RESUMO
The biology and diversity of glomerular parietal epithelial cells (PECs) are important for understanding podocyte regeneration and crescent formation. Although protein markers have revealed the morphological heterogeneity of PECs, the molecular characteristics of PEC subpopulations remain largely unknown. Here, we performed a comprehensive analysis of PECs using single-cell RNA sequencing (scRNA-seq) data. Our analysis identified five distinct PEC subpopulations: PEC-A1, PEC-A2, PEC-A3, PEC-A4 and PEC-B. Among these subpopulations, PEC- A1 and PEC-A2 were characterized as podocyte progenitors while PEC-A4 represented tubular progenitors. Further dynamic signaling network analysis indicated that activation of PEC-A4 and the proliferation of PEC-A3 played pivotal roles in crescent formation. Analyses suggested that upstream signals released by podocytes, immune cells, endothelial cells and mesangial cells serve as pathogenic signals and may be promising intervention targets in crescentic glomerulonephritis. Pharmacological blockade of two such pathogenic signaling targets, proteins Mif and Csf1r, reduced hyperplasia of the PECs and crescent formation in anti-glomerular basement membrane glomerulonephritis murine models. Thus, our study demonstrates that scRNA-seq-based analysis provided valuable insights into the pathology and therapeutic strategies for crescentic glomerulonephritis.
Assuntos
Glomerulonefrite , Nefropatias , Podócitos , Camundongos , Animais , Células Endoteliais/patologia , Células Epiteliais/metabolismo , Glomérulos Renais/patologia , Podócitos/patologia , Glomerulonefrite/patologia , Proteínas/metabolismo , Nefropatias/patologiaRESUMO
Podocyte injury critically contributes to the pathogenesis of obesity-related glomerulopathy (ORG). Recently, lipid accumulation and inflammatory responses have been found to be involved in podocyte injury. This study is to explore their role and relationship in podocyte injury of ORG. In animal experiments, the ORG mice developed proteinuria, podocyte injury, and hypertriglyceridemia, accompanied with deregulated lipid metabolism, renal ectopic lipid deposition, activation of NOD-like receptor protein 3 (NLRP3) inflammasome, and secretion of IL-1ß of the kidney. The expression of adipose differentiation-related protein (ADRP), CD36, sterol regulatory element-binding protein 1 (SREBP-1), and peroxisome proliferator-activated receptor α (PPARα) in renal tissue were increased. In in vitro cell experiments, after cultured podocytes were stimulated with leptin, similar to ORG mice, we found aggravated podocyte injury, formatted lipid droplet, increased expression of ADRP and CD36, activated NLRP3 inflammasome, and released IL-1ß. In addition, after blocking CD36 with inhibitor sulfo-N-succinimidyl oleate (SSO) or CD36 siRNA, activation of NLRP3 inflammasome and release of IL-1ß are downregulated, and podocyte injury was alleviated. However, after blocking NLRP3 with MCC950, although podocyte injury was alleviated and release of IL-1ß was decreased, there was no change in the expression of CD36, ADRP, and intracellular lipid droplets. Taken together, our study suggests that CD36-mediated lipid accumulation and activation of NLRP3 inflammasome may be one of the potential pathogeneses of ORG podocyte injury.
Assuntos
Antígenos CD36/metabolismo , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Obesidade/metabolismo , Podócitos/metabolismo , Animais , Western Blotting , Interleucina-1beta/metabolismo , Metabolismo dos Lipídeos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/fisiologiaRESUMO
[This corrects the article DOI: 10.1155/2019/3172647.].
RESUMO
Chronic aristolochic acid nephropathy (CAAN) is a chronic and progressive tubulointerstitial nephropathy characterized by extensive interstitial fibrosis. Aristolochic acid (AA) could induce overexpression of transforming growth factor-ß1 (TGF-ß1) in a human renal proximal tubule epithelial cells line (HKC), which has been implicated in the pathogenesis of CAAN. The present studies in HKC cells showed 1) AA could activate JNK in time- and dose-dependent manners and JNK inhibitor SP600125 could inhibit AA-induced TGF-ß1 promoter activity and TGF-ß1 synthesis; 2) AA-induced JNK activation and TGF-ß1 synthesis were significantly inhibited by kinase-inactive mutants of MEKK4, MKK4, or MKK7; 3) AA could upregulate luciferase activity derived by a wild-type TGF-ß1 promoter, but not by an AP-1 binding-deficient TGF-ß1 promoter; and 4) AA could upregulate expression of c-Fos, phospho-c-Jun, and phospho-ATF2. The above data suggest AA-induced TGF-ß1 overexpression in HKC cells may be mainly mediated by the JNK signaling pathway. Both the upstream kinases of JNK including MEKK4, MKK4, and MKK7, and the downstream transcription factor of JNK, AP-1, may also participate in this process.
Assuntos
Ácidos Aristolóquicos/farmacologia , Células Epiteliais/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Túbulos Renais Proximais/metabolismo , Fator de Transcrição AP-1/metabolismo , Fator de Crescimento Transformador beta1/biossíntese , Linhagem Celular , Células Cultivadas , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Humanos , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/efeitos dos fármacos , MAP Quinase Quinase 4/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Fosforilação/efeitos dos fármacosRESUMO
OBJECTIVE: To establish a new rat model of chronic cyclosporine A nephrotoxicity and explore its features. METHODS: Totally 24 male SD rats were equally randomized divided into 3 groups: sham-adrenalectomized (sham-ADX) group, ADX group and ADX plus cyclosporine A (CsA) group. Rats in ADX and CsA group first underwent adrenalectomy, followed by the administration of placebo or dexamethasone, respectively. Rats in sham-ADX group received sham adrenalectomy and distilled water as control. Six weeks later, all rats were sacrificed and the following indicators were evaluated: urine protein excretion, creatinine clearance, aldosterone level in serum and urine, aldosterone level and its synthase CYP11B2 gene expression in kidney, serum natrium and potassium, urine natrium and potassium excretion, and tubulointerstitial fibrosis by masson trichrome stain. RESULTS: In ADX and CsA group, serum and urine aldosterone were undetectable on the second post-operative day, with other observations including natriuresis, hyponatremia, decreased urine potassium excretion, and hyperpotassemia, suggesting that adrenals were removed intact and the adrenalectomy was successful. Rats in CsA group showed increased urine protein, decreased creatinine clearance and tubulointerstitial fibrosis, suggesting that a model of chronic CsA nephrotoxicity was successfully established. At the endpoint, serum potassium, serum aldosterone, urine potassium and urine aldosterone excretion partially retrieved. Natrium in serum and urine was not significant different between ADX group/CsA group and sham-ADX group. Local renal aldosterone and its gene expression were remarkably upregulated. CONCLUSIONS: We successfully established a new rat model of chronic CsA nephrotoxicity by adrenalectomy without low sodium diet. After adrenalectomy, local renal aldosterone in kidney may compensate for circulatory aldosterone deficit to maintain electrolyte balance.
Assuntos
Injúria Renal Aguda/induzido quimicamente , Ciclosporina/toxicidade , Modelos Animais de Doenças , Adrenalectomia , Aldosterona/metabolismo , Animais , Imunossupressores/toxicidade , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
We reported a large Chinese family diagnosed with autosomal dominant tubulointerstitial kidney disease caused by MUC1 mutation (ADTKD-MUC1). Cytosine duplication within a string of 7 cytosines in the variable-number tandem repeats (VNTR) region of the MUC1 gene was detected by long-read single-molecule real-time (SMRT) sequencing. MUC1 frameshift protein (MUC1fs) was found to be expressed in renal tubules and urinary exfoliated cells by pathological examination. The family, which consisted of 5 generations including 137 individuals, was followed for 5 years. Genetic testing was performed in thirty-four individuals, 17 of whom carried MUC1 mutations. The ADTKD-MUC1-affected individuals had an elevated incidence of hyperuricaemia without gout attack. Within five years, higher baseline levels of urinary α1-microglobulin were detected in affected individuals with rapidly progressing renal failure than in affected individuals with stable renal function, and the increases manifested even before increases in serum creatinine. This study demonstrates that SMRT sequencing is an effective method for the identification of MUC1 mutations. The pathological examination of MUC1fs expression in renal tissue and urinary exfoliated cells can contribute to early screening of family members suspected to be affected. It is suggested that affected individuals with elevated urinary α1-microglobulin levels should be closely monitored for renal function.
Assuntos
Povo Asiático/genética , Mucina-1/genética , Rim Policístico Autossômico Dominante/diagnóstico , Adulto , Idoso , Estudos de Casos e Controles , China , Feminino , Mutação da Fase de Leitura , Testes Genéticos , Humanos , Hiperuricemia/diagnóstico , Hiperuricemia/etiologia , Rim/diagnóstico por imagem , Rim/fisiopatologia , Masculino , Pessoa de Meia-Idade , Linhagem , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/patologia , Sequências de Repetição em Tandem/genética , Ultrassonografia , Ácido Úrico/urina , Sequenciamento do ExomaRESUMO
OBJECTIVE: To investigate whether aristolochic acid can be transported into human kidney proximal tubular cell (HKC) and its potential mechanism. METHODS: Intracellular aristolochic acid was measured by liquid chromatography-tandem mass spectrometry. The release of lactate dehydrogenase (LDH) induced by aristolochic acid in the presence of organic anion transporter inhibitor (probenecid) or organic cation transporter inhibitor (tetraethylammonium) was evaluated. The effects of probenecid on aristolochic acid induced connective tissue growth factor (CTGF) mRNA and protein expression were also examined by real time polymerase chain reaction and Western blot, respectively. RESULTS: Aristolochic acid was detected in the suspension of the denatured HKC after incubation with aristolochic acid sodium salt. The release of LDH from HKC, which was induced by 60 mg/L aristolochic acid sodium salt, was significantly inhibited by 1 mmol/L probenecid (P < 0.01), but not by 1 mmol/L tetraethylammonium. The increased CTGF mRNA and protein expression in HKC stimulated by 40 mg/L aristolochic acid sodium salt was significantly down-regulated by 1 mmol/L probenecid (P < 0.05), with an inhibition rate of 16% and 21%, respectively. CONCLUSION: Aristolochic acid can be transported into HKC by organic anion transport system, and then exerts its biological effects.
Assuntos
Ácidos Aristolóquicos/metabolismo , Rim/fisiologia , Transportadores de Ânions Orgânicos/metabolismo , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Células Epiteliais/metabolismo , HumanosRESUMO
OBJECTIVE: To investigate the antagonizing effect of Hirsutella sinensis (HS) on renal tubular epithelial-myofibroblast transdifferentiation (TEMT) and its possible pathogenic mechanism in rats with chronic aristolochic acid nephropathy (CAAN). METHODS: Eighteen male Sprague-Dawley rats were equally divided into 3 groups, the model (M) group, the intervention (I) group and the control (C) group. The 24 h urinary protein (UP) in rats was measured before intervention and at the end of the 1st, 4th, 8th, and 12th week, and creatinine clearance rate (CCr) was measured before intervention and at the end of the 12th week respectively. All rats were sacrificed at the end of the 12th week, their kidney was taken for examining the degree of fibrosis in renal interstitial with Masson's stain and determining mRNA and protein expressions of transforming growth factor-beta1 (TGF-beta1), Snail, alpha-smooth muscle actin (alpha-SMA) and cytokeratin in renal tissue by Real-time RT-PCR and immunohistochemistry staining, respectively. RESULTS: Compared with the C group, CCr was significantly lower, while 24 h UP was higher; the relative area of interstitial fibrosis was significantly larger in the M group; besides, the mRNA and protein expressions of TGF-beta1, Snail and alpha-SMA were significantly up-regulated (P < 0.01 or P < 0.05), and those of cytokeratin were significantly down-regulated (P < 0.01) in renal tissue of the M group. While in the I group, all the above-mentioned abnormalities were restored to some extent (P < 0.05) and showed significant difference (all P < 0.05) as compared with those in the M group. CONCLUSION: HS can downregulate TGF-beta1 and Snail expressions in renal tissue, antagonize TEMT and renal interstitial fibrosis, and improve renal function in CAAN rats.
Assuntos
Ácidos Aristolóquicos/toxicidade , Cordyceps/química , Medicamentos de Ervas Chinesas/uso terapêutico , Nefropatias/induzido quimicamente , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador alfa/metabolismo , Actinas/genética , Actinas/metabolismo , Animais , Transdiferenciação Celular , Doença Crônica , Fibroblastos/efeitos dos fármacos , Nefropatias/metabolismo , Túbulos Renais/patologia , Masculino , Fitoterapia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genética , Fator de Crescimento Transformador alfa/genéticaRESUMO
OBJECTIVE: Cryoglobulinemia often causes systemic vasculitis, thereby damaging to skin and internal organs including kidneys, even life-threatening. This review aimed to introduce the advances in understanding, detection, and treatment of this disease in recent years, with a particular concern to clinical practice. DATA SOURCES: All the data in this review were from the English or Chinese literature in the PubMed and China National Knowledge Infrastructure databases as of March 2019. STUDY SELECTION: This review selected important original articles, meaningful reviews, and some reports on cryoglobulinemia published in recent years and in history, as well as the guidelines for treatment of underlying diseases which lead to cryoglobulinemia. RESULTS: Diagnosis of cryoglobulinemia relies on serum cryoglobulin test, in which to ensure that the blood sample temperature is not less than 37°C in the entire pre-analysis phase is the key to avoid false negative results. Cryoglobulinemic vasculitis (Cryo Vas), including cryoglobulinemic glomerulonephritis (Cryo GN), usually occurs in types II and III mixed cryoglobulinemia, and can also be seen in type I cryoglobulinemia caused by monoclonal IgG3 or IgG1. Skin purpura, positive serum rheumatoid factor, and decreased serum levels of C4 and C3 are important clues for prompting types II and III Cryo Vas. Renal biopsy is an important means for diagnosis of Cryo GN, while membranous proliferative GN is the most common pathological type of Cryo GN. In recent years, great advances have been made in the treatment of Cryo Vas and its underlying diseases, and this review has briefly introduced these advances. CONCLUSIONS: Laboratory examinations of serum cryoglobulins urgently need standardization. The recent advances in the diagnosis and treatment of Cryo Vas and GN need to be popularized among the clinicians in related disciplines.
Assuntos
Crioglobulinemia/sangue , Glomerulonefrite/sangue , Animais , Complemento C3 , Complemento C4 , Crioglobulinemia/metabolismo , Crioglobulinemia/patologia , Crioglobulinas/metabolismo , Glomerulonefrite/metabolismo , Glomerulonefrite/patologia , Humanos , Vasculite/sangue , Vasculite/metabolismo , Vasculite/patologiaRESUMO
RATIONALE: IgG4-related disease (IgG4-RD) is a systemic autoimmune disease and mixed cryoglobulinemia may be caused by autoimmune diseases. However, so far only 1 case of IgG4-RD complicated with mixed cryoglobulinemia is reported. Our case further confirms the close relationship between these 2 diseases. PATIENT CONCERNS: A 55-year-old female was admitted because of dry mouth and teeth falling off. DIAGNOSES: The patient was diagnosed as IgG4-related sialadenitis (IgG4-RS) complicated with type III mixed cryoglobulinemia. IgG4-RS was confirmed by elevated serum IgG4 levels and diffuse IgG4 plasmocyte infiltration and storiform fibrosis in the interstitium of labial gland. Type III mixed cryoglobulinemia was confirmed by positive serum cryoglobulins and no monoclonal immunoglobulin in serum and urine. INTERVENTIONS AND OUTCOMES: After treatment with prednisone and cyclophosphamide, serum cryoglobulins rapidly turned negative with the remission of IgG4-RS. LESSONS: Type III mixed cryoglobulinemia can be caused by IgG4-RS, and the underlying mechanisms need to be further explored.
Assuntos
Crioglobulinemia/complicações , Doença Relacionada a Imunoglobulina G4/complicações , Sialadenite/complicações , Crioglobulinemia/tratamento farmacológico , Feminino , Humanos , Doença Relacionada a Imunoglobulina G4/tratamento farmacológico , Imunossupressores/uso terapêutico , Pessoa de Meia-Idade , Sialadenite/tratamento farmacológicoRESUMO
BACKGROUND/AIMS: It is known that chronic low-grade inflammation contributes to the initiation and development of both diabetes and diabetic nephropathy (DN), so we designed this study to investigate the role of P2X7R and NLRP3 inflammasome in DN pathogenesis and the antagonistic effects of artificially cultivated Ophiocordyceps sinensis (ACOS). METHODS: A rat model of DN caused by high-fat-diet feeding and low-dose streptozotocin injection and a mouse podocyte injury model induced by high-glucose (HG) stimulation were established, and the intervention effects of ACOS on them were observed. The biological parameters of serum and urine and the pathological manifestations of kidney tissue were examined. The expression of mRNA and protein of P2X7R and NLRP3 inflammasome (NLRP3, ASC, and caspase-1) and downstream effectors (IL-1ß and IL-18), as well as podocyte-associated molecules, was determined by real-time quantitative PCR and Western blot assay, respectively. RESULTS: The DN rats showed to have developed insulin resistance, elevated fasting blood glucose, increased urinary protein excretion, and serum creatinine level as well as corresponding glomerular pathological alterations including podocyte damages. ACOS significantly antagonized the above changes. The experiments in vivo and in vitro both displayed that the mRNA and protein expression of P2X7R, NLRP3, ASC, caspase1 (procaspase-1 mRNA in the gene level and active caspase-1 subunit P10 in the protein level), IL-1ß, and IL-18 was significantly upregulated and the mRNA and protein expression of podocyte-associated molecules was significantly changed (downregulation of nephrin, podocin, and WT-1 expression and upregulation of desmin expression) indicating podocyte injury in the kidney tissue of DN rats and in the HG-stressed mouse podocytes, respectively. ACOS also significantly antagonized all the above changes. CONCLUSION: Our research work suggests that P2X7R and NLRP3 inflammasome are involved in the pathogenesis of DN, and ACOS can effectively inhibit the high expression of P2X7R and the activation of NLRP3 inflammasome, which may contribute to the therapeutic effects of Ophiocordyceps sinensis.
Assuntos
Cordyceps , Nefropatias Diabéticas/terapia , Inflamassomos/metabolismo , Medicina Tradicional Chinesa , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Podócitos/patologia , Receptores Purinérgicos P2X7/metabolismo , Animais , Apoptose/fisiologia , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Resistência à Insulina , Masculino , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Podócitos/metabolismo , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Receptores Purinérgicos P2X7/genéticaRESUMO
Obesity-related glomerulopathy (ORG) is morphologically characterized by glomerulomegaly with or without observable focal segmental glomerulosclerosis under light microscope, with decreased podocyte density and number, and with increased footprocess width observed under electron microscope. The severity of podocyte injury is correlated with the degree of proteinuria and renal dysfunction. However, the pathogenesis of ORG is not well understood. The aim of the present study was to explore the possible pathogenic role of aldosterone (ALDO) in ORG. In the in vivo animal experiments, body weight, Lee's obesity index, abdominal fat index, urinary protein excretion, average glomerular diameter were significantly increased, the mRNA and protein expression of podocyteassociated molecules including nephrin, podocin, podoplanin and podocalyxin were significantly reduced, and the Wnt/ßcatenin signaling pathway was activated in ORG model mice compared with the Control mice, whereas the administration of spironolactone significantly ameliorated these effects. In the in vitro experiments on cultured podocytes, the mRNA and protein expression levels of the aforementioned podocyteassociated molecules were significantly downregulated and the Wnt/ßcatenin signaling pathway was activated following ALDO stimulation, whereas eplerenone significantly attenuated all the above effects. Dickkopfrelated protein 1 (DKK1), an inhibitor of Wnt/ßcatenin signaling pathway, also reduced the effects of ALDO exposure on the expression of podocyteassociated molecules. The present study hypothesized that ALDO may be involved in the pathogenesis of ORG through the activation of Wnt/ßcatenin signaling pathway in podocytes.
Assuntos
Aldosterona/farmacologia , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/metabolismo , Animais , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Eplerenona , Glomerulonefrite/etiologia , Glomerulonefrite/patologia , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Córtex Renal/efeitos dos fármacos , Córtex Renal/metabolismo , Córtex Renal/patologia , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/complicações , Obesidade/patologia , Podócitos/citologia , Podócitos/efeitos dos fármacos , Podócitos/metabolismo , Sialoglicoproteínas/genética , Sialoglicoproteínas/metabolismo , Espironolactona/análogos & derivados , Espironolactona/farmacologia , Proteínas Wnt/genética , beta Catenina/genéticaRESUMO
OBJECTIVE: To study the protective effects of Hirsutella sinensis on renal interstitial fibrosis in chronic aristolochic acid nephropathy (CAAN). METHODS: Eighteen male SD rats were divided into 3 equal groups: model group, given the extract of Aristolochia manshuriensis Kom (AmK) by gavage in the morning everyday for 12 weeks, intervention group, given the extract of Amk in the morning and suspension of Hirsutella sinensis in the afternoon by gavage once a day for 12 weeks, and control group, receiving tap water only by gavage. Bodyweight, urinary glucose, 24 h urinary protein excretion, and serum creatinine (SCr) were measured at the ends of the 1st, 4th, 8th, and 12th weeks respectively. At the end of the 12th week, all the rats were sacrificed with their kidneys taken out to undergo pathological examination. RT-PCR and immunohistochemistry were used to detect the mRNA and protein expression of transforming growth factor-beta1 (TGF-beta1), connective tissue growth factor (CTGF), plasminogen activator inhibitor-1 (PAI-1), tissue inhibitor of metalloproteinase-1 (TIMP-1), and type I collagen (ColI) in the kidney tissues. RESULTS: Since the 1st week, the urinary protein excretion and SCr levels in the model group were significantly higher than those in the control group (P < 0.01 or 0.05). At the end of the 12th week, the relative area of interstitial fibrosis of the model group was significantly enlarged (P < 0.01). The mRNA expression levels of TGF-beta1, CTGF, PAI-1, TIMP-1, and ColI in the model group were up-regulated by 4.19, 2.66, 6.12, 3.09, and 7.03 times respectively, and their protein expression levels were up-regulated by 2.31, 3.53, 3.17, 3.18, and 6.87 times respectively (all P < 0.01). By the end of the 12th week, the urinary protein excretion, SCr level and the relative area of interstitial fibrosis in the intervention group were all significantly lower than those in the model group (all P < 0.05). The mRNA expression levels of TGF-beta, CTGF, PAI-1, TIMP-1, and ColIof the intervention group were all significantly lower than those of the model group (all P < 0.05) with the inhibition rates of 45%, 41%, 47%, 48%, and the protein expression levels of TGF-beta, CTGF, PAI-1, TIMP-1, and ColI of the intervention group were all significantly lower than those of the model group (all P < 0.05) with the inhibition rates of 38%, 39%, 49%, 46%, and 61% respectively. CONCLUSION: Hirsutella sinensis can inhibit the production of TGF-beta1 and CTGF, factors that promote the extracellular matrix (ECM) synthesis and TIMP-1 and PAI-1, factors that antagonize ECM degradation in kidney tissues, thus alleviating renal interstitial fibrosis and improving renal function in CAAN.
Assuntos
Produtos Biológicos/uso terapêutico , Hypocreales/química , Rim/efeitos dos fármacos , Nefrite Intersticial/prevenção & controle , Animais , Ácidos Aristolóquicos , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Doença Crônica , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Fator de Crescimento do Tecido Conjuntivo , Modelos Animais de Doenças , Fibrose , Expressão Gênica/efeitos dos fármacos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Rim/metabolismo , Rim/patologia , Masculino , Nefrite Intersticial/induzido quimicamente , Nefrite Intersticial/patologia , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
Autophagy plays an essential role in cellular homeostasis in kidney. Previous studies have found that aristolochic acid (AA) can induce autophagy of renal tubular epithelial cells and epithelial-to-myofibroblast transition (EMT). However, the relationship between AA-induced autophagy and EMT is unclear. Our results showed that, after AA stimulation, the appearance of autophagy preceded EMT. Autophagy of HKC cells began to increase gradually from the 3rd hour, reached the peak at 12th hour, and then weakened gradually until 36th hour; the EMT process of HKC continued to increase from 6th hour to 36th hour after AA stimulation. The enhancement of autophagy using autophagy inducers, rapamycin or serum-free medium, led to an aggravation of EMT and upregulated expression of fibronectin, a component of extracellular matrix, in AA-treated HKC cells. In contrast, the inhibition of autophagy by autophagy inhibitor, 3-methyladenine, or by knockdown of Beclin 1 led to an attenuation of EMT and downregulated expression of fibronectin in AA-treated HKC cells. Taken together, our study suggests that, after AA stimulation, two types of cell responses of HKC cells, autophagy and EMT, will successively appear, and autophagy can promote EMT of HKC.
RESUMO
OBJECTIVE: Our previous study have demonstrated that the expression of transforming growth factor-beta1 (TGF-beta1) by HKC could be up-regulated by aldosterone (ALDO) in vitro. The present study was designed to evaluate the role of MAPK/ERK1/2 phosphorylation in mediating the synthesis of TGF-beta1 in renal tubular epithelial cells that was activated by aldosterone. METHODS: The following tests were performed in vitro: (1) HKC were pretreated with different concentrations of specific ERK1/2, JNK and P38 MAPK pathway inhibitors for 4h, then HKC were stimulated with 10(-7) mol/L ALDO for 48 h, finally enzyme-linked immunosorbent assay (ELISA) were performed to detect TGF-beta1 expression; (2) HKC were stimulated with ALDO at different concentrations and times, then western blot assay was performed to detect the expression of phosphorylated and total ERK1/2 in the cell lysate of HKC. (3) HKC which were co-stimulated with 10(-7) mol/L ALDO and different concentrations of spironolactone or specific glucocorticoid hormone receptor inhibitor RU486 for 30min, then western blot assay was performed to detect the expression of phosphorylated and total ERK1/2 in the cell lysate of HKC. RESULTS: (1) the production in 15 and 25 micromol/L U0126 incubated groups was (87 +/- 11) pg/ml and (75 +/- 19) pg/ml respectively, which was significantly decreased compared with that in 10(-7) mol/L ALDO incubated group (P < 0.05), however, the amount of TGF-beta1 in these groups were still significant higher than that in the control group (P < 0.05). The production of TGF-beta1 in the groups which were incubated with SP600125 and SB203580 did not appear significant decrease compared with that in 10(-7) mol/L ALDO incubated group (P > 0.05), the production of TGF-beta1 in these groups was also significant higher than that in the control group (P < 0.05). (2) The Phos/Total ERK1/2 ratio was increased in a dose-dependent manner. After HKC were stimulated with 10(-9) - 10(-7) mol/L ALDO for 30 min. Phos/TotalERK1/2 ratio was 0.67 +/- 0.06 and 0.80 +/- 0.05 respectively, which was significantly increased (vs 0 mol/L ALDO, P < 0.05 or 0.01). The expression of Phos/Total ERK1/2 ratio also had a positive correlations with the production of TGF-beta1 (R = 0.793, P < 0.01). With 10(-7) mol/L ALDO stimulated at different times, the Phos/Total ERK1/2 ratio was also significantly increased (vs 0 h, P < 0.05 or 0.01), which began to be increased and reached the peak at 15 min, the relatively ratio was 0.84 +/- 0.06, and waned till 240 min, and returned to normal level at 360 min. (3) After co-stimulated with 10(-7) mol/L ALDO and different concentration of spironolactone, Phos/TotalERK1/2 ratio was significantly decreased along with the concentrations of spironolactone, the relatively ratio of in 10(-9) - 10(-7) was 0.62 +/- 0.08 and 0.60 +/- 0.04 separately (vs 0 mol/L ALDO, or 0.01), but they were still significantly higher than that in control group (P < 0.05). The Phos/TotalERK1/2 ratio was not changed significantly along with the concentrations of RU486 (P > 0.05). CONCLUSION: The effect of aldosterone in up-regulating the expression of TGF-beta1 in HKC is mediated, at least in part, by MAPK/ERK1/2 pathway. Aldosterone may exert this effect on the conditions of binding to the mineralocorticoid receptor first.
Assuntos
Aldosterona/farmacologia , Células Epiteliais/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta1/biossíntese , Antracenos/farmacologia , Western Blotting , Butadienos/farmacologia , Células Cultivadas , Relação Dose-Resposta a Droga , Células Epiteliais/citologia , Células Epiteliais/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Humanos , Imidazóis/farmacologia , Túbulos Renais Proximais/citologia , Mifepristona/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Nitrilas/farmacologia , Fosforilação/efeitos dos fármacos , Piridinas/farmacologia , Fatores de TempoRESUMO
OBJECTIVE: To investigate the expression and regulation of alpha 1 anti-trypsin (AAT) in tubular epithelial cells. METHODS: The expression of AAT in tubular epithelial cell line HKC was detected with indirect immunofluorescence assay and confirmed by reverse transcription polymerase chain reaction (PCR) in transcription level respectively, and PCR product was sequenced. In order to observe the response of HCK to LPS, the different concentrations of LPS (0, 0.5, 1.0, 2.0 microg/ml) were used in the research and the regulation of AAT in HKC was semi-quantitatively detected with Western Blot and Real-Time PCR respectively. RESULTS: Indirect immunofluorescence staining showed that AAT was positive in HKC, but negative in renal interstitial fibroblast. By PCR, a significant messenger RNA band of AAT was found in HKC, but not in renal interstitial fibroblast. DNA sequencing indicated that the sequence of PCR product is consistent with AAT mRNA sequence in Gene Bank. Real-time PCR showed that the expression of AAT mRNA was up-regulated (fluorescence intensity ratio is 3.43 +/- 0.88 versus 1.22 +/- 0.20; P < 0.01) in HKC stimulated with 2.0 microg/ml LPS for 4 h, and Western Blot showed that the synthesis of AAT significantly increased (band density ratio is 0.88 +/- 0.12 versus 0.59 +/- 0.05; P < 0.01) in HKC stimulated with 2.0 microg/ml LPS for 8 h, as compared with unstimulated HKC. 0.5, 1.0 g/ml of LPS has no effect on the expression of AAT mRNA and AAT protein in HKC. CONCLUSIONS: HKC express AAT and the expression can be up-regulated by LPS.
Assuntos
Células Epiteliais/metabolismo , Túbulos Renais/metabolismo , alfa 1-Antitripsina/biossíntese , Linhagem Celular , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Humanos , Túbulos Renais/citologia , Lipopolissacarídeos/farmacologia , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
OBJECTIVE: To investigate the inhibitory effect of Hirsutella sinensis (HS) on epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells induced by aristolochic acid (AA) and its possible mechanism. METHODS: 18 male Sprague-Dawley rats were randomly and equally divided into the following 3 groups: AA group, AA+HS group and control group. Urinary protein excretion and creatinine clearance (CCr) were measured. All rats were sacrificed at the end of 12th week. The pathological examination of renal tissue was performed and the mRNA and protein expression of transforming growth factor-ß1 (TGF-ß1), α-smooth muscle actin (α-SMA), cytokeratin-18 and Snail in renal cortex were determined by real time quantitative PCR and immunohistochemical staining respectively. In addition, human renal proximal tubule epithelial cells line (HKC) was divided into the following 4 groups: AA group, AA+HS group, HS control group and control group. The above mRNA and protein expression in HKC was determined by real time quantitative PCR and Western blot respectively. RESULTS: (1) CCr was significantly decreased, and the urinary protein excretion and relative area of renal interstitial fibrosis were significantly increased in the rats of AA and AA+HS group compared to those in control group (P<0.05 or P<0.01); all the above abnormalities significantly lightened in the rats of AA+HS group compared to those in AA group (P<0.05). (2) The mRNA and protein expression of TGF-ß1, α-SMA and Snail was significantly up-regulated and the expression of cytokeratin-18 was significantly down-regulated in the rat renal cortex as well as in the cultured HKC cells in AA and AA+HS groups compared to those in control group (P<0.05 or P<0.01); all the above abnormalities significantly alleviated in AA+HS group compared to those in AA group (P<0.05 or P<0.01). (3) Knockdown endogenous Snail expression by siRNA could ameliorate AA-induced EMT of HKC cells, while overexpression of Snail by plasmid transfection diminished the antagonistic effect of HS on AA-induced EMT. These results suggest Snail might be a potential target of HS effect. CONCLUSION: HS is able to antagonize, to some extent, tubular EMT and renal interstitial fibrosis caused by AA, which might be related to its inhibitory effects on the TGF-ß1 and Snail expression.
Assuntos
Ácidos Aristolóquicos/efeitos adversos , Ascomicetos/fisiologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Túbulos Renais/citologia , Fatores de Transcrição/genética , Fator de Crescimento Transformador beta1/genética , Actinas/genética , Actinas/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Queratina-18/genética , Queratina-18/metabolismo , Córtex Renal/efeitos dos fármacos , Córtex Renal/metabolismo , Córtex Renal/patologia , Nefropatias/etiologia , Nefropatias/metabolismo , Nefropatias/patologia , Nefropatias/fisiopatologia , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Ratos , Fatores de Transcrição da Família Snail , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta1/metabolismoRESUMO
BACKGROUND AND OBJECTIVE: The fact that mineralocorticoid receptor antagonists reduce structural and functional alterations induced by cyclosporine A (CsA) indicates that aldosterone plays a key role in chronic CsA nephrotoxicity. We and other researchers have reported local renal aldosterone synthesis. To investigate local renal aldosterone's role in chronic CsA nephrotoxicity, we evaluated the effect of eplerenone (Epl) on renal structural damage and renal dysfunction in adrenalectomized (ADX) rats, and assessed whether the therapeutic benefit was associated with reduction of transforming growth factor-ß1 (TGF-ß1), connective tissue growth factor (CTGF), plasminogen activator inhibitor type 1 (PAI-1) and collagen I (COL-I) expression. METHODS: Male Sprague-Dawley rats fed a normal-sodium diet were divided in four groups: sham-ADX, ADX, CsA, or Epl. Rats in the ADX, CsA and Epl groups were adrenalectomized first. Aldosterone, sodium and potassium levels in serum and urine were measured on the second day. Two weeks later, vehicle (sham-ADX and ADX group), CsA (25mg/kg/d), or CsA and Epl (100 mg/ kg/d) combination was administrated, respectively. After six weeks, urinary protein, creatinine clearance (Ccr), tubulointerstitial fibrosis (TIF), aldosterone level in kidney, and renal aldosterone synthase CYP11B2, COL-I, TGF-ß1, CTGF and PAI-1 gene expression levels were determined. RESULTS: On the second day after surgery, adrenalectomized rats showed undetectable aldosterone with natriuresis, hyponatremia, decreased urinary potassium excretion and hyperpotassemia. CsA reduced Ccr, induced urinary proteins and up-regulated COL-I, TGF-ß1, CTGF and PAI-1 gene expression with a significant development of TIF. Eplerenone administration prevented TIF and COL-I, TGF-ß1 and PAI-1 up-regulation but did not improve renal function. CONCLUSION: Our results suggest local renal aldosterone is an important mediator of renal injury induced by CsA.
Assuntos
Aldosterona/metabolismo , Ciclosporina/efeitos adversos , Nefropatias/induzido quimicamente , Rim/patologia , Espironolactona/análogos & derivados , Adrenalectomia , Aldosterona/sangue , Aldosterona/urina , Animais , Doença Crônica , Citocromo P-450 CYP11B2/genética , Citocromo P-450 CYP11B2/metabolismo , Modelos Animais de Doenças , Eletrólitos/sangue , Eletrólitos/urina , Eplerenona , Fibrose , Rim/efeitos dos fármacos , Rim/fisiopatologia , Nefropatias/sangue , Nefropatias/fisiopatologia , Nefropatias/urina , Masculino , Potássio/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Sódio/metabolismo , Espironolactona/farmacologiaRESUMO
The present study investigated the effects of curcumin, one of the most important active ingredients of turmeric, on podocyte injury in vitro and obesity-related glomerulopathy (ORG) in vivo. Cellular experiments in vitro showed that curcumin significantly antagonized leptin-induced downregulation of the mRNA and protein expression of podocyte-associated molecules including nephrin, podocin, podoplanin, and podocalyxin. Animal experiments in vivo showed that curcumin significantly reduced the body weight, Lee's index, abdominal fat index, urinary protein excretion, and average glomerular diameter and significantly upregulated the mRNA and protein expressions of the above podocyte-associated molecules in ORG mice. Furthermore, the experiments in vitro and in vivo both displayed that curcumin could downregulate the mRNA and protein expressions of Wnt1, Wnt2b, Wnt6, and ß-catenin and upregulate the phosphorylation level of ß-catenin protein in podocytes and renal tissue. In conclusion, curcumin is able to alleviate the harmful reaction of leptin on podocytes and reduce the severity of ORG. The above protective effects are associated with the inhibition of Wnt/ß-catenin signaling activation in podocytes.