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1.
Eur Surg Res ; 58(5-6): 193-203, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28433992

RESUMO

BACKGROUND: The pig is one of the most frequently used large animal models for biomedical research, especially in the field of translational research and surgical models. While standard livestock breeds are used in short-term and acute studies, minipig breeds are the preferred breeds in long-term and chronic studies due to their limited growth and body weight. OBJECTIVE: In consideration of the 3R principle (refinement, reduction, replacement) and the increasing demand, the aim of this study was to generate a new, robust, non-specific-pathogen-free minipig breed, the Aachen minipig. METHODS: Phenotype, genotype, and hematological as well as clinical chemistry parameters were characterized, and reference values of the Aachen minipig were generated and compared to the values in the commonly used Göttingen minipig. Organ weights of the heart, kidney, liver, lung, spleen, and brain were determined using a laboratory balance. Blood samples were collected for hematology and clinical chemistry. Assessment of genetic diversity was performed by microsatellite markers. Nasal swabs were collected from 11 individual minipigs representing 6 races for DNA extraction. DNA was quantified and the identity and origin of the Aachen minipigs at the genomic level was determined by microsatellites. RESULTS: The Aachen minipig established here is based on the Mini-LEWE breed and consists of the Vietnamese potbelly pig, the Schwäbisch Hällisch Landpig, the German Landrace, and the Minnesota minipig. Relative organ weights (lung, heart, kidneys, brain), hematology (hemoglobin, hematocrit, platelet count, mean corpuscular hemoglobin concentration, segmented neutrophils, lymphocytes, eosinophils, basophils), and clinical chemistry parameters (sodium, calcium, chloride, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transferase, lactate dehydrogenase, triglycerides, blood urea nitrogen, creatinine, total bilirubin, total protein, creatine kinase) of the Aachen minipigs and the Göttingen minipigs were not significantly different. Significant differences where only seen in relative organ weights (liver, spleen), hematology (red blood cell count, mean corpuscular volume, mean corpuscular hemoglobin, white blood cell count, banded neutrophils, monocytes), and clinical chemistry parameters (inorganic phosphorus, potassium, glucose, cholesterol, albumin, amylase). CONCLUSION: The Aachen minipig is a suitable model for research due to its similarity to other minipig breeds, especially the Göttingen minipig. The reference values established in this study may be used for the comparison of scientific data and encourage the use of the Aachen minipig as an animal model for biomedical research.


Assuntos
Modelos Animais , Porco Miniatura/fisiologia , Animais , Suínos
2.
Kidney Int ; 84(5): 920-30, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23677246

RESUMO

Podocytes are highly specialized epithelial cells with complex actin cytoskeletal architecture crucial for maintenance of the glomerular filtration barrier. The mammalian Rho GTPases Rac1 and Cdc42 are molecular switches that control many cellular processes, but are best known for their roles in the regulation of actin cytoskeleton dynamics. Here, we employed podocyte-specific Cre-lox technology and found that mice with deletion of Rac1 display normal podocyte morphology without glomerular dysfunction well into adulthood. Using the protamine sulfate model of acute podocyte injury, podocyte-specific deletion of Rac1 prevented foot process effacement. In a long-term model of chronic hypertensive glomerular damage, however, loss of Rac1 led to an exacerbation of albuminuria and glomerulosclerosis. In contrast, mice with podocyte-specific deletion of Cdc42 had severe proteinuria, podocyte foot process effacement, and glomerulosclerosis beginning as early as 10 days of age. In addition, slit diaphragm proteins nephrin and podocin were redistributed, and cofilin was dephosphorylated. Cdc42 is necessary for the maintenance of podocyte structure and function, but Rac1 is entirely dispensable in physiological steady state. However, Rac1 has either beneficial or deleterious effects depending on the context of podocyte impairment. Thus, our study highlights the divergent roles of Rac1 and Cdc42 function in podocyte maintenance and injury.


Assuntos
Injúria Renal Aguda/enzimologia , Neuropeptídeos/metabolismo , Podócitos/enzimologia , Insuficiência Renal/enzimologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Fatores de Despolimerização de Actina/metabolismo , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/genética , Injúria Renal Aguda/patologia , Albuminúria/metabolismo , Animais , Forma Celular , Acetato de Desoxicorticosterona , Modelos Animais de Doenças , Genótipo , Hipertensão/induzido quimicamente , Hipertensão/enzimologia , Hipertensão/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nefrectomia , Neuropeptídeos/deficiência , Neuropeptídeos/genética , Fenótipo , Fosforilação , Podócitos/patologia , Protaminas , Insuficiência Renal/etiologia , Insuficiência Renal/genética , Insuficiência Renal/patologia , Transdução de Sinais , Fatores de Tempo , Proteína cdc42 de Ligação ao GTP/deficiência , Proteína cdc42 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/deficiência , Proteínas rac1 de Ligação ao GTP/genética
3.
PLoS One ; 5(10): e13451, 2010 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-20976140

RESUMO

Proteinuria is the most important predictor of outcome in glomerulonephritis and experimental data suggest that the tubular cell response to proteinuria is an important determinant of progressive fibrosis in the kidney. However, it is unclear whether proteinuria is a marker of disease severity or has a direct effect on tubular cells in the kidneys of patients with glomerulonephritis. Accordingly we studied an in vitro model of proteinuria, and identified 231 "albumin-regulated genes" differentially expressed by primary human kidney tubular epithelial cells exposed to albumin. We translated these findings to human disease by studying mRNA levels of these genes in the tubulo-interstitial compartment of kidney biopsies from patients with IgA nephropathy using microarrays. Biopsies from patients with IgAN (n = 25) could be distinguished from those of control subjects (n = 6) based solely upon the expression of these 231 "albumin-regulated genes." The expression of an 11-transcript subset related to the degree of proteinuria, and this 11-mRNA subset was also sufficient to distinguish biopsies of subjects with IgAN from control biopsies. We tested if these findings could be extrapolated to other proteinuric diseases beyond IgAN and found that all forms of primary glomerulonephritis (n = 33) can be distinguished from controls (n = 21) based solely on the expression levels of these 11 genes derived from our in vitro proteinuria model. Pathway analysis suggests common regulatory elements shared by these 11 transcripts. In conclusion, we have identified an albumin-regulated 11-gene signature shared between all forms of primary glomerulonephritis. Our findings support the hypothesis that albuminuria may directly promote injury in the tubulo-interstitial compartment of the kidney in patients with glomerulonephritis.


Assuntos
Glomerulonefrite/genética , Proteinúria/genética , Albuminas/genética , Biópsia , Perfilação da Expressão Gênica , Glomerulonefrite/patologia , Humanos , Rim/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Proteinúria/patologia
4.
Am J Pathol ; 174(6): 2073-85, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19465643

RESUMO

Although chronic kidney disease (CKD) is common, only a fraction of CKD patients progress to end-stage renal disease. Molecular predictors to stratify CKD populations according to their risk of progression remain undiscovered. Here we applied transcriptional profiling of kidneys from transforming growth factor-beta1 transgenic (Tg) mice, characterized by heterogeneity of kidney disease progression, to identify 43 genes that discriminate kidneys by severity of glomerular apoptosis before the onset of tubulointerstitial fibrosis in 2-week-old animals. Among the genes examined, 19 showed significant correlation between mRNA expression in uninephrectomized left kidneys at 2 weeks of age and renal disease severity in right kidneys of Tg mice at 4 weeks of age. Gene expression profiles of human orthologs of the 43 genes in kidney biopsies were highly significantly related (R(2) = 0.53; P < 0.001) to the estimated glomerular filtration rates in patients with CKD stages I to V, and discriminated groups of CKD stages I/II and III/IV/V with positive and negative predictive values of 0.8 and 0.83, respectively. Protein expression patterns for selected genes were successfully validated by immunohistochemistry in kidneys of Tg mice and kidney biopsies of patients with IgA nephropathy and CKD stages I to V, respectively. In conclusion, we developed novel mRNA and protein expression signatures that predict progressive renal fibrosis in mice and may be useful molecular predictors of CKD progression in humans.


Assuntos
Perfilação da Expressão Gênica , Nefropatias/genética , Nefropatias/patologia , Animais , Análise por Conglomerados , Progressão da Doença , Expressão Gênica , Humanos , Imuno-Histoquímica , Nefropatias/metabolismo , Camundongos , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Risco , Transcrição Gênica , Fator de Crescimento Transformador beta1/genética
5.
Transplantation ; 87(2): 222-6, 2009 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-19155976

RESUMO

Gene expression profiling has emerged as a powerful strategy to define transcriptional mechanism activated in organ transplantation. We performed a pilot feasibility study of mRNA-based pancreas transplant biopsy stratification. The mRNAs expression of 32 genes, observed in renal transplant dysfunction, and 10 pancreas-specific genes were evaluated in 26 pancreas transplant biopsy specimens by quantitative real-time polymerase chain reaction using TaqMan Low Density Array technology. Unsupervised 2D hierarchical clustering segregated the biopsies in two main cluster branches, A and B. Six of seven patients (85.7%) in cluster A and 6 of 19 (31.6%) in cluster B retained functioning pancreas allograft. CD20/MS4A1 mRNA and protein, in addition to CD 3 protein, were detected in four specimens in cluster B. Three of those four pancreas transplants were subsequently lost. Our study demonstrates the potential association of gene expression with clinical outcome of pancreas transplants and justifies further studies in an independent cohort.


Assuntos
Perfilação da Expressão Gênica , Rejeição de Enxerto/genética , Sobrevivência de Enxerto/genética , Transplante de Pâncreas , Pâncreas/química , Pâncreas/cirurgia , RNA Mensageiro/análise , Adulto , Biópsia , Análise por Conglomerados , Estudos de Viabilidade , Perfilação da Expressão Gênica/métodos , Rejeição de Enxerto/imunologia , Sobrevivência de Enxerto/imunologia , Humanos , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Pâncreas/imunologia , Pâncreas/patologia , Projetos Piloto , Reação em Cadeia da Polimerase , Valor Preditivo dos Testes , Estudos Retrospectivos , Fatores de Tempo , Transplante Homólogo , Resultado do Tratamento
6.
Diabetes ; 58(2): 469-77, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19017763

RESUMO

OBJECTIVE: Glomerular mesangial expansion and podocyte loss are important early features of diabetic nephropathy, whereas tubulointerstitial injury and fibrosis are critical for progression of diabetic nephropathy to kidney failure. Therefore, we analyzed the expression of genes in glomeruli and tubulointerstitium in kidney biopsies from diabetic nephropathy patients to identify pathways that may be activated in humans but not in murine models of diabetic nephropathy that fail to progress to glomerulosclerosis, tubulointerstitial fibrosis, and kidney failure. RESEARCH DESIGN AND METHODS: Kidney biopsies were obtained from 74 patients (control subjects, early and progressive type 2 diabetic nephropathy). Glomerular and tubulointerstitial mRNAs were microarrayed, followed by bioinformatics analyses. Gene expression changes were confirmed by real-time RT-PCR and immunohistological staining. Samples from db/db C57BLKS and streptozotocin-induced DBA/2J mice, commonly studied murine models of diabetic nephropathy, were analyzed. RESULTS: In human glomeruli and tubulointerstitial samples, the Janus kinase (Jak)-signal transducer and activator of transcription (Stat) pathway was highly and significantly regulated. Jak-1, -2, and -3 as well as Stat-1 and -3 were expressed at higher levels in patients with diabetic nephropathy than in control subjects. The estimated glomerular filtration rate significantly correlated with tubulointerstitial Jak-1, -2, and -3 and Stat-1 expression (R(2) = 0.30-0.44). Immunohistochemistry found strong Jak-2 staining in glomerular and tubulointerstitial compartments in diabetic nephropathy compared with control subjects. In contrast, there was little or no increase in expression of Jak/Stat genes in the db/db C57BLKS or diabetic DBA/2J mice. CONCLUSIONS: These data suggest a direct relationship between tubulointerstitial Jak/Stat expression and progression of kidney failure in patients with type 2 diabetic nephropathy and distinguish progressive human diabetic nephropathy from nonprogressive murine diabetic nephropathy.


Assuntos
Nefropatias Diabéticas/genética , Nefropatias Diabéticas/metabolismo , Expressão Gênica , Adulto , Animais , Western Blotting , Feminino , Humanos , Imuno-Histoquímica , Janus Quinase 1/genética , Janus Quinase 1/metabolismo , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Janus Quinase 3/genética , Janus Quinase 3/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo
7.
J Am Soc Nephrol ; 19(8): 1491-9, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18385421

RESUMO

Mutations in the NPHS2 gene, which encodes podocin, are responsible for some cases of sporadic and familial autosomal recessive steroid-resistant nephrotic syndrome. Inter- and intrafamilial variability in the progression of renal disease among patients bearing NPHS2 mutations suggests a potential role for modifier genes. Using a mouse model in which the podocin gene is constitutively inactivated, we sought to identify genetic determinants of the development and progression of renal disease as a result of the nephrotic syndrome. We report that the evolution of renal disease as a result of nephrotic syndrome in Nphs2-null mice depends on genetic background. Furthermore, the maternal environment significantly interacts with genetic determinants to modify survival and progression of renal disease. Quantitative trait locus mapping suggested that these genetic determinants may be encoded for by genes on the distal end of chromosome 3, which are linked to proteinuria, and on the distal end of chromosome 7, which are linked to a composite trait of urea, creatinine, and potassium. These loci demonstrate epistatic interactions with other chromosomal regions, highlighting the complex genetics of renal disease progression. In summary, constitutive inactivation of podocin models the complex interactions between maternal and genetically determined factors on the progression of renal disease as a result of nephrotic syndrome in mice.


Assuntos
Meio Ambiente , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Síndrome Nefrótica/genética , Animais , Progressão da Doença , Feminino , Genômica , Rim/patologia , Masculino , Camundongos , Síndrome Nefrótica/patologia , Fenótipo
8.
J Am Soc Nephrol ; 19(5): 904-14, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18287563

RESUMO

Apoptotic cell death contributes to diabetic nephropathy (DN), but its role is not well understood. The tubulointerstitium from DN biopsy specimens was microdissected, and expression profiles of genes related to apoptosis were analyzed. A total of 112 (25%) of 455 cell death-related genes were found to be significantly differentially regulated. Among those that showed the greatest changes in regulation were two death receptors, OPG (the gene encoding osteoprotegerin) and Fas, and the death ligand TRAIL. Glomerular and proximal tubular TRAIL expression, assessed by immunohistochemistry, was higher in DN kidneys than controls and was associated with clinical and histologic severity of disease. In vitro, proinflammatory cytokines but not glucose alone regulated TRAIL expression in the human proximal tubular cell line HK-2. TRAIL induced tubular cell apoptosis in a dosage-dependant manner, an effect that was more marked in the presence of high levels of glucose and proinflammatory cytokines. TRAIL also activated NF-kappaB, and inhibition of NF-kappaB sensitized cells to TRAIL-induced apoptosis. It is proposed that TRAIL-induced cell death could play an important role in the progression of human DN.


Assuntos
Apoptose/fisiologia , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/fisiopatologia , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Apoptose/efeitos dos fármacos , Biópsia , Linhagem Celular , Sobrevivência Celular/fisiologia , Nefropatias Diabéticas/patologia , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/fisiologia , Feminino , Regulação da Expressão Gênica , Glucose/farmacologia , Humanos , Imuno-Histoquímica , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/fisiologia , Ligantes , Masculino , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Osteoprotegerina/genética , Osteoprotegerina/metabolismo , RNA Mensageiro/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo
9.
BMC Cancer ; 8: 23, 2008 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-18215331

RESUMO

BACKGROUND: Osteosarcoma is the most frequent bone tumor in childhood and adolescence. Patients with primary metastatic disease have a poor prognosis. It is therefore important to better characterize the biology of this tumor to define new prognostic markers or therapeutic targets for tailored therapy. Chemokines and their receptors have been shown to be involved in the development and progression of malignant tumors. They are thought to be active participants in the biology of osteosarcoma. The function of specific chemokines and their receptors is strongly associated with the biological context and microenvironment of their expression. In this report we characterized the expression of a series of chemokine receptors in the complex environment that defines osteosarcoma. METHODS: The overall level of chemokine receptor mRNA expression was determined using TaqMan RT-PCR of microdissected archival patient biopsy samples. Expression was then verified at the protein level by immunohistochemistry using a series of receptor specific antibody reagents to elucidate the cellular association of expression. RESULTS: Expression at the RNA level was found for most of the tested receptors. CCR1 expression was found on infiltrating mononuclear and polynuclear giant cells in the tumor. Cells associated with the lining of intratumoral vessels were shown to express CCR4. Infiltrating mononuclear cells and tumor cells both showed expression of the receptor CCR5, while CCR7 was predominantly expressed by the mononuclear infiltrate. CCR10 was only very rarely detected in few scattered infiltrating cells. CONCLUSION: Our data elucidate for the first time the cellular context of chemokine receptor expression in osteosarcoma. This is an important issue for better understanding potential chemokine/chemokine receptor function in the complex biologic processes that underlie the development and progression of osteosarcoma. Our data support the suggested involvement of chemokines and their receptors in diverse aspects of the biology of osteosarcoma, but also contradict aspects of previous reports describing the expression of these receptors in this tumor.


Assuntos
Neoplasias Ósseas/metabolismo , Regulação Neoplásica da Expressão Gênica/fisiologia , Osteossarcoma/metabolismo , Receptores de Quimiocinas/biossíntese , Adolescente , Adulto , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Criança , Feminino , Humanos , Masculino , Osteossarcoma/genética , Osteossarcoma/patologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores de Quimiocinas/genética , Células-Tronco/citologia , Células-Tronco/metabolismo
10.
Nat Med ; 14(1): 55-63, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18084301

RESUMO

Podocyte dysfunction, represented by foot process effacement and proteinuria, is often the starting point for progressive kidney disease. Therapies aimed at the cellular level of the disease are currently not available. Here we show that induction of urokinase receptor (uPAR) signaling in podocytes leads to foot process effacement and urinary protein loss via a mechanism that includes lipid-dependent activation of alphavbeta3 integrin. Mice lacking uPAR (Plaur-/-) are protected from lipopolysaccharide (LPS)-mediated proteinuria but develop disease after expression of a constitutively active beta3 integrin. Gene transfer studies reveal a prerequisite for uPAR expression in podocytes, but not in endothelial cells, for the development of LPS-mediated proteinuria. Mechanistically, uPAR is required to activate alphavbeta3 integrin in podocytes, promoting cell motility and activation of the small GTPases Cdc42 and Rac1. Blockade of alphavbeta3 integrin reduces podocyte motility in vitro and lowers proteinuria in mice. Our findings show a physiological role for uPAR signaling in the regulation of kidney permeability.


Assuntos
Regulação da Expressão Gênica , Rim/metabolismo , Podócitos/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Movimento Celular , Técnicas de Transferência de Genes , Humanos , Integrina alfaVbeta3/metabolismo , Rim/patologia , Lipopolissacarídeos/metabolismo , Microdomínios da Membrana , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Transdução de Sinais
11.
Biol Chem ; 388(5): 497-506, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17516845

RESUMO

Mesangial cells are thought to be important mediators of glomerular inflammation and fibrosis. Studies have established a direct role for nitric oxide (NO) in the regulation of gene expression in mesangial cells. Representational difference analysis was used to investigate changes in gene expression elicited by the treatment of S-nitroso-L-glutathione in rat mesangial cells. Seven upregulated and 11 downregulated genes were identified. Four out of 11 downregulated genes (connective tissue growth factor, thrombospondin-1, collagen type I alpha1 and collagen type I alpha2) are known to be linked to inflammation and fibrosis. Results were verified across species in mesangial cells treated with a series of NO donors using Northern blot analysis, quantitative real-time PCR and protein analysis methods. Induction of endogenous NO production by cytokine stimulation also triggered regulation of the genes. One example gene, connective tissue growth factor, was studied at the promoter level. Promoter-reporter gene studies in mesangial cells demonstrated that NO acts at the transcriptional level to suppress gene expression. Our results reveal a complex role of NO in regulating gene expression in mesangial cells and suggest an antifibrotic potential for NO.


Assuntos
Regulação para Baixo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Células Mesangiais/metabolismo , Células Mesangiais/patologia , Óxido Nítrico/metabolismo , Animais , Biglicano , Biopterinas/análogos & derivados , Biopterinas/farmacologia , Células Cultivadas , Colágeno/genética , Fator de Crescimento do Tecido Conjuntivo , Ativação Enzimática/efeitos dos fármacos , Fibrose/genética , Fibrose/metabolismo , Fibrose/patologia , Proteínas Imediatamente Precoces/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Interferon gama/farmacologia , Células Mesangiais/efeitos dos fármacos , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/metabolismo , Regiões Promotoras Genéticas/genética , Proteoglicanas/genética , Estabilidade de RNA , RNA Mensageiro/genética , Ratos , Trombospondina 1/metabolismo
12.
J Am Soc Nephrol ; 18(6): 1765-76, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17475821

RESUMO

Diabetic nephropathy (DN) is a frequent complication in patients with diabetes. Although the majority of DN models and human studies have focused on glomeruli, tubulointerstitial damage is a major feature of DN and an important predictor of renal dysfunction. This study sought to investigate molecular markers of pathogenic pathways in the renal interstitium of patients with DN. Microdissected tubulointerstitial compartments from biopsies with established DN and control kidneys were subjected to expression profiling. Analysis of candidate genes, potentially involved in DN on the basis of common hypotheses, identified 49 genes with significantly altered expression levels in established DN in comparison with controls. In contrast to some rodent models, the growth factors vascular endothelial growth factor A (VEGF-A) and epidermal growth factor (EGF) showed a decrease in mRNA expression in DN. This was validated on an independent cohort of patients with DN by real-time reverse transcriptase-PCR. Immunohistochemical staining for VEGF-A and EGF also showed a reduced expression in DN. The decrease of renal VEGF-A expression was associated with a reduction in peritubular capillary densities shown by platelet-endothelial cell adhesion molecule-1/CD31 staining. Furthermore, a significant inverse correlation between VEGF-A and proteinuria, as well as EGF and proteinuria, and a positive correlation between VEGF-A and hypoxia-inducible factor-1alpha mRNA was found. Thus, in human DN, a decrease of VEGF-A, rather than the reported increase as described in some rodent models, may contribute to the progressive disease. These findings and the questions about rodent models in DN raise a note of caution regarding the proposal to inhibit VEGF-A to prevent progression of DN.


Assuntos
Nefropatias Diabéticas , Análise de Sequência com Séries de Oligonucleotídeos , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Adolescente , Adulto , Idoso , Biomarcadores/metabolismo , Biópsia , Capilares/patologia , Capilares/fisiologia , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Túbulos Renais/patologia , Túbulos Renais/fisiologia , Masculino , Pessoa de Meia-Idade , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Proteinúria/genética , Proteinúria/metabolismo , Proteinúria/patologia , RNA Mensageiro/metabolismo , Especificidade da Espécie
13.
J Am Soc Nephrol ; 18(1): 29-36, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17167110

RESUMO

Injury to podocytes and their slit diaphragms typically leads to marked proteinuria. Mutations in the TRPC6 gene that codes for a slit diaphragm-associated, cation-permeable ion channel have been shown recently to co-segregate with hereditary forms of progressive kidney failure. Herein is shown that induced expression of wild-type TRPC6 is a common feature of human proteinuric kidney diseases, with highest induction observed in membranous nephropathy. Cultured podocytes that are exposed to complement upregulate TRPC6 protein. Stimulation of receptor-operated channels in puromycin aminonucleoside-treated podocytes leads to increased calcium influx in a time- and dosage-dependent manner. Mechanistically, it is shown that TRPC6 is functionally connected to the podocyte actin cytoskeleton, which is rearranged upon overexpression of TRPC6. Transient in vivo gene delivery of TRPC6 into mice leads to expression of TRPC6 protein at the slit diaphragm and causes proteinuria. These studies suggest the involvement of TRPC6 in the pathology of nongenetic forms of proteinuric disease.


Assuntos
Nefropatias/metabolismo , Proteinúria/metabolismo , Canais de Cátion TRPC/biossíntese , Animais , Células Cultivadas , Expressão Gênica , Humanos , Nefropatias/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Podócitos/metabolismo , Proteinúria/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Canais de Cátion TRPC/genética , Canal de Cátion TRPC6 , Transfecção
14.
Diabetes ; 55(11): 2993-3003, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17065335

RESUMO

Diabetic nephropathy (DN) is the leading cause of end-stage renal failure and a major risk factor for cardiovascular mortality in diabetic patients. To evaluate the multiple pathogenetic factors implicated in DN, unbiased mRNA expression screening of tubulointerstitial compartments of human renal biopsies was combined with hypothesis-driven pathway analysis. Expression fingerprints obtained from biopsies with histological diagnosis of DN (n = 13) and from control subjects (pretransplant kidney donors [n = 7] and minimal change disease [n = 4]) allowed us to segregate the biopsies by disease state and stage by the specific expression signatures. Functional categorization showed regulation of genes linked to inflammation in progressive DN. Pathway mapping of nuclear factor-kappaB (NF-kappaB), a master transcriptional switch in inflammation, segregated progressive from mild DN and control subjects by showing upregulation of 54 of 138 known NF-kappaB targets. The promoter regions of regulated NF-kappaB targets were analyzed using ModelInspector, and the NF-kappaB module NFKB_IRFF_01 was found to be specifically enriched in progressive disease. Using this module, the induction of eight NFKB_IRFF_01-dependant genes was correctly predicted in progressive DN (B2M, CCL5/RANTES, CXCL10/IP10, EDN1, HLA-A, HLA-B, IFNB1, and VCAM1). The identification of a specific NF-kappaB promoter module activated in the inflammatory stress response of progressive DN has helped to characterize upstream pathways as potential targets for the treatment of progressive renal diseases such as DN.


Assuntos
Nefropatias Diabéticas/genética , Regulação da Expressão Gênica , NF-kappa B/genética , Transcrição Gênica , Biópsia , Cadáver , Nefropatias Diabéticas/patologia , Progressão da Doença , Humanos , Inflamação/genética , Inflamação/fisiopatologia , Rim/citologia , Rim/patologia , Doadores Vivos , Regiões Promotoras Genéticas , RNA Mensageiro/genética , Valores de Referência , Doadores de Tecidos
15.
Nephrol Dial Transplant ; 21(11): 3062-73, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16954173

RESUMO

BACKGROUND: How microbial infections exacerbate immune complex glomerulonephritis remains speculative. Toll-like receptors (TLRs) may be involved in this phenomenon, because TLRs have potent immunostimulatory functions when exposed to selected pathogen-associated molecules. METHODS: We addressed this issue by characterizing the expression of TLR1-9 in MRLlpr/lpr mice that spontaneously develop immune complex glomerulonephritis as part of a systemic lupus-like autoimmune syndrome. RESULTS: Five-week-old healthy MRLlpr/lpr mice expressed TLR3 mRNA in kidneys at comparable levels as in the spleen, while all other TLRs were expressed at low levels in the kidney. In 20-week-old nephritic MRLlpr/lpr mice, renal mRNA levels had increased for TLR1-9. Renal TLR mRNA originated at least in part from glomeruli as evidenced by real-time RT-PCR from laser capture microdissected glomeruli. Immunostaining for TLR3, TLR7 and TLR9 revealed their expression by F4/80-positive infiltrating macrophages in 20-week-old nephritic MRLlpr/lpr mice. In addition, TLR3 localized to glomerular mesangial cells. Cultured mesangial cells expressed TLR1-4 and TLR6, while murine macrophages expressed TLR1-9. TNF-alpha and IFN-gamma induced TLR2, TLR3 and TLR6 mRNA in mesangial cells, while they down-regulated TLR1-9 mRNA in macrophages. Stimulation of both cell types with ligands for TLR1-4, TLR5, TLR7 and TLR9 induced IL-6 production consistent with their respective TLR expression patterns. TNF-alpha and IFN-gamma enhanced ligand-induced IL-6 production in both cell types irrespective of their modulatory effect on respective TLR mRNA levels. CONCLUSION: Thus, cell-type-specific expression and regulation of TLRs may be involved in infection-associated exacerbation of immune complex glomerulonephritis of MRLlpr/lpr mice.


Assuntos
Doenças do Complexo Imune/metabolismo , Nefrite Lúpica/metabolismo , Receptores Toll-Like/biossíntese , Animais , Linhagem Celular , Doenças do Complexo Imune/imunologia , Doenças do Complexo Imune/patologia , Nefrite Lúpica/imunologia , Nefrite Lúpica/patologia , Camundongos , Camundongos Endogâmicos MRL lpr , Especificidade de Órgãos/genética , Especificidade de Órgãos/imunologia , RNA Mensageiro/biossíntese , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo
16.
Clin Exp Nephrol ; 10(2): 91-8, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16791393

RESUMO

The increase in progressive kidney disease, resulting in a constantly rising prevalence of endstage renal disease (ESRD), urgently warrants the development of more effective strategies to diagnose, prevent, and intervene in renal disease. Histological information obtained by renal biopsies (RBx) is a cornerstone of the current management of kidney disease. Renal tissue can provide critical information on the disease process not available by nontissue-based approaches. However, insight gained by conventional histopathology remains limited and additional strategies to define renal disease on a molecular level are required. The sequencing of the human genome, together with recent advances in genome-wide profiling techniques, has provided the framework for a comprehensive analysis of renal disease-associated transcriptional programs. In this review, strategies to apply these technological advances towards the analysis of RBx will be described, with special emphasis on their potential impact on clinical management, but also on their inherent limitations. Finally, an outlook towards the emerging proteomic studies of renal disease will be given.


Assuntos
Perfilação da Expressão Gênica , Nefropatias/genética , Antígenos CD20/genética , Biópsia , Bases de Dados de Ácidos Nucleicos , Europa (Continente) , Perfilação da Expressão Gênica/métodos , Biblioteca Gênica , Glomerulonefrite Membranosa/genética , Humanos , Nefropatias/metabolismo , Nefropatias/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Proteômica/tendências , Reação em Cadeia da Polimerase Via Transcriptase Reversa
17.
J Am Soc Nephrol ; 17(5): 1334-44, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16611717

RESUMO

Alterations in glomerular podocyte cell-cell and cell-matrix contacts are key events in progressive glomerular failure. Integrin-linked kinase (ILK) has been implicated in podocyte cell-matrix interaction and is induced in proteinuria. For evaluation of ILK function in vivo, mice with a Cre-mediated podocyte-specific ILK inactivation were generated. These mice seemed normal at birth but developed progressive focal segmental glomerulosclerosis and died in terminal renal failure. The first ultrastructural lesions that are seen at onset of albuminuria are glomerular basement membrane (GBM) alterations with a significant increase in true harmonic mean GBM thickness. Podocyte foot process effacement and loss of slit diaphragm followed with progression to unselective proteinuria. No significant reduction of slit membrane molecules (podocin and nephrin), key GBM components (fibronectin, laminins, and collagen IV isoforms), or podocyte integrins could be observed at onset of proteinuria. However, alpha3-integrins were relocalized into a granular pattern along the GBM, consistent with altered integrin-mediated matrix assembly in ILK-deficient podocytes. As the increased GBM thickness precedes structural podocyte lesions and key components of the GBM were expressed at comparable levels to controls, these data suggest an essential role of ILK for the close interconnection of GBM structure and podocyte function.


Assuntos
Membrana Celular/metabolismo , Membrana Celular/patologia , Glomerulosclerose Segmentar e Focal/metabolismo , Glomerulosclerose Segmentar e Focal/patologia , Podócitos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Células Cultivadas , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas Serina-Treonina Quinases/genética , Análise de Sobrevida , Taxa de Sobrevida
18.
Proc Natl Acad Sci U S A ; 103(15): 5682-7, 2006 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-16581909

RESUMO

Shared transcription factor binding sites that are conserved in distance and orientation help control the expression of gene products that act together in the same biological context. New bioinformatics approaches allow the rapid characterization of shared promoter structures and can be used to find novel interacting molecules. Here, these principles are demonstrated by using molecules linked to the unique functional unit of the glomerular slit diaphragm. An evolutionarily conserved promoter model was generated by comparative genomics in the proximal promoter regions of the slit diaphragm-associated molecule nephrin. Phylogenetic promoter fingerprints of known elements of the slit diaphragm complex identified the nephrin model in the promoter region of zonula occludens-1 (ZO-1). Genome-wide scans using this promoter model effectively predicted a previously unrecognized slit diaphragm molecule, cadherin-5. Nephrin, ZO-1, and cadherin-5 mRNA showed stringent coexpression across a diverse set of human glomerular diseases. Comparative promoter analysis can identify regulatory pathways at work in tissue homeostasis and disease processes.


Assuntos
Proteínas de Membrana/genética , Podócitos/fisiologia , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Animais , Antígenos CD , Caderinas/genética , Sequência Conservada , Evolução Molecular , Regulação da Expressão Gênica , Humanos , Junções Intercelulares/genética , Camundongos , Modelos Genéticos , Fosfoproteínas/genética , Ratos , Vertebrados , Proteína da Zônula de Oclusão-1
19.
Curr Opin Nephrol Hypertens ; 15(2): 123-9, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16481877

RESUMO

PURPOSE OF REVIEW: The progression of chronic kidney disease to terminal renal failure remains a major challenge in nephrology. Definition of the dynamic differences in gene regulation, protein interaction and protein function in this process might allow the development of rationally designed management strategies for the individual patient. Current approaches to identifying the molecular markers required to implement this 'personalized medicine' concept in progressive renal failure will be presented in this review. RECENT FINDINGS: In small populations, molecular fingerprints derived from renal biopsies have allowed the definition of distinct patient subgroups. These parameters could be shown to correlate with the response to available therapies and, in chronic transplant failure, with the therapeutic toxicity of cyclosporine. Urine analysis for mRNA and protein markers is rapidly evolving as a non-invasive approach for molecular patient monitoring. As only a small fraction of these fingerprints have been evaluated in independent populations, studies to test marker sets in diverse cohorts for their clinical applicability are warranted. SUMMARY: The genome-wide tools discussed in this review might define the molecular mechanism active in each single patient with progressive kidney disease. Reflecting the individuality of the disease process could result in a tailored therapy for the unique human being, contrasting with the 'one-size-fits-all' therapies currently employed in nephrology.


Assuntos
Regulação da Expressão Gênica , Genoma , Falência Renal Crônica/genética , Feminino , Perfilação da Expressão Gênica , Marcadores Genéticos , Rejeição de Enxerto/genética , Sobrevivência de Enxerto/genética , Humanos , Falência Renal Crônica/diagnóstico , Falência Renal Crônica/epidemiologia , Falência Renal Crônica/cirurgia , Transplante de Rim/efeitos adversos , Transplante de Rim/métodos , Masculino , Biologia Molecular , RNA Mensageiro/análise , Sensibilidade e Especificidade , Índice de Gravidade de Doença
20.
Am J Pathol ; 168(2): 370-85, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16436653

RESUMO

Hepatitis C virus (HCV) infection is frequently complicated by glomerulonephritis with immune complexes containing viral RNA. We examined the potential influence of Toll-like receptors (TLRs), specifically TLR3 recognition of viral dsRNA exemplified by polyriboinosinic:polyribocytidylic acid [poly(I:C) RNA]. Normal human kidney stained positive for TLR3 on mesangial cells (MCs), vascular smooth muscle cells, and collecting duct epithelium. Cultured MCs have low TLR3 mRNA levels with predominant intracellular protein localization, which was increased by tumor necrosis factor-alpha, interleukin (IL)-1beta, interferon (IFN)-gamma, and the TLR3 ligand poly(I:C) RNA. Poly(I:C) RNA stimulation of MCs increased mRNA and protein synthesis of IL-6, IL-1beta, M-CSF, IL-8/CXCL8, RANTES/CCL5, MCP-1/CCL2, and ICAM-I; it also increased anti-proliferative and proapoptotic effects, the latter of which was decreased by inhibiting caspase-8. In microdissected glomeruli of normal and non-HCV membranoproliferative glomerulonephritis biopsies, TLR3 mRNA expression was low. In contrast TLR3 mRNA expression was significantly increased in hepatitis C-positive glomerulonephritis and was associated with enhanced mRNA for RANTES/CCL5 and MCP-1/CCL2. We hypothesize that immune complexes containing viral RNA activate mesangial TLR3 during HCV infection, thereby contributing to chemokine/cytokine release and effecting proliferation and apoptosis. Thus, TLR3 expression on renal cells, and especially MCs, may establish a link between viral infections and glomerular diseases.


Assuntos
Glomerulonefrite/metabolismo , Glomerulonefrite/virologia , Hepacivirus/genética , Hepatite C/complicações , Receptor 3 Toll-Like/metabolismo , Apoptose , Proliferação de Células , Células Cultivadas , Citocinas/metabolismo , Epitélio/metabolismo , Epitélio/virologia , Glomerulonefrite/imunologia , Hepacivirus/patogenicidade , Humanos , Interferon gama/farmacologia , Interleucina-1/farmacologia , Rim/citologia , Rim/metabolismo , Rim/virologia , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/metabolismo , Túbulos Renais Coletores/virologia , Ligantes , Células Mesangiais/metabolismo , Células Mesangiais/virologia , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/virologia , Poli I-C/metabolismo , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Receptor 3 Toll-Like/genética , Fator de Necrose Tumoral alfa/farmacologia
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