networks. The most highly scored network highlighted the roles for oxidative stress and mitochondrial dysfunction pathways. Functional analyses of these networks provided evidence for activation of transforming growth factor beta (TGFβ) signaling in ROP-Os/+ kidneys despite similar expression of the TGFβ ligand between the tested strains.
These data demonstrate the complex dysregulation of normal cellular functions in this animal model of FSGS and suggest that therapies directed at multiple levels will be needed to effectively treat human kidney diseases.
An abnormal neutrophil subset has been identified in the PBMC fractions from lupus patients. We have proposed that these “low density granulocytes” (LDGs) play an important role in lupus pathogenesis by damaging endothelial cells and synthesizing increased levels of proinflammatory cytokines and type I interferons. To directly establish LDGs as a distinct neutrophil subset, their gene array profiles were compared to those of autologous normal density neutrophils and control neutrophils. LDGs significantly overexpress mRNA of various immunostimulatory bactericidal proteins and alarmins, relative to lupus and control neutrophils. In contrast, gene profiles of lupus normal density neutrophils do not differ from those of controls. LDGs have heightened capacity to synthesize extracellular traps (NETs) which display increased externalization of bactericidal, immunostimulatory proteins and autoantigens, including LL-37, IL-17, and double-stranded DNA (dsDNA). Through NETosis, LDGs have increased capacity to kill endothelial cells and to stimulate IFN-α synthesis by pDCs. Affected skin and kidneys from lupus patients are infiltrated by netting neutrophils, which expose LL-37 and ds-DNA. Tissue NETosis is associated with increased anti-dsDNA in sera. These results expand the potential pathogenic roles of aberrant lupus neutrophils and suggest that dysregulation of NET formation and its subsequent responses may play a prominent deleterious role.
A better understanding of the molecular mechanisms underlying the development and progression of diabetic neuropathy (DN) is essential for the design of mechanism-based therapies. We examined changes in global gene expression to define pathways regulated by diabetes in peripheral nerve.
RESEARCH DESIGN AND METHODS
Microarray data for 24-week-old BKS db/db and db/+ mouse sciatic nerve were analyzed to define significantly differentially expressed genes (DEGs); DEGs were further analyzed to identify regulated biological processes and pathways. Expression profile clustering was performed to identify coexpressed DEGs. A set of coexpressed lipid metabolism genes was used for promoter sequence analysis.
Gene expression changes are consistent with structural changes of axonal degeneration. Pathways regulated in the db/db nerve include lipid metabolism, carbohydrate metabolism, energy metabolism, peroxisome proliferator–activated receptor signaling, apoptosis, and axon guidance. Promoter sequences of lipid metabolism–related genes exhibit evidence of coregulation of lipid metabolism and nervous system development genes.
Our data support existing hypotheses regarding hyperglycemia-mediated nerve damage in DN. Moreover, our analyses revealed a possible coregulation mechanism connecting hyperlipidemia and axonal degeneration.
Renal infiltration with mononuclear cells is associated with poor prognosis in SLE. A renal macrophage/dendritic cell signature is associated with onset of nephritis in NZB/W mice and immune modulating therapies can reverse this signature and the associated renal damage despite ongoing immune complex deposition. In nephritic NZB/W mice renal F4/80hi/CD11cint macrophages are located throughout the interstitium whereas F4/80lo/CD11chi dendritic cells accumulate in perivascular lymphoid aggregates. We show here that F4/80hi/CD11cint renal macrophages have a Gr1lo/Ly6Clo/VLA4lo/MHCIIhi/CD43lo/CD62Llo phenotype, different to that described for inflammatory macrophages. At nephritis onset F4/80hi/CD11cint cells upregulate cell surface CD11b, acquire cathepsin and MMP activity and accumulate large numbers of autophagocytic vacuoles; these changes reverse after induction of remission. Latex bead labeling of peripheral blood Gr1lo monocytes indicates that these are the source of F4/80hi/CD11cint macrophages. CD11chi/MHCIIlo dendritic cells are found in the kidneys only after proteinuria onset, turnover rapidly, and disappear rapidly after remission induction. Gene expression profiling of the F4/80hi/CD11cint population displays increased expression of pro-inflammatory, regulatory and tissue repair/degradation associated genes at nephritis onset that reverses with remission induction. Our findings suggest that mononuclear phagocytes with an aberrant activation profile contribute to tissue damage in lupus nephritis by mediating both local inflammation and excessive tissue remodeling.
Previous work shows that gene associations and network properties common between pairs of diseases can provide molecular evidence of comorbidity, but relationships among diseases may extend to larger groups. Formal concept analysis allows the study of multiple diseases based on a concept lattice whose structure indicates gene set commonality. We use the concept lattice for gene associations to evaluate the complexity of the relationships among diseases, and to identify concepts whose gene sets are candidates for further functional analysis. For this, we define a heuristic on the lattice structure that allows the identification of concepts whose gene sets indicate strong relationships among the included diseases, which are distinguished from other diseases in the family. Applying this approach to a family of renal diseases we demonstrate that this approach finds gene sets that may be promising for studying common (and differing) mechanism among a family of comorbid or phenotypically related diseases.
Chronic kidney disease (CKD) is a significant public health problem, and progression to end-stage renal disease leads to dramatic increases in morbidity and mortality. The mechanisms underlying progression of disease are poorly defined, and current noninvasive markers incompletely correlate with disease progression. Therefore, there is a great need for discovering novel markers for CKD. We utilized a glycoproteomic profiling approach to test the hypothesis that the urinary glycoproteome profile from subjects with CKD would be distinct from healthy controls. N-linked glycoproteins were isolated and enriched from the urine of healthy controls and subjects with CKD. This strategy identified several differentially expressed proteins in CKD, including a diverse array of proteins with endopeptidase inhibitor activity, protein binding functions, and acute-phase/immune-stress response activity supporting the proposal that inflammation may play a central role in CKD. Additionally, several of these proteins have been previously linked to kidney disease implicating a mechanistic role in disease pathogenesis. Collectively, our observations suggest that the human urinary glycoproteome may serve as a discovery source for novel mechanism-based biomarkers of CKD.
Systemic lupus erythematosus (SLE) is characterized by increased vascular risk due to premature atherosclerosis independent of traditional risk factors. We previously proposed that Interferon-α (IFN-α) plays a crucial role in premature vascular damage in SLE. IFN-α alters the balance between endothelial cell apoptosis and vascular repair mediated by endothelial progenitor cells (EPCs) and myeloid circulating angiogenic cells (CACs). Here we demonstrate that IFN-α promotes an antiangiogenic signature in SLE and control EPCs/CACs, characterized by transcriptional repression of IL-1α and β, IL-1 receptor 1 and vascular endothelial growth factor A (VEGF-A) and upregulation of IL-1 receptor antagonist (IL-1RN) and the decoy receptor IL1-R2. IL-1β promotes significant improvement in the functional capacity of lupus EPCs/CACs, therefore abrogating the deleterious effects of IFN-α. The beneficial effects from IL-1 are mediated, at least in part, by increases in EPC/CAC proliferation, decreases in EPC/CAC apoptosis, and by preventing the skewing of CACs towards non-angiogenic pathways. IFN-α induces STAT2 and 6 phosphorylation in EPCs/CACs and JAK inhibition abrogates the transcriptional antiangiogenic changes induced by IFN-α in these cells. Immunohistochemistry of renal biopsies from patients with lupus nephritis, but not ANCA-positive vasculitis, showed this pathway to be operational in vivo, with increased IL-1RN , downregulation of VEGF-A and glomerular and blood vessel decreased capillary density, compared to controls. Our study introduces a novel putative pathway by which type I IFNs may interfere with vascular repair in SLE through repression of IL-1-dependent pathways. This could promote atherosclerosis and loss of renal function in this disease.
The number of kidney podocytes is reduced in diabetic nephropathy. Advanced glycation end products (AGEs) accumulate in patients with diabetes and promote the apoptosis of podocyte by activating the forkhead box O4 (Foxo4) transcription factor to increase the expression of a pro-apoptosis gene, Bcl2l11. Using chromatin immunoprecipitation we demonstrate that AGE-modified bovine serum albumin (AGE-BSA) enhances Foxo4 binding to a forkhead binding element in the promoter of Bcl2lll. AGE-BSA also increases the acetylation of Foxo4. Lysine acetylation of Foxo4 is required for Foxo4 binding and transcription of Bcl2l11 in podocytes treated with AGE-BSA. The expression of a protein deacetylase that targets Foxo4 for deacetylation, sirtuin (Sirt1), is down regulated in cultured podocytes by AGE-BSA treatment and in glomeruli of diabetic patients. SIRT1 over expression in cultured murine podocytes prevents AGE-induced apoptosis. Glomeruli isolated from diabetic db/db mice have increased acetylation of Foxo4, suppressed expression of Sirt1, and increased expression of Bcl2l11 compared to non-diabetic littermates. Together, our data provide evidence that alteration of Foxo4 acetylation and down regulation of Sirt1 expression in diabetes promote podocyte apoptosis. Strategies to preserve Sirt1 expression or reduce Foxo4 acetylation could be used to prevent podocyte loss in diabetes.
Diabetic nephropathy (DN) is among the most lethal complications that occur in type 1 and type 2 diabetics. Podocyte dysfunction is postulated to be a critical event associated with proteinuria and glomerulosclerosis in glomerular diseases including DN. However, molecular mechanisms of podocyte dysfunction in the development of DN are not well understood. Here we have shown that activity of mTOR complex 1 (mTORC1), a kinase that senses nutrient availability, was enhanced in the podocytes of diabetic animals. Further, podocyte-specific mTORC1 activation induced by ablation of an upstream negative regulator (PcKOTsc1) recapitulated many DN features, including podocyte loss, glomerular basement membrane thickening, mesangial expansion, and proteinuria in nondiabetic young and adult mice. Abnormal mTORC1 activation caused mislocalization of slit diaphragm proteins and induced an epithelial-mesenchymal transition–like phenotypic switch with enhanced ER stress in podocytes. Conversely, reduction of ER stress with a chemical chaperone significantly protected against both the podocyte phenotypic switch and podocyte loss in PcKOTsc1 mice. Finally, genetic reduction of podocyte-specific mTORC1 in diabetic animals suppressed the development of DN. These results indicate that mTORC1 activation in podocytes is a critical event in inducing DN and suggest that reduction of podocyte mTORC1 activity is a potential therapeutic strategy to prevent DN.
Chronic glomerular diseases, associated with renal failure and cardiovascular morbidity, represent a major health issue. However, they remain poorly understood. Here we have reported that tightly controlled mTOR activity was crucial to maintaining glomerular podocyte function, while dysregulation of mTOR facilitated glomerular diseases. Genetic deletion of mTOR complex 1 (mTORC1) in mouse podocytes induced proteinuria and progressive glomerulosclerosis. Furthermore, simultaneous deletion of both mTORC1 and mTORC2 from mouse podocytes aggravated the glomerular lesions, revealing the importance of both mTOR complexes for podocyte homeostasis. In contrast, increased mTOR activity accompanied human diabetic nephropathy, characterized by early glomerular hypertrophy and hyperfiltration. Curtailing mTORC1 signaling in mice by genetically reducing mTORC1 copy number in podocytes prevented glomerulosclerosis and significantly ameliorated the progression of glomerular disease in diabetic nephropathy. These results demonstrate the requirement for tightly balanced mTOR activity in podocyte homeostasis and suggest that mTOR inhibition can protect podocytes and prevent progressive diabetic nephropathy.