RESUMO
Background Diabetic nephropathy (DN) is a leading cause of ESRD in the United States, but the molecular mechanisms mediating the early stages of DN are unclear.Methods To assess global changes that occur in early diabetic kidneys and to identify proteins potentially involved in pathogenic pathways in DN progression, we performed proteomic analysis of diabetic and nondiabetic rat glomeruli. Protein S (PS) among the highly upregulated proteins in the diabetic glomeruli. PS exerts multiple biologic effects through the Tyro3, Axl, and Mer (TAM) receptors. Because increased activation of Axl by the PS homolog Gas6 has been implicated in DN progression, we further examined the role of PS in DN.Results In human kidneys, glomerular PS expression was elevated in early DN but suppressed in advanced DN. However, plasma PS concentrations did not differ between patients with DN and healthy controls. A prominent increase of PS expression also colocalized with the expression of podocyte markers in early diabetic kidneys. In cultured podocytes, high-glucose treatment elevated PS expression, and PS knockdown further enhanced the high-glucose-induced apoptosis. Conversely, PS overexpression in cultured podocytes dampened the high-glucose- and TNF-α-induced expression of proinflammatory mediators. Tyro3 receptor was upregulated in response to high glucose and mediated the anti-inflammatory response of PS. Podocyte-specific PS loss resulted in accelerated DN in streptozotocin-induced diabetic mice, whereas the transient induction of PS expression in glomerular cells in vivo attenuated albuminuria and podocyte loss in diabetic OVE26 mice.Conclusions Our results support a protective role of PS against glomerular injury in DN progression.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Podócitos/metabolismo , Podócitos/patologia , Proteína S/metabolismo , Albuminúria/genética , Animais , Apoptose/efeitos dos fármacos , Proteínas de Ligação ao Cálcio , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células Cultivadas , Diabetes Mellitus Experimental/complicações , Nefropatias Diabéticas/sangue , Inativação Gênica , Glucose/farmacologia , Humanos , Camundongos , NF-kappa B/metabolismo , Proteína S/genética , Proteômica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Mensageiro/metabolismo , Ratos , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , c-Mer Tirosina Quinase/genética , c-Mer Tirosina Quinase/metabolismo , Receptor Tirosina Quinase AxlRESUMO
A recent clinical study showed that combination therapy consisting of mycophenolate mofetil, tacrolimus and steroids was shown to be more effective in achieving complete remission in patients with severe forms of lupus nephritis than conventional therapy consisting of intravenous cyclophosphamide and steroids. To explore the underlying molecular and cellular mechanisms of increased efficacy of the combination therapy regimen, we employed a mouse model of lupus nephritis, MRL/lpr mice, and treated them with monotherapies of prednisone, mycophenolate mofetil, or tacrolimus, or with their combination. Consistent with previous clinical findings, combination therapy markedly improved renal outcome compared to the monotherapies in mice with lupus nephritis. Transcriptomic analysis of their kidneys revealed distinct molecular pathways that were differentially regulated in combination therapy versus monotherapies. Combination therapy not only provided additive immunosuppressive effects, but also induced gene expression and molecular pathways to confer enhanced renoprotection. Specifically, combination therapy inhibited TLR7 expression in the kidneys of mice with lupus nephritis; combination of tacrolimus and mycophenolate mofetil led to better stabilization of the podocyte actin cytoskeleton through the reciprocal regulation of RhoA and Rac1 activities. Combination therapy strongly suppressed the IL-6/Stat3 pathway. These findings were further validated in renal biopsy samples from patients with lupus nephritis before and after treatments with mycophenolate mofetil, tacrolimus or combination therapy. Thus, our study further supports the earlier clinical finding and further provides insights into the molecular basis for increased efficacy of combination therapy.
Assuntos
Perfilação da Expressão Gênica/métodos , Glucocorticoides/farmacologia , Imunossupressores/farmacologia , Rim/efeitos dos fármacos , Nefrite Lúpica/tratamento farmacológico , Ácido Micofenólico/farmacologia , Prednisona/farmacologia , Tacrolimo/farmacologia , Transcriptoma/efeitos dos fármacos , Animais , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/genética , Citoesqueleto/metabolismo , Modelos Animais de Doenças , Sinergismo Farmacológico , Quimioterapia Combinada , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Rim/metabolismo , Rim/fisiopatologia , Nefrite Lúpica/genética , Nefrite Lúpica/metabolismo , Nefrite Lúpica/fisiopatologia , Camundongos Endogâmicos MRL lpr , Podócitos/efeitos dos fármacos , Podócitos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
Proliferation of glomerular epithelial cells, including podocytes, is a key histologic feature of crescentic glomerulonephritis. We previously found that retinoic acid (RA) inhibits proliferation and induces differentiation of podocytes by activating RA receptor-α (RARα) in a murine model of HIV-associated nephropathy. Here, we examined whether RA would similarly protect podocytes against nephrotoxic serum-induced crescentic glomerulonephritis and whether this effect was mediated by podocyte RARα. RA treatment markedly improved renal function and reduced the number of crescentic lesions in nephritic wild-type mice, while this protection was largely lost in mice with podocyte-specific ablation of Rara (Pod-Rara knockout). At a cellular level, RA significantly restored the expression of podocyte differentiation markers in nephritic wild-type mice, but not in nephritic Pod-Rara knockout mice. Furthermore, RA suppressed the expression of cell injury, proliferation, and parietal epithelial cell markers in nephritic wild-type mice, all of which were significantly dampened in nephritic Pod-Rara knockout mice. Interestingly, RA treatment led to the coexpression of podocyte and parietal epithelial cell markers in a small subset of glomerular cells in nephritic mice, suggesting that RA may induce transdifferentiation of parietal epithelial cells toward a podocyte phenotype. In vitro, RA directly inhibited the proliferation of parietal epithelial cells and enhanced the expression of podocyte markers. In vivo lineage tracing of labeled parietal epithelial cells confirmed that RA increased the number of parietal epithelial cells expressing podocyte markers in nephritic glomeruli. Thus, RA attenuates crescentic glomerulonephritis primarily through RARα-mediated protection of podocytes and in part through the inhibition of parietal epithelial cell proliferation and induction of their transdifferentiation into podocytes.
Assuntos
Proliferação de Células/efeitos dos fármacos , Glomerulonefrite/tratamento farmacológico , Podócitos/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Receptor alfa de Ácido Retinoico/metabolismo , Tretinoína/farmacologia , Animais , Autoanticorpos/administração & dosagem , Autoanticorpos/imunologia , Biomarcadores/metabolismo , Biópsia , Cápsula Glomerular/citologia , Cápsula Glomerular/efeitos dos fármacos , Cápsula Glomerular/fisiologia , Transdiferenciação Celular/efeitos dos fármacos , Células Cultivadas , Glomerulonefrite/imunologia , Glomerulonefrite/patologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Podócitos/patologia , Podócitos/fisiologia , Substâncias Protetoras/uso terapêutico , Receptor alfa de Ácido Retinoico/genética , Tretinoína/uso terapêuticoRESUMO
Both the incidence and prevalence of chronic kidney disease are increasing in the elderly population. Although aging is known to induce kidney injury, the underlying molecular mechanisms remain unclear. Sirtuin 1 (Sirt1), a longevity gene, is known to protect kidney cell injury from various cellular stresses. In previous studies, we showed that the podocyte-specific loss of Sirt1 aggravates diabetic kidney injury. However, the role of Sirt1 in aging-induced podocyte injury is not known. Therefore, in this study we sought to determine the effects of podocyte-specific reduction of Sirt1 in age-induced kidney injury. We employed the inducible podocyte-specific Sirt1 knockdown mice that express shRNA against Sirt1 (Pod-Sirt1RNAi) and control mice that express shRNA for luciferase (Pod-LuciRNAi). We found that reduction of podocyte Sirt1 led to aggravated aging-induced glomerulosclerosis and albuminuria. In addition, urinary level of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a marker of oxidative stress, was markedly increased in aged Pod-Sirt1RNAi mice compared with aged Pod-LuciRNAi mice. Although podocyte-specific markers decreased in aged mice compared with the young controls, the decrease was further exacerbated in aged Pod-Sirt1RNAi compared with Pod-LuciRNAi mice. Interestingly, expression of cellular senescence markers was significantly higher in the glomeruli of Pod-Sirt1RNAi mice than Pod-LuciRNAi mice, suggesting that cellular senescence may contribute to podocyte loss in aging kidneys. Finally, we confirmed that Pod-Sirt1RNAi glomeruli were associated with reduced activation of the transcription factors peroxisome proliferator-activated receptor (PPAR)-α coactivador-1 (PGC1α)/PPARγ, forkhead box O (FOXO)3, FOXO4, and p65 NF-κB, through SIRT1-mediated deacetylation. Together, our data suggest that SIRT1 may be a potential therapeutic target to treat patients with aging-related kidney disease.
Assuntos
Envelhecimento/metabolismo , Albuminúria/enzimologia , Podócitos/enzimologia , Insuficiência Renal Crônica/enzimologia , Sirtuína 1/deficiência , 8-Hidroxi-2'-Desoxiguanosina , Acetilação , Fatores Etários , Envelhecimento/genética , Envelhecimento/patologia , Albuminúria/genética , Albuminúria/patologia , Animais , Proteínas de Ciclo Celular , Senescência Celular , Desoxiguanosina/análogos & derivados , Desoxiguanosina/urina , Proteína Forkhead Box O3/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Técnicas de Silenciamento de Genes , Genótipo , Glomerulonefrite/enzimologia , Glomerulonefrite/genética , Glomerulonefrite/patologia , Camundongos , Estresse Oxidativo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Fenótipo , Podócitos/patologia , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/patologia , Transdução de Sinais , Sirtuína 1/genética , Fator de Transcrição RelA/metabolismoRESUMO
Maintenance of mitochondrial structure and function is critical for preventing podocyte apoptosis and eventual glomerulosclerosis in the kidney; however, the transcription factors that regulate mitochondrial function in podocyte injury remain to be identified. Here, we identified Krüppel-like factor 6 (KLF6), a zinc finger domain transcription factor, as an essential regulator of mitochondrial function in podocyte apoptosis. We observed that podocyte-specific deletion of Klf6 increased the susceptibility of a resistant mouse strain to adriamycin-induced (ADR-induced) focal segmental glomerulosclerosis (FSGS). KLF6 expression was induced early in response to ADR in mice and cultured human podocytes, and prevented mitochondrial dysfunction and activation of intrinsic apoptotic pathways in these podocytes. Promoter analysis and chromatin immunoprecipitation studies revealed that putative KLF6 transcriptional binding sites are present in the promoter of the mitochondrial cytochrome c oxidase assembly gene (SCO2), which is critical for preventing cytochrome c release and activation of the intrinsic apoptotic pathway. Additionally, KLF6 expression was reduced in podocytes from HIV-1 transgenic mice as well as in renal biopsies from patients with HIV-associated nephropathy (HIVAN) and FSGS. Together, these findings indicate that KLF6-dependent regulation of the cytochrome c oxidase assembly gene is critical for maintaining mitochondrial function and preventing podocyte apoptosis.
Assuntos
Glomerulosclerose Segmentar e Focal/metabolismo , Infecções por HIV/complicações , Rim/metabolismo , Fatores de Transcrição Kruppel-Like/fisiologia , Mitocôndrias/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Animais , Apoptose , Sítios de Ligação , Células Cultivadas , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Glomerulosclerose Segmentar e Focal/patologia , Glomerulosclerose Segmentar e Focal/virologia , Infecções por HIV/metabolismo , HIV-1/fisiologia , Humanos , Rim/patologia , Fator 6 Semelhante a Kruppel , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Chaperonas Moleculares , Podócitos/fisiologia , Regiões Promotoras GenéticasRESUMO
Profiling of temporal changes of gene expression in the same kidney over the course of renal disease progression is challenging because repeat renal biopsies are rarely indicated in clinical practice. Here, we profiled the temporal change in renal transcriptome of HIV-1 transgenic mice (Tg26), an animal model for human HIV-associated nephropathy (HIVAN), and their littermates at three different time points (4, 8, and 12 weeks of age) representing early, middle, and late stages of renal disease by serial kidney biopsy. We analyzed both static levels of gene expression at three stages of disease and dynamic changes in gene expression between different stages. Analysis of static and dynamic changes in gene expression revealed that up-regulated genes at the early and middle stages are mostly involved in immune response and inflammation, whereas down-regulated genes mostly related to fatty acid and retinoid metabolisms. We validated the expression of a selected panel of genes that are up-regulated at the early stage (CCL2, CCL5, CXCL11, Ubd, Anxa1, and Spon1) by real-time PCR. Among these up-regulated genes, Spon1, which is a previously identified candidate gene for hypertension, was found to be up-regulated in kidney of human with diabetic nephropathy. Immunostaining of human biopsy samples demonstrated that protein expression of Spon1 was also markedly increased in kidneys of patients with both early and late HIVAN and diabetic nephropathy. Our studies suggest that analysis of both static and dynamic changes of gene expression profiles in disease progression avails another layer of information that could be utilized to gain a more comprehensive understanding of disease progression and identify potential biomarkers and drug targets.
Assuntos
Progressão da Doença , Perfilação da Expressão Gênica , HIV-1/genética , Córtex Renal/metabolismo , Animais , HIV-1/fisiologia , Córtex Renal/virologia , Nefropatias/genética , Nefropatias/virologia , Camundongos , Camundongos Transgênicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos TestesRESUMO
Our previous in vitro studies suggested that cyclic AMP (cAMP) signaling prevents adriamycin (ADR) and puromycin aminonucleoside (PAN)-induced apoptosis in podocytes. As cAMP is an important second messenger and plays a key role in cell proliferation, differentiation and cytoskeleton formation via protein kinase A (PKA) or exchange protein directly activated by cAMP (Epac) pathways, we sought to determine the role of PKA or Epac signaling in cAMP-mediated protection of podocytes. In the ADR nephrosis model, we found that forskolin, a selective activator of adenylate cyclase, attenuated albuminuria and improved the expression of podocyte marker WT-1. When podocytes were treated with pCPT-cAMP (a selective cAMP/PKA activator), PKA activation was increased in a time-dependent manner and prevented PAN-induced podocyte loss and caspase 3 activation, as well as a reduction in mitochondrial membrane potential. We found that PAN and ADR resulted in a decrease in Mfn1 expression and mitochondrial fission in podocytes. pCPT-cAMP restored Mfn1 expression in puromycin or ADR-treated podocytes and induced Drp1 phosphorylation, as well as mitochondrial fusion. Treating podocytes with arachidonic acid resulted in mitochondrial fission, podocyte loss and cleaved caspase 3 production. Arachidonic acid abolished the protective effects of pCPT-cAMP on PAN-treated podocytes. Mdivi, a mitochondrial division inhibitor, prevented PAN-induced cleaved caspase 3 production in podocytes. We conclude that activation of cAMP alleviated murine podocyte caused by ADR. PKA signaling resulted in mitochondrial fusion in podocytes, which at least partially mediated the effects of cAMP.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/genética , AMP Cíclico/metabolismo , Mitocôndrias/metabolismo , Nefrose/metabolismo , Podócitos/metabolismo , Transdução de Sinais , Animais , Apoptose , Ácido Araquidônico/farmacologia , Caspase 3/genética , Caspase 3/metabolismo , Linhagem Celular Transformada , Colforsina/farmacologia , AMP Cíclico/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Doxorrubicina/farmacologia , Dinaminas/genética , Dinaminas/metabolismo , Ativação Enzimática , Ativadores de Enzimas/farmacologia , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Regulação da Expressão Gênica , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Dinâmica Mitocondrial/efeitos dos fármacos , Nefrose/induzido quimicamente , Nefrose/genética , Nefrose/patologia , Fosforilação , Podócitos/efeitos dos fármacos , Podócitos/patologia , Puromicina Aminonucleosídeo/farmacologia , Quinazolinonas/farmacologia , Proteínas WT1/genética , Proteínas WT1/metabolismoRESUMO
Nuclear factor (NF)-κB and signal transducer and activator of transcription 3 (STAT3) play a critical role in diabetic nephropathy (DN). Sirtuin-1 (SIRT1) regulates transcriptional activation of target genes through protein deacetylation. Here, we determined the roles of Sirt1 and the effect of NF-κB (p65) and STAT3 acetylation in DN. We found that acetylation of p65 and STAT3 was increased in both mouse and human diabetic kidneys. In human podocytes, advanced glycation end products (AGEs) induced p65 and STAT3 acetylation and overexpression of acetylation-incompetent mutants of p65 and STAT3 abrogated AGE-induced expression of NF-κB and STAT3 target genes. Inhibition of AGE formation in db/db mice by pyridoxamine treatment attenuated proteinuria and podocyte injury, restored SIRT1 expression, and reduced p65 and STAT3 acetylation. Diabetic db/db mice with conditional deletion of SIRT1 in podocytes developed more proteinuria, kidney injury, and acetylation of p65 and STAT3 compared with db/db mice without SIRT1 deletion. Treatment of db/db mice with a bromodomain and extraterminal (BET)-specific bromodomain inhibitor (MS417) which blocks acetylation-mediated association of p65 and STAT3 with BET proteins, attenuated proteinuria, and kidney injury. Our findings strongly support a critical role for p65 and STAT3 acetylation in DN. Targeting protein acetylation could be a potential new therapy for DN.
Assuntos
Nefropatias Diabéticas/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Fator de Transcrição STAT3/metabolismo , Sirtuína 1/metabolismo , Fator de Transcrição RelA/metabolismo , Acetilação , Animais , Células Cultivadas , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/metabolismo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Podócitos/metabolismo , Sirtuína 1/genéticaRESUMO
Podocytes are kidney cells with specialized morphology that is required for glomerular filtration. Diseases, such as diabetes, or drug exposure that causes disruption of the podocyte foot process morphology results in kidney pathophysiology. Proteomic analysis of glomeruli isolated from rats with puromycin-induced kidney disease and control rats indicated that protein kinase A (PKA), which is activated by adenosine 3',5'-monophosphate (cAMP), is a key regulator of podocyte morphology and function. In podocytes, cAMP signaling activates cAMP response element-binding protein (CREB) to enhance expression of the gene encoding a differentiation marker, synaptopodin, a protein that associates with actin and promotes its bundling. We constructed and experimentally verified a ß-adrenergic receptor-driven network with multiple feedback and feedforward motifs that controls CREB activity. To determine how the motifs interacted to regulate gene expression, we mapped multicompartment dynamical models, including information about protein subcellular localization, onto the network topology using Petri net formalisms. These computational analyses indicated that the juxtaposition of multiple feedback and feedforward motifs enabled the prolonged CREB activation necessary for synaptopodin expression and actin bundling. Drug-induced modulation of these motifs in diseased rats led to recovery of normal morphology and physiological function in vivo. Thus, analysis of regulatory motifs using network dynamics can provide insights into pathophysiology that enable predictions for drug intervention strategies to treat kidney disease.
Assuntos
Nefropatias/metabolismo , Rim/metabolismo , Podócitos/metabolismo , Transdução de Sinais , Animais , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Expressão Gênica , Redes Reguladoras de Genes , Immunoblotting , Rim/patologia , Rim/fisiopatologia , Nefropatias/induzido quimicamente , Nefropatias/genética , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Microscopia Eletrônica , Podócitos/patologia , Podócitos/ultraestrutura , Proteômica/métodos , Puromicina , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Our understanding of the pathogenesis of most primary glomerular diseases, including IgA nephropathy, membranous nephropathy and focal segmental glomerulosclerosis, is limited. Advances in molecular technology now permit genome-wide, high-throughput characterization of genes and gene products from biological samples. Comprehensive examinations of the genome, transcriptome, proteome and metabolome (collectively known as omics analyses), have been applied to the study of IgA nephropathy, membranous nephropathy and focal segmental glomerulosclerosis in both animal models and human patients. However, most omics studies of primary glomerular diseases, with the exception of large genomic studies, have been limited by inadequate sample sizes and the lack of kidney-specific data sets derived from kidney biopsy samples. Collaborative efforts to develop a standardized approach for prospective recruitment of patients, scheduled monitoring of clinical outcomes, and protocols for sampling of kidney tissues will be instrumental in uncovering the mechanisms that drive these diseases. Integration of molecular data sets with the results of clinical and histopathological studies will ultimately enable these diseases to be characterized in a comprehensive and systematic manner, and is expected to improve the diagnosis and treatment of these diseases.
Assuntos
Estudo de Associação Genômica Ampla/métodos , Glomerulonefrite , Rim/patologia , Modelos Biológicos , Biologia de Sistemas/métodos , Animais , Perfilação da Expressão Gênica , Glomerulonefrite/genética , Glomerulonefrite/metabolismo , Glomerulonefrite/patologia , Humanos , Rim/metabolismoRESUMO
Activation of signal transducer and activator of transcription (STAT)3 correlates with proliferation of extracapillary glomerular epithelial cells and the extent of renal injury in glomerulonephritis. To delineate the role of STAT3 in glomerular epithelial cell proliferation, we examined the development of nephrotoxic serum-induced glomerulonephritis in mice with and without podocyte-restricted STAT3 deletion. Mice with STAT3 deletion in podocytes developed less crescents and loss of renal function compared with those without STAT3 deletion. Proliferation of glomerular cells, loss of podocyte markers, and recruitment of parietal epithelial cells were found in nephritic mice without STAT3 deletion, but mitigated in nephritic mice with podocyte STAT3 deletion. Glomerular expression of pro-inflammatory STAT3 target genes was significantly reduced in nephritic mice with, compared with those without, podocyte STAT3 deletion. However, the extent of glomerular immune complex deposition was not different. Podocytes with STAT3 deletion were resistant to interleukin-6-induced STAT3 phosphorylation and pro-inflammatory STAT3 target gene expression. Thus, podocyte STAT3 activation is critical for the development of crescentic glomerulonephritis.
Assuntos
Glomerulonefrite/prevenção & controle , Podócitos/metabolismo , Fator de Transcrição STAT3/deficiência , Soro , Albuminúria/metabolismo , Animais , Complexo Antígeno-Anticorpo/metabolismo , Apoptose , Linhagem Celular , Proliferação de Células , Complemento C3/metabolismo , Modelos Animais de Doenças , Fator de Crescimento Epidérmico/metabolismo , Glomerulonefrite/induzido quimicamente , Glomerulonefrite/genética , Glomerulonefrite/metabolismo , Glomerulonefrite/patologia , Glomerulonefrite/fisiopatologia , Imunoglobulinas/metabolismo , Mediadores da Inflamação/metabolismo , Interleucina-6/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Podócitos/patologia , Cultura Primária de Células , Fator de Transcrição STAT3/genética , Transdução de Sinais , Fatores de TempoRESUMO
IgA nephropathy (IgAN) encompasses different pathological entities, all of which are characterized by the mesangial deposition of IgA. Aberrantly glycosylated IgA molecules appear to play a major role in the pathogenesis of IgAN. Both genetic and environmental factors contribute to the formation of IgG antibodies that binds to aberrantly glycosylated IgA molecules and the glomerular deposition of circulating polymeric IgA complexes. Mesangial cells serve as primary glomerular cell type injured in IgAN with a variety of pathological changes leading to progressive renal dysfunction. Recent evidence suggests that indirect podocyte injury also contributes to glomerular damage observed in IgAN. These mechanisms were demonstrated in cultured podocytes exposed to mesangial cell-conditioned medium of human IgAN. Large-scale biopsy series of human kidneys also suggest that podocyte injury plays a role in IgAN. In this review, we discuss the recent advances that have helped elucidate the mechanisms of podocyte injury in IgAN and how these mechanisms correlate with clinically important outcomes in human studies.
Assuntos
Glomerulonefrite por IGA/etiologia , Podócitos/patologia , Comunicação Celular , Ativação do Complemento , Humanos , Imunoglobulina A/metabolismo , Células Mesangiais/fisiologia , Podócitos/fisiologiaRESUMO
The Connectivity Map database contains microarray signatures of gene expression derived from approximately 6000 experiments that examined the effects of approximately 1300 single drugs on several human cancer cell lines. We used these data to prioritize pairs of drugs expected to reverse the changes in gene expression observed in the kidneys of a mouse model of HIV-associated nephropathy (Tg26 mice). We predicted that the combination of an angiotensin-converting enzyme (ACE) inhibitor and a histone deacetylase inhibitor would maximally reverse the disease-associated expression of genes in the kidneys of these mice. Testing the combination of these inhibitors in Tg26 mice revealed an additive renoprotective effect, as suggested by reduction of proteinuria, improvement of renal function, and attenuation of kidney injury. Furthermore, we observed the predicted treatment-associated changes in the expression of selected genes and pathway components. In summary, these data suggest that the combination of an ACE inhibitor and a histone deacetylase inhibitor could have therapeutic potential for various kidney diseases. In addition, this study provides proof-of-concept that drug-induced expression signatures have potential use in predicting the effects of combination drug therapy.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Rim/efeitos dos fármacos , Animais , Benzazepinas/farmacologia , Linhagem Celular Tumoral , Sinergismo Farmacológico , Quimioterapia Combinada , Perfilação da Expressão Gênica , Humanos , Ácidos Hidroxâmicos/farmacologia , Nefropatias/tratamento farmacológico , Masculino , Camundongos , VorinostatRESUMO
OBJECTIVE: HIV-1 gene expression in kidney epithelial cells is thought to be responsible for the pathogenesis of HIV-1-associated nephropathy (HIVAN). Signal transducer and activator of transcription (STAT) 3 signaling is activated in podocytes of patients with HIVAN and drives the dedifferentiation and proliferation of podocytes in culture. We confirm here that deletion of podocyte STAT3 is sufficient to mitigate the glomerular as well as tubulointerstitial findings of HIVAN. METHODS: To demonstrate the functional role of podocyte STAT3 in the pathogenesis of HIVAN we compared the development of HIVAN in Tg26 HIV-transgenic mice with and without deletion of STAT3 in the podocyte. RESULTS: Tg26 mice with podocyte-specific STAT3 deletion developed significantly less weight loss, albuminuria, and renal function impairment compared to Tg26 mice without STAT3 deletion. Tg26 mice with podocyte STAT3 deletion also had significantly less glomerular collapse, sclerosis, epithelial cell hyperplasia, podocyte dedifferentiation, and proinflammatory STAT3 target gene expression; and tubulointerstitial changes of HIVAN, including tubular atrophy, degeneration, apoptosis, and lymphocyte infiltration, were also significantly reduced compared to Tg26 mice without STAT3 deletion. CONCLUSION: Development of glomerular as well as tubulointerstitial injuries in the Tg26 HIVAN model is dependent on podocyte STAT3 expression. Inhibition of STAT3 could be a potential adjunctive therapy for the treatment of HIVAN.
Assuntos
Nefropatia Associada a AIDS/patologia , Infecções por HIV/patologia , Glomérulos Renais/patologia , Podócitos/metabolismo , Fator de Transcrição STAT3/metabolismo , Nefropatia Associada a AIDS/etiologia , Nefropatia Associada a AIDS/genética , Albuminúria , Animais , Western Blotting , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Células Epiteliais/patologia , Deleção de Genes , Perfilação da Expressão Gênica , Infecções por HIV/complicações , Infecções por HIV/genética , Humanos , Glomérulos Renais/metabolismo , Camundongos , Camundongos Transgênicos , MicroRNAs , Podócitos/patologia , Reação em Cadeia da Polimerase em Tempo Real , Fator de Transcrição STAT3/genética , Esclerose , Transdução de Sinais/genética , Redução de PesoRESUMO
Kidney fibrosis is a common process that leads to the progression of various types of kidney disease. We used an integrated computational and experimental systems biology approach to identify protein kinases that regulate gene expression changes in the kidneys of human immunodeficiency virus (HIV) transgenic mice (Tg26 mice), which have both tubulointerstitial fibrosis and glomerulosclerosis. We identified homeo-domain interacting protein kinase 2 (HIPK2) as a key regulator of kidney fibrosis. HIPK2 was upregulated in the kidneys of Tg26 mice and in those of patients with various kidney diseases. HIV infection increased the protein concentrations of HIPK2 by promoting oxidative stress, which inhibited the seven in absentia homolog 1 (SIAH1)-mediated proteasomal degradation of HIPK2. HIPK2 induced apoptosis and the expression of epithelial-to-mesenchymal transition markers in kidney epithelial cells by activating the p53, transforming growth factor ß (TGF-ß)-SMAD family member 3 (Smad3) and Wnt-Notch pathways. Knockout of HIPK2 improved renal function and attenuated proteinuria and kidney fibrosis in Tg26 mice, as well as in other murine models of kidney fibrosis. We therefore conclude that HIPK2 is a potential target for anti-fibrosis therapy.
Assuntos
Proteínas de Transporte/metabolismo , Fibrose/fisiopatologia , Nefropatias/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Regulação para Cima/fisiologia , Análise de Variância , Animais , Apoptose/genética , Diferenciação Celular/genética , Células Cultivadas , Creatinina/urina , Modelos Animais de Doenças , Transição Epitelial-Mesenquimal , Perfilação da Expressão Gênica , Glutationa/metabolismo , Dissulfeto de Glutationa/metabolismo , HIV/genética , Infecções por HIV/metabolismo , Infecções por HIV/patologia , Humanos , Hidroxiprolina/metabolismo , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , NF-kappa B/metabolismo , Proteínas Nucleares , Análise de Sequência com Séries de Oligonucleotídeos , Estresse Oxidativo/fisiologia , Proteínas Serina-Treonina Quinases/deficiência , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/genética , Proteína Smad3/metabolismo , Fatores de Tempo , Transfecção , Fator de Crescimento Transformador beta1/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases , Regulação para Cima/genética , Via de Sinalização Wnt/genéticaRESUMO
Retinoic acid decreases proteinuria and glomerulosclerosis in several animal models of kidney disease by protecting podocytes from injury. Our recent in vitro studies suggest that all-trans retinoic acid induces podocyte differentiation by activating the retinoic acid receptor-α (RARα)/cAMP/PKA/CREB pathway. When used in combination with all-trans retinoic acid, an inhibitor of phosphodiesterase 4 further enhanced podocyte differentiation by increasing intracellular cAMP. Additionally, we found that Am580, a specific RARα agonist, has similar renal protective effects as all-trans retinoic acid in a rederived colony of HIV-1 transgenic mice with rapidly progressive renal failure (HIV-Tg) that mimics human HIV-associated nephropathy. Treatment with either the inhibitor of phosphodiesterase 4, roflumilast, or Am580 significantly reduced proteinuria, attenuated kidney injury, and improved podocyte differentiation in these HIV-Tg mice. Additional renal protective effects were found when roflumilast was combined with Am580. Consistent with the in vitro data, glomeruli from HIV-Tg mice treated with both Am580 and roflumilast had more active phosphorylated CREB than with either agent alone. Thus, phosphodiesterase 4 inhibitors could be used in combination with RARα agonists to provide additional renal protection.
Assuntos
Nefropatia Associada a AIDS/prevenção & controle , Aminopiridinas/farmacologia , Benzamidas/farmacologia , Benzoatos/farmacologia , Infecções por HIV/tratamento farmacológico , HIV-1/genética , Túbulos Renais/efeitos dos fármacos , Inibidores da Fosfodiesterase 4/farmacologia , Insuficiência Renal/prevenção & controle , Tetra-Hidronaftalenos/farmacologia , Nefropatia Associada a AIDS/genética , Nefropatia Associada a AIDS/metabolismo , Nefropatia Associada a AIDS/patologia , Nefropatia Associada a AIDS/virologia , Animais , Proteína de Ligação a CREB/metabolismo , Diferenciação Celular/efeitos dos fármacos , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ciclopropanos/farmacologia , Citoproteção , Modelos Animais de Doenças , Progressão da Doença , Quimioterapia Combinada , Feminino , Infecções por HIV/complicações , Infecções por HIV/genética , Infecções por HIV/virologia , HIV-1/patogenicidade , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Túbulos Renais/virologia , Masculino , Camundongos , Camundongos Transgênicos , Fosforilação , Proteinúria/prevenção & controle , Proteinúria/virologia , Receptores do Ácido Retinoico/agonistas , Receptores do Ácido Retinoico/metabolismo , Insuficiência Renal/genética , Insuficiência Renal/metabolismo , Insuficiência Renal/patologia , Insuficiência Renal/virologia , Receptor alfa de Ácido Retinoico , Transdução de Sinais/efeitos dos fármacos , Fatores de TempoRESUMO
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.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Fatores de Transcrição Forkhead/metabolismo , Proteínas de Membrana/metabolismo , Podócitos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Acetilação , Animais , Proteínas Reguladoras de Apoptose/genética , Proteína 11 Semelhante a Bcl-2 , Western Blotting , Bovinos , Proteínas de Ciclo Celular , Linhagem Celular , Células Cultivadas , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologia , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Fatores de Transcrição Forkhead/genética , Expressão Gênica/efeitos dos fármacos , Produtos Finais de Glicação Avançada/química , Produtos Finais de Glicação Avançada/farmacologia , Humanos , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Podócitos/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas Proto-Oncogênicas/genética , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Soroalbumina Bovina/química , Soroalbumina Bovina/farmacologia , Sirtuína 1/genética , Sirtuína 1/metabolismoRESUMO
HIV-1 Nef induces podocyte proliferation and dedifferentiation by activating the Stat3 and MAPK1,2 pathways. Activation of Stat3 also occurs in human kidneys affected by HIV-associated nephropathy (HIVAN), but its contribution to the development of HIVAN is unknown. Here, we generated HIV-1 transgenic mice (Tg26) with either 75% Stat3 activity (Tg26-SA/+) or 25% Stat3 activity (Tg26-SA/-). The kidneys of Tg26-SA/+ mice, but not Tg26-SA/- mice, showed increased Stat3 phosphorylation. The Tg26-SA/+ phenotype was not different from Tg26 mice, but Tg26-SA/- mice developed significantly less proteinuria, glomerulosclerosis, and tubulointerstitial injury. Tg26-SA/+ mice exhibited reduced expression of podocyte differentiation markers and increased expression of VEGF and proliferation markers as compared to Tg26-SA/- mice. Primary podocytes isolated from Tg26-SA/+ mice showed increased Stat3 phosphorylation and reduced expression of podocyte differentiation markers. The tubulointerstitial compartment and isolated tubules of Tg26-SA/+ mice also had increased Stat3 phosphorylation and expression of Stat3 target genes. We confirmed that the expression of the HIV-1 transgene and reduction of Stat3 activity did not affect T and B cell development. In conclusion, Stat3 plays a critical role in the pathogenesis of HIVAN.
Assuntos
Nefropatia Associada a AIDS/etiologia , Fator de Transcrição STAT3/fisiologia , Animais , Antígenos CD4/genética , Proliferação de Células , HIV-1/genética , Rim/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/análise , Podócitos/patologia , Fator A de Crescimento do Endotélio Vascular/análise , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/análiseRESUMO
Recent studies suggest that Stat3, a transcription factor that mediates cytokine signaling, plays a critical role in the pathogenesis of diabetic nephropathy. Complete Stat3 gene knockout is embryonic lethal; therefore, we crossed Stat3+/- mice with Stat3 mutant mice (SA/SA) that lack full Stat3 activity. This strategy generated Stat3SA/- mice (25% activity) and Stat3SA/+ mice (75% activity), which were made diabetic using streptozotocin in order to define the role of Stat3 in diabetic kidney disease. While the glomerular number was not different between these two groups of mice, the diabetic SA/- mice had significantly less proteinuria, mesangial expansion, glomerular cell proliferation, and macrophage infiltration than the diabetic SA/+ mice. The reduction in Stat3 activity did not affect glomerular hyperfiltration seen after the induction of diabetes, as it was increased to the same degree in both groups of mice. Phosphorylation of Stat3 was markedly increased in the glomeruli of diabetic SA/+ mice compared to diabetic SA/- mice. The expression of inflammatory markers, IL-6, MCP-1, and activated NF-kappaB; type IV collagen, TGF-beta, and ICAM-1 mRNA; or type IV collagen and TGF-beta protein, were all found to be significantly less in glomeruli isolated from diabetic SA/- mice, as compared with diabetic SA/+ mice. Our study shows that Stat3 plays a critical role in the regulation of inflammation and abnormal matrix synthesis at an early stage of DN.