RESUMO
Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited cause of kidney failure and characterized by the formation of multiple fluid-filled cysts in the kidneys. It is believed that environmental factors may play an important role in the disease progression. However, the molecular identity of autocrine/paracrine factors influencing cyst formation is largely unknown. In this study, we identified transforming growth factor-ß2 (TGF-ß2) secreted by normal human kidney (NHK) and ADPKD cells as an inhibitor of cystogenesis in 3D culture system using ADPKD cells from human kidneys. TGF-ß2 was identified in conditioned media (CM) of NHK and ADPKD cells as a latent factor activated by heat in vitro. While all TGF-ß isoforms recombinant proteins (TGF-ß1, -ß2, or -ß3) displayed a similar inhibitory effect on cyst formation, TGF-ß2 was the predominant isoform detected in CM. The involvement of TGF-ß2 in the suppression of cyst formation was demonstrated by using a TGF-ß2 specific blocking antibody and a TGF-ß receptor I kinase inhibitor. TGF-ß2 inhibited cyst formation by a mechanism other than activation of p38 mitogen-activated protein (MAP) kinase that mediated cell death in ADPKD cells. Further, we found that TGF-ß2 modulated expression of various genes involved in cell-cell and cell-matrix interactions and extracellular matrix proteins that may play a role in the regulation of cystogenesis. Collectively, our results suggest that TGF-ß2 secreted by renal epithelial cells may be an inhibitor of cystogenesis influencing the progression of ADPKD.
Assuntos
Rim Policístico Autossômico Dominante/metabolismo , Fator de Crescimento Transformador beta2/metabolismo , Anticorpos Bloqueadores/farmacologia , Proliferação de Células , Células Cultivadas , Meios de Cultivo Condicionados , Progressão da Doença , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Expressão Gênica , Humanos , Rim/citologia , Rim/efeitos dos fármacos , Rim/metabolismo , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/patologia , Rim Policístico Autossômico Dominante/prevenção & controle , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Proteínas Recombinantes/farmacologia , Fator de Crescimento Transformador beta2/antagonistas & inibidores , Fator de Crescimento Transformador beta2/genética , Fator de Crescimento Transformador beta2/farmacologiaRESUMO
BACKGROUND: Cystatin C (CyC) concentration has been suggested as a marker of middle-molecule accumulation, hemodialysis (HD) adequacy and for estimating residual renal function (RRF), but it has not been studied in pediatric HD. High CyC is associated with increased cardiovascular disease (CVD). We investigated CyC kinetics and the effect of RRF on CyC in a pediatric HD population. METHODS: A total of 21 HD sessions and 20 interdialytic periods were analyzed in seven patients, age 5-19 years, of whom four were anuric (A) and three were non-anuric (NA). CyC was measured before (preHD) and after (postHD) three standard HD sessions in 1 week and prior to the first session of the following week. RESULTS: We found no difference (p=0.67) in CyC concentration between preHD CyC (9.85 ± 2.15 mg/l; A vs. NA, p=0.37) and postHD CyC (10.04 ± 2.83 mg/l; A vs NA, p=0.28). The weekly average preHD CyC median concentration was 10.14 mg/l (A vs. NA, p=0.87) and correlated with age (r=0.808, p=0.03) and height measurement (r=0.799, p=0.03), but not with RRF, single-pool Kt/V, ultrafiltration, HD duration or blood liters processed. CONCLUSIONS: Cystatin C is very elevated in children on HD. It does not rise between HD sessions, is not removed by standard HD and remains at steady state; therefore, elimination is extrarenal. Low RRF does not affect CyC elimination. CyC increases with age and height. If a high CyC concentration can be proven to have a causative role in the development of CVD, routine intensified HD regimens in children may be indicated for its removal.
Assuntos
Cistatina C/sangue , Falência Renal Crônica/terapia , Rim/fisiopatologia , Diálise Renal , Adolescente , Fatores Etários , Biomarcadores/sangue , Criança , Pré-Escolar , Humanos , Falência Renal Crônica/sangue , Falência Renal Crônica/fisiopatologia , Testes de Função Renal , Cinética , Modelos Lineares , Projetos Piloto , Resultado do Tratamento , Regulação para Cima , Adulto JovemRESUMO
Previously, we demonstrated that prostaglandin E(2) (PGE(2)) induced cAMP and cyst formation through PGE(2) receptor-2 (EP2) activity in human autosomal-dominant polycystic kidney disease (ADPKD) epithelial cells. In this study, we determined the role of EP2 and EP4 receptors in mediating PGE(2) stimulation of cAMP signaling and cystogenesis in mouse renal epithelial cells using the inner medullary collecting duct-3 (IMCD-3) cell line. In contrast to human ADPKD cells, using novel EP2 and EP4 antagonists, we found that IMCD-3 cells expressed functional EP4 but not EP2, which stimulated cAMP formation and led to cyst formation in 3D culture system. The involvement of EP4 receptors in IMCD-3 cells was further supported by the specific effect of EP4 siRNA that inhibited PGE(2)-induced cystogenesis. We also observed different cellular localization of EP2 or EP4 receptors in IMCD-3 transfected cells. Collectively, our results suggest an important role of different expression of EP2 or EP4 receptors in the regulation of cystogenesis.
Assuntos
Cistos/metabolismo , Dinoprostona/farmacologia , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Animais , Western Blotting , Linhagem Celular , AMP Cíclico/metabolismo , Cistos/induzido quimicamente , Camundongos , Reação em Cadeia da Polimerase , Receptores de Prostaglandina E Subtipo EP2/genética , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Receptores de Prostaglandina E Subtipo EP4/genéticaRESUMO
BACKGROUND/AIMS: Progressive renal fibrotic disease is accompanied by the massive accumulation of myofibroblasts as defined by alpha smooth muscle actin (alphaSMA) expression. We quantitated gene expression using real-time RT-PCR analysis during conversion of primary cultured human renal tubular cells (RTC) to myofibroblasts after treatment with transforming growth factor-beta1 (TGF-beta1). We report herein the limitations of commonly used reference genes for mRNA quantitation. METHODS: We determined the expression of alphaSMA and megakaryoblastic leukemia-1 (MKL1), a transcriptional regulator of alphaSMA, by quantitative real-time PCR using three common internal controls, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cyclophilin A and 18S rRNA. RESULTS: Expression of GAPDH mRNA and cyclophilin A mRNA, and to a lesser extent, 18S rRNA levels varied over time in culture and with exposure to TGF-beta1. Thus, depending on which reference gene was used, TGF-beta1 appeared to have different effects on expression of MKL1 and alphaSMA. CONCLUSIONS: RTC converting to myofibroblasts in primary culture is a valuable system to study renal fibrosis in humans. However, variability in expression of reference genes with TGF-beta1 treatment illustrates the need to validate mRNA quantitation with multiple reference genes to provide accurate interpretation of fibrosis studies in the absence of a universal internal standard for mRNA expression.
Assuntos
Sistemas Computacionais , Fibroblastos/patologia , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Miócitos de Músculo Liso/patologia , Controle de Qualidade , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Actinas/genética , Actinas/metabolismo , Diferenciação Celular , Células Cultivadas , Sistemas Computacionais/normas , Ciclofilina A/genética , Fibrose , Expressão Gênica , Gliceraldeído-3-Fosfato Desidrogenases/genética , Humanos , Túbulos Renais/efeitos dos fármacos , MAP Quinase Quinase Quinases/genética , Músculo Liso/metabolismo , RNA Ribossômico 18S/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Fator de Crescimento Transformador beta/farmacologia , Fator de Crescimento Transformador beta1RESUMO
BETA2/NeuroD, a basic helix-loop-helix transcription factor, is expressed in pancreatic endocrine cells during development and regulates insulin gene expression. We demonstrated previously that the endocrine pancreas of BETA2/NeuroD-deficient mice undergoes massive apoptosis and, consequently, animals die of diabetes shortly after birth. Here we show that a significant fraction of BETA2-deficient mice in a new genetic background can survive diabetes and live to adulthood through the process of beta-cell neogenesis. Morphometric examination indicates that pancreatic beta-, but not alpha-cell mass, was restored to a level comparable to that of wild-type animals. However, the newly formed islet cells cannot form mature islets of Langerhans, indicating an indispensable role of BETA2 in morphogenesis of normal islet structure. Furthermore, immunohistochemical examinations revealed that newly formed beta-cells of BETA2/NeuroD-deficient mice come from two sources: either directly budding from the pancreatic ductal tree or from the preexisting beta-cells in the residual endocrine pancreas. Our results indicate that beta-cell neogenesis in our BETA2/NeuroD-deficient mice contributes to their survival, and these mice may provide a useful model for studying the mechanism of beta-cell regeneration.
Assuntos
Proteínas de Ligação a DNA/deficiência , Ilhotas Pancreáticas/fisiologia , Transativadores/deficiência , Animais , Apoptose , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Cruzamentos Genéticos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Diabetes Mellitus/genética , Diabetes Mellitus/mortalidade , Teste de Tolerância a Glucose , Sequências Hélice-Alça-Hélice , Imuno-Histoquímica , Ilhotas Pancreáticas/embriologia , Ilhotas Pancreáticas/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitose , Morfogênese , Ductos Pancreáticos/patologia , Regeneração , Transativadores/genética , Transativadores/fisiologiaRESUMO
Middle-molecules (MM) are not monitored in children on hemodialysis (HD), but are accumulated and increase the risk of cardiovascular disease and mortality. Molecular properties of Cystatin C (CyC), 13 kDa, potentially make it a preferred MM marker over Beta-2-Microglobulin (B2M), 12 kDa. We compared CyC and B2M kinetics to investigate if CyC can be used as preferred MM marker. CyC (mg/L) and B2M (µg/mL) were measured in 21 low-flux HD sessions in seven children. Blood samples were taken at HD start (pre), 1 and 2 hours into HD and at end of HD (post) for all sessions and 60 minutes after the first HD (Eq). PreCyC (9.85 ± 2.15) did not differ (P > 0.05) from postCyC (10.04 ± 2.83). PostB2M (38.87 ± 7.12) was higher (P < 0.05) than preHD B2M (33.27 ± 7.41). There was no change in CyC at 1 and 2 hours into HD, while B2M progressively increased. CyC or B2M changes did not significantly correlate with spKt/V (2.09 ± 0.86), ultrafiltration (4.61 ± 1.98%) or HD duration (218 ± 20 minutes). EqCyC was not different from postCyC (11.07 ± 3.14 vs. 10.71 ± 2.85, P > 0.05), while EqB2M was lower than postB2M (36.48 ± 7.68 vs. 41.09 ± 8.99, P < 0.05). MMs as represented by B2M and CyC are elevated in children on standard HD. Intensified HD modalities would be needed for their removal. B2M is affected by the dialytic process with a rise during HD independent of ultrafiltration and decrease 1 hour after, while CyC remains unchanged. We suggest that CyC be used as preferred marker of MM removal and as a marker of adequacy of intensified HD regimens.
Assuntos
Cistatina C/sangue , Falência Renal Crônica/sangue , Falência Renal Crônica/terapia , Diálise Renal/métodos , Microglobulina beta-2/sangue , Adolescente , Fatores Etários , Biomarcadores/sangue , Criança , Cistatina C/metabolismo , Feminino , Humanos , Masculino , Microglobulina beta-2/metabolismoRESUMO
Transforming growth factor-beta1 (TGF-beta1) is known to induce epithelial-mesenchymal transition in the kidney, a process involved in tubulointerstitial fibrosis. We hypothesized that a coactivator of the serum response factor (SRF), megakaryoblastic leukemia factor-1 (MKL1), stimulates alpha-smooth muscle actin (alpha-SMA) transcription in primary cultures of renal tubular epithelial cells (RTC), which convert into myofibroblasts on treatment with TGF-beta1. Herein, we study the effect of MKL1 expression on alpha-SMA in these cells. We demonstrate that TGF-beta1 stimulation of alpha-SMA transcription is mediated through CC(A/T)(6)-rich GG elements known to bind to SRF. These elements also mediate the MKL1 effect that dramatically activates alpha-SMA transcription in serum-free media. MKL1 fused to green fluorescent protein localizes to the nucleus and induces alpha-SMA expression regardless of treatment with TGF-beta1. Using proteasome inhibitors, we also demonstrate that the proteolytic ubiquitin pathway regulates MKL1 expression. These data indicate that MKL1 overexpression is sufficient to induce alpha-SMA expression. Inhibition of endogenous expression of MKL1 by small interfering RNA abolishes TGF-beta1 stimulation of alpha-SMA expression. Therefore, MKL1 is also absolutely required for TGF-beta1 stimulation of alpha-SMA expression. Western blot and immunofluorescence analysis show that overexpressed and endogenous MKL1 are located in the nucleus in non-stimulated RTC. Chromatin immunoprecipitation assay demonstrates that TGF-beta1 induces binding of endogenous SRF and MKL1 to the alpha-SMA promoter in chromatin. Since MKL1 constitutes a potent factor regulating alpha-SMA expression, modulation of endogenous MKL1 expression or activity may have a profound effect on myofibroblast formation and function in the kidney.
Assuntos
Actinas/biossíntese , Proteínas de Ligação a DNA/fisiologia , Células Epiteliais/metabolismo , Rim/metabolismo , Músculo Liso/metabolismo , Proteínas de Fusão Oncogênica/fisiologia , Fator de Crescimento Transformador beta1/fisiologia , Western Blotting , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Células Cultivadas , DNA/biossíntese , DNA/genética , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Fibroblastos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Humanos , Rim/citologia , Túbulos Renais/citologia , Túbulos Renais/metabolismo , Microscopia de Fluorescência , Proteínas de Fusão Oncogênica/antagonistas & inibidores , Proteínas de Fusão Oncogênica/genética , Regiões Promotoras Genéticas/genética , RNA Interferente Pequeno/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransativadoresRESUMO
Autosomal-dominant polycystic kidney disease (ADPKD) is characterized by formation of cysts from tubular epithelial cells. Previous studies indicate that secretion of prostaglandin E2 (PGE2) into cyst fluid and production of cAMP underlie cyst expansion. However, the mechanism by which PGE2 directly stimulates cAMP formation and modulates cystogenesis is still unclear, because the particular E-prostanoid (EP) receptor mediating the PGE2 effect has not been characterized. Our goal is to define the PGE2 receptor subtype involved in ADPKD. We used a three-dimensional cell-culture system of human epithelial cells from normal and ADPKD kidneys in primary cultures to demonstrate that PGE2 induces cyst formation. Biochemical evidence gathered by using real-time RT-PCR mRNA analysis and immunodetection indicate the presence of EP2 receptor in cystic epithelial cells in ADPKD kidney. Pharmacological evidence obtained by using PGE2-selective analogs further demonstrates that EP2 mediates cAMP formation and cystogenesis. Functional evidence for a role of EP2 receptor in mediating cAMP signaling was also provided by inhibiting EP2 receptor expression with transfection of small interfering RNA in cystic epithelial cells. Our results indicate that PGE2 produced in cyst fluid binds to adjacent EP2 receptors located on the apical side of cysts and stimulates EP2 receptor expression. PGE2 binding to EP2 receptor leads to cAMP signaling and cystogenesis by a mechanism that involves protection of cystic epithelial cells from apoptosis. The role of EP2 receptor in mediating the PGE2 effect on stimulating cyst formation may have direct pharmacological implications for the treatment of polycystic kidney disease.