RESUMO
TGF-ß is considered an important cytokine in the development of interstitial fibrosis in chronic kidney disease. The TGF-ß co-receptor endoglin (ENG) tends to be upregulated in kidney fibrosis. ENG has two membrane bound isoforms generated via alternative splicing. Long-ENG was shown to enhance the extent of renal fibrosis in an unilateral ureteral obstruction mouse model, while short-ENG inhibited renal fibrosis. Here we aimed to achieve terminal intron retention of endoglin using antisense-oligo nucleotides (ASOs), thereby shifting the ratio towards short-ENG to inhibit the TGF-ß1-mediated pro-fibrotic response. We isolated mRNA from kidney biopsies of patients with chronic allograft disease (CAD) (n = 12) and measured total ENG and short-ENG mRNA levels. ENG mRNA was upregulated 2.3 fold (p < 0.05) in kidneys of CAD patients compared to controls, while the percentage short-ENG of the total ENG mRNA was significantly lower (1.8 fold; p < 0.05). Transfection of ASOs that target splicing regulatory sites of ENG into TK173 fibroblasts led to higher levels of short-ENG (2 fold; p < 0.05). In addition, we stimulated these cells with TGF-ß1 and measured a decrease in upregulation of ACTA2, COL1A1 and FN1 mRNA levels, and protein expression of αSMA, collagen type I, and fibronectin. These results show a potential for ENG ASOs as a therapy to reduce interstitial fibrosis in CKD.
Assuntos
Endoglina , Fibrose , Íntrons , Rim , Oligonucleotídeos Antissenso , Fator de Crescimento Transformador beta1 , Humanos , Endoglina/metabolismo , Endoglina/genética , Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos Antissenso/genética , Íntrons/genética , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta1/genética , Rim/metabolismo , Rim/patologia , Masculino , Fibronectinas/metabolismo , Fibronectinas/genética , Feminino , Actinas/metabolismo , Actinas/genética , Pessoa de Meia-Idade , Animais , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Processamento Alternativo , Fibroblastos/metabolismo , Fibroblastos/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Camundongos , Linhagem CelularRESUMO
Preeclampsia is a pregnancy-specific syndrome characterized by renal dysfunction and high blood pressure. When evaluated with light microscopy, the renal lesion of preeclampsia is marked by endothelial cell swelling and the appearance of bloodless glomeruli. However, regarding the pathobiology of renal damage in preeclampsia, attention recently has shifted from the glomerular endothelial cells to the podocytes. The angiogenic imbalance in preeclampsia plays a key role in the development of both podocyte and endothelial damage in the glomerular filtration barrier. Here, we review the latest studies on the role of podocytes in the development of renal damage in preeclampsia and on podocytes as potential targets for diagnosis, treatment, and prevention of long-term complications of preeclampsia.
Assuntos
Podócitos/patologia , Pré-Eclâmpsia/fisiopatologia , Animais , Feminino , Taxa de Filtração Glomerular , Humanos , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Glomérulos Renais/fisiopatologia , Pré-Eclâmpsia/metabolismo , Medicina de Precisão , Gravidez , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
A growing body of evidence suggests that complement dysregulation plays a role in the pathogenesis of preeclampsia. The kidney is one of the major organs affected in preeclampsia. Because the kidney is highly susceptible to complement activation, we hypothesized that preeclampsia is associated with renal complement activation. We performed a nationwide search for renal autopsy material in the Netherlands using a computerized database (PALGA). Renal tissue was obtained from 11 women with preeclampsia, 25 pregnant controls, and 14 nonpregnant controls with hypertension. The samples were immunostained for C4d, C1q, mannose-binding lectin, properdin, C3d, C5b-9, IgA, IgG, and IgM. Preeclampsia was significantly associated with renal C4d-a stable marker of complement activation-and the classical pathway marker C1q. In addition, the prevalence of IgM was significantly higher in the kidneys of the preeclamptic women. No other complement markers studied differed between the groups. Our findings in human samples were validated using a soluble fms-like tyrosine kinase 1 mouse model of preeclampsia. The kidneys in the soluble fms-like tyrosine kinase 1-injected mice had significantly more C4 deposits than the control mice. The association between preeclampsia and renal C4d, C1q, and IgM levels suggests that the classical complement pathway is involved in the renal injury in preeclampsia. Moreover, our finding that soluble fms-like tyrosine kinase 1-injected mice develop excess C4 deposits indicates that angiogenic dysregulation may play a role in complement activation within the kidney. We suggest that inhibiting complement activation may be beneficial for preventing the renal manifestations of preeclampsia.
Assuntos
Complemento C4b/análise , Via Clássica do Complemento , Glomérulos Renais/imunologia , Fragmentos de Peptídeos/análise , Pré-Eclâmpsia/imunologia , Adulto , Animais , Biomarcadores , Doença Crônica , Fator D do Complemento/análise , Proteínas do Sistema Complemento/análise , Modelos Animais de Doenças , Feminino , Humanos , Hipertensão/imunologia , Hipertensão/patologia , Imunoglobulinas/análise , Glomérulos Renais/química , Lectinas de Ligação a Manose/análise , Camundongos , Países Baixos/epidemiologia , Pré-Eclâmpsia/mortalidade , Pré-Eclâmpsia/patologia , Gravidez , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/toxicidadeRESUMO
Preeclampsia is associated with increased levels of the circulating antiangiogenic factor sFlt-1 and with an excessive shedding of placenta-derived multinucleated syncytial aggregates into the maternal circulation. However, it remains unclear whether these aggregates are transcriptionally active in the maternal organs and can, therefore, contribute to the systemic manifestations of preeclampsia. In this study, we measured placental soluble fms-like tyrosine kinase-1 (sFlt-1) mRNA levels in preeclamptic- and control placentas and performed RNA in situ hybridization to localize the main placental expression site of sFlt-1 mRNA. Because the maternal lung is the first capillary bed that circulating syncytial aggregates traverse, we studied the presence and persistence of placental material in lungs of preeclamptic and control subjects. To confirm the placental origin of these aggregates in maternal lungs, immunohistochemistry for the placenta-specific marker hCG (human chorionic ghonadotropin) and Y chromosome in situ hybridization were performed. Using human placental tissue, we found that syncytial knots are the principal site of expression of the antiangiogenic factor sFlt-1. In addition, autopsy material obtained from women with preeclampsia (n=9) showed significantly more placenta-derived syncytial aggregates in the lungs than in control subjects (n=26). Importantly, these aggregates still contained the antiangiogenic factor sFlt-1 after their entrapment in the maternal lungs. The current study confirms the important role of syncytial knots in placental sFlt-1 mRNA production. In addition, it shows a significant association between preeclampsia and larger quantities of sFlt-1 containing syncytial aggregates in maternal lungs, suggesting that the transfer of syncytial aggregates to the maternal compartment may contribute to the systemic endothelial dysfunction that characterizes preeclampsia.
Assuntos
Pulmão/metabolismo , Placenta/metabolismo , Pré-Eclâmpsia/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Adulto , Gonadotropina Coriônica/metabolismo , Cromossomos Humanos Y/metabolismo , Feminino , Humanos , Gravidez , RNA Mensageiro/metabolismo , Transcrição Gênica , Trofoblastos/metabolismo , Útero/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genéticaAssuntos
Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/diagnóstico , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/tratamento farmacológico , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/complicações , Anticorpos Anticitoplasma de Neutrófilos/análise , Diagnóstico Diferencial , Humanos , Medicina , PrognósticoRESUMO
OBJECTIVE: The 5-5 homozygous CNDP1 (carnosinase) genotype is associated with a reduced risk of diabetic nephropathy. We investigated whether this association is sex specific and independent of susceptibility for type 2 diabetes. RESEARCH DESIGN AND METHODS: Three separate groups of 114, 90, and 66 patients with type 2 diabetes and diabetic nephropathy were included in this study and compared with 93 patients with type 2 diabetes for >15 years without diabetic nephropathy and 472 population control subjects. The diabetes control group was used to determine an association in the three patient groups separately, and the population control group was used to estimate the genotype risk [odds ratio (CI)] for the population in a pooled analysis. The population control subjects were also compared with 562 patients with type 2 diabetes without diabetic nephropathy to determine whether the association was independent of type 2 diabetes. The CNDP1 genotype was determined by fragment analysis after PCR amplification. RESULTS: The frequency of the 5-5 homozygous genotype was 28, 36, and 41% in the three diabetic nephropathy patient groups and 43 and 42% in the diabetic and population control subjects, respectively. The 5-5 homozygous genotype occurred significantly less frequently in women in all three patient groups compared with diabetic control subjects. The genotype risk for the population was estimated to be 0.5 (0.30-0.68) in women and 1.2 (0.77-1.69) in men. The 562 patients with type 2 diabetes without diabetic nephropathy did not differ from the general population (P = 0.23). CONCLUSIONS: This study suggests that the association between the CNDP1 gene and diabetic nephropathy is sex specific and independent of susceptibility for type 2 diabetes.
Assuntos
Diabetes Mellitus Tipo 2/genética , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/prevenção & controle , Dipeptidases/genética , Idade de Início , Idoso , Feminino , Frequência do Gene , Predisposição Genética para Doença , Genótipo , Homozigoto , Humanos , Leucina , Masculino , Pessoa de Meia-Idade , Valores de Referência , Sequências Repetitivas de Aminoácidos/genética , Caracteres Sexuais , População Branca/genéticaRESUMO
OBJECTIVE: In patients with systemic lupus erythematosus (SLE) and lupus nephritis, the presence of antiphospholipid antibodies (aPL) is considered to be an indication of increased risk of thrombotic microangiopathy, a serious complication of SLE. Previous studies have demonstrated a critical role for activation of the classical pathway of complement that leads to thrombotic injury in the presence of aPL. This study was undertaken to investigate whether C4d deposition in lupus nephritis is related to circulating aPL and the presence of renal microthrombi. METHODS: Deposition patterns of C4d in 44 renal biopsy samples obtained from 38 patients with biopsy-proven lupus nephritis were determined by staining with a polyclonal anti-C4d antibody. A phosphotungstic acid-hematoxylin stain was used to identify fibrin microthrombi. Clinical data (serum creatinine levels and presence or absence of aPL) were obtained and correlated with findings in the renal biopsy specimens. Patients were categorized as having aPL (n = 20) or not having aPL (n = 18). RESULTS: A strong relationship between the intensity of glomerular C4d staining and the presence of microthrombi was found (P < 0.002). Intense glomerular C4d deposition was present in 7 of 8 biopsy samples showing renal microthrombi. Neither C4d deposition nor the presence of microthrombi was correlated with aPL status. CONCLUSION: Our findings suggest that activation of the classical pathway of complement plays a pathogenic role in the development of renal tissue injury leading to thrombosis, irrespective of the type of circulating antibodies present. Immunodetection of glomerular C4d deposition in renal biopsy samples could be a convenient method of identifying patients at risk of thrombotic microangiopathy.
Assuntos
Complemento C4b/metabolismo , Glomérulos Renais/metabolismo , Nefrite Lúpica/diagnóstico , Fragmentos de Peptídeos/metabolismo , Trombose/diagnóstico , Doenças Vasculares/diagnóstico , Adolescente , Adulto , Idoso , Anticorpos Antifosfolipídeos/sangue , Biomarcadores/metabolismo , Biópsia , Complemento C1q/metabolismo , Complemento C3/metabolismo , Feminino , Humanos , Imunoglobulina A/metabolismo , Imunoglobulina G/metabolismo , Imunoglobulina M/metabolismo , Glomérulos Renais/patologia , Nefrite Lúpica/metabolismo , Nefrite Lúpica/patologia , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Trombose/metabolismo , Trombose/patologia , Doenças Vasculares/metabolismo , Doenças Vasculares/patologiaRESUMO
To evaluate changes during the development of proteinuria, podocyte morphology and protein expression were evaluated in spontaneously proteinuric, Dahl salt-sensitive (Dahl SS) rats. Dahl SS rats on a low-salt diet were compared with spontaneously hypertensive rats (SHR) at age 2, 4, 6, 8, and 10 weeks. Blood pressure, urinary protein excretion, urinary albumin excretion, and podocyte morphology were evaluated. In addition, the expression of 11 podocyte-related proteins was determined by analyzing protein and mRNA levels. In Dahl SS rats, proteinuria became evident around week 5, increasing thereafter. SHR rats remained non-proteinuric. Dahl SS rats showed widespread foot process effacement at 10 weeks. At < or =8 weeks, expression and distribution of the podocyte proteins was similar between the two strains, except for the protein podoplanin. At 4 weeks, podoplanin began decreasing in the glomeruli of Dahl SS rats in a focal and segmental fashion. Podoplanin loss increased progressively and correlated with albuminuria (r = 0.8, P < 0.001). Double labeling experiments revealed increased expression of the podocyte stress marker desmin in glomerular areas where podoplanin was lost. Dahl SS rats did not show podoplanin gene mutations or decreased mRNA expression. Thus, podocyte morphology and the expression and distribution of most podocyte-specific proteins were normal in young Dahl SS rats, despite marked proteinuria. Our study suggests that decreased expression of podoplanin plays a role in the decrease of glomerular permselectivity.
Assuntos
Glicoproteínas de Membrana/biossíntese , Podócitos/patologia , Proteinúria/metabolismo , Fatores Etários , Animais , Pressão Sanguínea , Contagem de Células , Desmina/metabolismo , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Masculino , Glicoproteínas de Membrana/genética , Permeabilidade , Podócitos/metabolismo , Proteinúria/patologia , Proteinúria/fisiopatologia , RNA Mensageiro/biossíntese , Ratos , Ratos Endogâmicos Dahl , Ratos Endogâmicos SHRRESUMO
BACKGROUND: A major drawback of allogeneic hepatocyte transplantation is the lack of sustained survival of the transplanted cells in the recipient liver parenchyma. The purpose of this study was to determine the effect of the presence or absence of hepatic extracellular matrix (ECM) molecules on hepatocyte survival and function following hepatocyte isolation for transplantation purposes, and the role of beta1-integrin molecules therein. METHODS: Hepatocytes, either untreated or treated with anti-beta1 integrin antibodies or RGD peptides, were seeded on wells precoated with collagen type I, type IV, laminin, fibronectin or polyhydroxyethylmehacrylate. The extent of attachment and apoptosis was evaluated. RESULTS: When hepatocytes were added into wells precoated with either fibronectin, or collagen type IV, rapid spreading and prolonged survival occurred, in contrast to hepatocytes that were seeded in wells precoated with collagen type I or polyhydroxyethylmehacrylate. Pretreatment of the cells with anti-beta1-integrin antibodies resulted in reduction of cell attachment to laminin, fibronectin, collagen I, and collagen IV. Synthetic RGD (arginine-glycine-aspartate)-peptides and anti-beta1 antibodies inhibited apoptosis of cultured hepatocytes. CONCLUSIONS: Our findings indicate that embedding of hepatocytes within their normal liver ECM surroundings maintains their survival. When detached from their natural surrounding hepatocytes enter into apoptosis, unless treated with anti-beta1-integrin antibodies or RGD peptides. This knowledge will allow improvement of hepatocyte transplantation efficiency.
Assuntos
Matriz Extracelular/fisiologia , Hepatócitos/fisiologia , Integrina beta1/fisiologia , Fígado/metabolismo , Animais , Anoikis , Anticorpos Monoclonais/farmacologia , Apoptose/efeitos dos fármacos , Caspase 3 , Caspases/metabolismo , Adesão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Colágeno Tipo IV/farmacologia , Fragmentação do DNA , Eletroforese , Feminino , Fibronectinas/farmacologia , Imuno-Histoquímica , Integrina beta1/imunologia , Oligopeptídeos/farmacologia , Ratos , Ratos Endogâmicos BNRESUMO
The goal of this study was to improve a procedure for the extraction and storage of RNA from minute quantities of human renal tissue in clinical practice, using kidney biopsies and cadaveric donor kidneys unsuitable for transplantation. Collagen alpha1(IV) mRNA was analyzed as a measure for RNA integrity. The results show that at least 3 h may pass between microdissecting the renal tissue and the onset of cDNA synthesis without degradation of the glomerular mRNA. To extract the glomerular mRNA, microdissected glomeruli were incubated in a permeabilization solution. Treating glomeruli with collagenase IV before permeabilization had a deteriorating effect on the mRNA yield. The addition of reverse transcription mixture to the permeabilization solution in the presence of the glomeruli resulted in the highest cDNA yields. Storage of glomerular tissue in the presence of Nonidet P-40-based buffer for 1 wk at -70 degrees C did not significantly affect the mRNA, but storage for 2 or 4 wk resulted in deterioration of the mRNA by approximately 40 and 95%, respectively. Furthermore, three methods for total RNA isolation from microdissected interstitial tissue were compared. An approximately 2.5 times higher yield of collagen alpha1(IV) mRNA was obtained with silica gel-based membrane spin technology than with a guanidine isothiocyanate/phenol chloroform or a lithium chloride/phenol chloroform method. Finally, this study shows for the first time reliable detection of collagen alpha1(IV) mRNA in biopsies that had been frozen for at least 10 yr at -70 degrees C. These experiments have helped to improve a procedure for the processing of glomerular and interstitial tissue acquired from human kidney biopsies for mRNA analysis. This method is suitable for implementation in routine clinical practice.