RESUMEN
Glomerular hyperfiltration plays a pathogenic role in the early stages of diabetic nephropathy. Experimental studies in laboratory animals suggest that nitric oxide (NO) might be involved in the pathogenesis of glomerular hyperfiltration. We performed a cross-sectional study to determine the relationship between diabetic glomerular hyperfiltration and the NO system. Normoalbuminuric (n=41), microalbuminuric (n=25), and macroalbuminuric (n=16) patients with type 2 diabetes were recruited in this study and compared with age-matched 84 non-diabetic control subjects. Creatinine clearance and urinary NO(2)(-)/NO(3)(-) excretion (urinary NOx) were measured, and the expression of endothelial cell nitric oxide synthase (ecNOS) was evaluated in human renal tissues. Glomerular hyperfiltration was present in 19 (37.5%) and nine (36.6%) of normoalbuminuric and microalbuminuric type 2 diabetic patients, respectively. The urinary NOx was significantly higher in normoalbuminuric patients compared with normal subjects. Creatinine clearance correlated significantly with urinary NOx in normoalbuminuric and microalbuminuric patients. Immunohistochemical staining intensities for ecNOS were significantly increased in glomerular endothelial cells of microalbuminuric type 2 diabetic patients as compared with the control subjects. These results suggest that NO may contribute to the pathogenesis of glomerular hyperfiltration in Japanese type 2 diabetic patients.
Asunto(s)
Albuminuria , Diabetes Mellitus Tipo 2/fisiopatología , Tasa de Filtración Glomerular/fisiología , Óxido Nítrico/fisiología , Adulto , Factores de Edad , Anciano , Animales , Glucemia/metabolismo , Presión Sanguínea , Creatinina/sangre , Creatinina/orina , Estudios Transversales , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/orina , Dihidrolipoamida Deshidrogenasa/análisis , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Hemoglobina Glucada/análisis , Humanos , Glomérulos Renales/enzimología , Masculino , Persona de Mediana Edad , Nitratos/orina , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo III , Nitritos/orina , Valores de ReferenciaRESUMEN
BACKGROUND: To elucidate the molecular mechanism of diabetic nephropathy, a high-density DNA filter array was employed to survey the gene expression profile of streptozotocin-induced diabetic CD-1 (ICR) mouse kidneys. METHODS: Ten-week-old CD-1 male mice were divided into four groups: (1) control, (2) unilaterally nephrectomized (UX) mice, (3) streptozotocin (STZ)-induced diabetic (STZ) mice, and (4) STZ mice with unilateral renal ablation (STZ-UX). Pathological changes were examined at 24 weeks after the induction. The gene expression profile was compared between the control and STZ mice by a Gene Discovery Array (GDA). RESULTS: The glomeruli in UX mouse kidney showed prominent glomerular hypertrophy, while the accumulation of mesangial matrix was minimal. Both STZ and STZ + UX mice had significant glomerular hypertrophy and glomerulosclerosis, and the lesions were not enhanced by renal ablation. By comparison between control and STZ mice, 16 clones that increased in expression with the induction of diabetes and 65 clones that decreased in diabetic kidneys were identified. The 37 known genes were related to glucose and lipid metabolism, ion transport, transcription factors, signaling molecules, and extracellular matrix-related molecules. The genes known to be involved in cell differentiation and organogenesis in various tissues (that is, Unc-18 homolog, POU domain transcription factor 2, lunatic fringe gene homolog, fibrous sheath component 1, Sox-17, fibulin 2, and MRJ) were found to be differentially expressed in the early phase of diabetic kidneys. CONCLUSIONS: Hyperglycemia is a major determinant of glomerulosclerosis in STZ-induced diabetic CD-1 mice, and the altered gene expression in the early phase of diabetic kidney may be critical for the development of diabetic nephropathy.
Asunto(s)
Nefropatías Diabéticas/genética , Perfilación de la Expresión Génica , Glomeruloesclerosis Focal y Segmentaria/genética , Riñón/fisiopatología , Animales , Diabetes Mellitus Experimental , Nefropatías Diabéticas/metabolismo , Nefropatías Diabéticas/patología , Expresión Génica , Glomeruloesclerosis Focal y Segmentaria/metabolismo , Glomeruloesclerosis Focal y Segmentaria/patología , Riñón/patología , Masculino , Ratones , Ratones Endogámicos ICR , Nefrectomía , Análisis de Secuencia por Matrices de Oligonucleótidos , Valores de ReferenciaRESUMEN
Collectrin, a novel homolog of angiotensin-converting enzyme-related carboxypeptidase (ACE2), was identified during polymerase chain reaction-based cDNA subtraction and up-regulated in 5/6 ablated kidneys at hypertrophic phase. Collectrin, with 222 amino acids, has an apparent signal peptide and a transmembrane domain; the sequence is conserved in mouse, rat, and human and shares 81.9% identity. Human collectrin has 47.8% identity with non-catalytic extracellular, transmembrane, and cytosolic domains of ACE2; however, unlike ACE and ACE2, collectrin lacks active dipeptidyl carboxypeptidase catalytic domains. The collectrin mRNA transcripts are expressed exclusively in the kidney. In situ hybridization reveals its mRNA expression in renal collecting ducts, and immunohistochemistry shows that it is localized to the luminal surface and cytoplasm of collecting ducts. Immunoprecipitation studies, using [35S]methionine-labeled renal cortical and inner medullar collecting duct cells, i.e. M-1 and mIMCD-3, indicate that the protein size is approximately 32 kDa. During the development of mouse kidney, mRNA signal is detectable at day 13 of gestation, and the protein product is observed in the ureteric bud branches. Its expression is progressively increased during later stages of the gestation extending into the neonatal periods and then is decreased in adult life. Up-regulated expression of collectrin in the hypertrophic kidneys after renal ablation and restricted spatio-temporal expression during development indicates a possible role(s)in the process of progressive renal failure and renal organogenesis.
Asunto(s)
Carboxipeptidasas/genética , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Túbulos Renales Colectores/enzimología , Riñón/enzimología , Glicoproteínas de Membrana/genética , Secuencia de Aminoácidos , Enzima Convertidora de Angiotensina 2 , Animales , Secuencia de Bases , Carboxipeptidasas/química , Carboxipeptidasas/metabolismo , Membrana Celular/enzimología , Citosol/enzimología , Embrión de Mamíferos , Desarrollo Embrionario y Fetal , Humanos , Riñón/embriología , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos , Peptidil-Dipeptidasa A , Señales de Clasificación de Proteína/genética , ARN Mensajero/genética , Ratas , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transcripción GenéticaRESUMEN
The Otsuka Long-Evans Tokushima fatty (OLETF) rat is an animal model of type 2 diabetes, characterized by abdominal obesity, insulin resistance, hypertension, and dyslipidemia. To elucidate the underlying molecular mechanism of obesity and its related complications, we used representational difference analysis and identified the genes more abundantly and specifically expressed in the visceral adipose tissue (VAT) of obese OLETF rats compared with the diabetes-resistant counterpart, that is, Long-Evans Tokushima Otsuka (LETO) rats. By Northern blot analysis, we confirmed the differential expression of 13 genes, including 3 novel genes. The upregulated expression of well-characterized lipid metabolic enzymes, such as lipoprotein lipase, phosphoenolpyruvate carboxykinase, and cholesterol esterase, were observed in VAT of OLETF rats. We demonstrated the differential expression of secreted proteins in VAT of OLETF rats, such as thrombospondin 1 and contrapsin-like protease inhibitor. In contrast to lipid enzymes, the secreted proteins revealed exclusive mRNA expression and they were not detected in VAT of LETO rats. Furthermore, the novel genes OL-16 and OL-64 were also expressed specifically in VAT of OLETF rats and were absent in that of LETO rats and other tissues, including subdermal and brown adipose tissues. The C-terminal partial amino acid sequence of OL-64 revealed that it showed approximately 40% homology with alpha(1)-antitrypsin and it seemed to be a new member of the serine proteinase inhibitor (SERPIN) gene family. VAT of OLEFT rats had a unique gene expression profile, and the accumulated VAT-specific known and novel secreted proteins may play a role(s) in the pathogenesis of obesity and its related complications.
Asunto(s)
Tejido Adiposo/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Ratones Obesos/genética , Ratas Endogámicas OLETF/genética , Vísceras/patología , Animales , Northern Blotting , ADN Complementario/genética , Diabetes Mellitus Tipo 2/genética , Masculino , Ratones , Ratones Obesos/metabolismo , Modelos Animales , Datos de Secuencia Molecular , Obesidad/genética , Obesidad/metabolismo , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , Ratas , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico , Distribución Tisular , Regulación hacia ArribaRESUMEN
BACKGROUND: Galectins are characterized by specific affinity for beta-galactoside sugars, and they play a role in diverse biological processes, including cell adhesion, cell proliferation, and apoptosis. Galectin-1, -3, and -9 have been implicated in modulating the immune response. METHODS: Nephrotoxic serum nephritis, which is characterized by crescent formation and glomerular influx of CD8+ cells into glomerular capillaries, was induced in Wistar Kyoto (WKY) rats by injecting rabbit antiglomerular basement membrane serum. Following induction, the rats were treated either with phosphate-buffered saline or dexamethasone, galectin-1, galectin-3, or galectin-9 on alternate days and were sacrificed at day 14. At day 8, splenic lymphocytes were isolated and employed for terminal deoxytransferase-mediated uridine triphosphate nick end-labeling (TUNEL) assay to assess the degree of apoptosis, and the kidneys were utilized to determine the extent of influx of CD4(+) and CD8(+) cells and glomerular damage. RESULTS: Dexamethasone induced a marked apoptosis of splenic CD4(+) and CD8(+) cells, and it inhibited the production of anti-rabbit IgG and the influx of CD8+ cells and macrophages into the renal glomeruli. Crescent formation and excretion of urinary proteins were also reduced. Galectin-9 failed to induce apoptosis in the CD4(+) cells; however, it induced apoptosis in the CD8(+) cells and inhibited the infiltration of CD8(+) cells. Although galectin-1 and -3 did not induce the apoptosis in the T cells, they inhibited the accumulation of macrophages in the renal glomeruli. Like dexamethasone, the galectins also reduced the crescentic formation, proliferation of glomerular cells, and excretion of urinary proteins. CONCLUSIONS: Galectin-9 selectively induces apoptosis of the activated CD8(+) cells, while the macrophage influx into the kidney is modulated by all three galectins. This finding raises an interesting possibility for the utility of galectins in the modulation of macrophages that are involved in immune-mediated glomerular diseases.
Asunto(s)
Adyuvantes Inmunológicos/uso terapéutico , Anticuerpos/inmunología , Antígenos de Diferenciación/uso terapéutico , Galectinas , Hemaglutininas/uso terapéutico , Sueros Inmunes/inmunología , Lectinas/uso terapéutico , Nefritis/tratamiento farmacológico , Nefritis/inmunología , Adyuvantes Inmunológicos/farmacocinética , Animales , Antígenos de Diferenciación/metabolismo , Apoptosis/efectos de los fármacos , Autoanticuerpos , Dexametasona/uso terapéutico , Femenino , Galectina 1 , Galectina 3 , Glucocorticoides/uso terapéutico , Hemaglutininas/metabolismo , Riñón/metabolismo , Glomérulos Renales/patología , Lectinas/metabolismo , Nefritis/patología , Nefritis/fisiopatología , Nefritis/orina , Proteinuria/orina , Ratas , Ratas Endogámicas WKY , Bazo/fisiopatología , Linfocitos T/fisiologíaRESUMEN
BACKGROUND: In diabetic and nondiabetic renal diseases, glomerular hyperfiltration is believed to play a central role in the subsequent progression of glomerulosclerosis and interstitial renal scarring. To identify genes involved in the process of hyperfiltration and hypertrophy, a polymerase chain reaction (PCR)-based subtraction method, that is, representational difference analysis of cDNA (cDNA-RDA), was employed. METHODS: Ten-week-old ICR mice were 5/6 nephrectomized and sham operated. After two weeks, mRNAs were isolated from control and remnant kidneys and were subjected to the cDNA-RDA procedure. RESULTS: We identified 10 known and 9 novel genes. Among 19 clones, 12 clones (8 known and 4 novel) showed 1.5- to 6-fold up-regulation by Northern blot analyses. The remaining seven clones were rarely expressed genes and were barely detected by Northern blot analyses, and their up-regulated expression was confirmed by Southern blot analysis using the PCR-amplified representative amplicons. The known genes included kidney androgen-regulated protein, major urinary protein, lysozyme M, metalloproteinase-3 tissue inhibitor, chaperonin 10, cytochrome oxidase I, epsilon-sarcoglycan, ribosomal protein S3a, G-proteingamma10 subunit, and splicing factor 9G8. All of the isolated known genes have not been reported to be up-regulated in the nephrectomized mouse kidney and suggest the possible role of androgen action, mitochondrial functions, matrix metabolism, cell-matrix interactions, and intracellular signaling events in the initiation of the progressive renal injury of the remnant kidney. Furthermore, cDNA-RDA facilitates the discovery of novel genes, including two kidney-specific genes. CONCLUSIONS: The isolated known and novel genes may be involved in the pathobiological process of initial hyperfiltration and hypertrophy of remnant kidney.
Asunto(s)
Pruebas Genéticas , Glomeruloesclerosis Focal y Segmentaria/genética , Riñón/fisiología , Nefrectomía , Animales , Northern Blotting , Southern Blotting , Cicatriz/patología , ADN Complementario/análisis , Diabetes Mellitus Experimental/genética , Nefropatías Diabéticas/genética , Expresión Génica/fisiología , Hipertrofia , Riñón/patología , Riñón/cirugía , Masculino , Ratones , Ratones Endogámicos ICR , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisisRESUMEN
Previously, we reported that the formation of focal adhesion accelerated by accumulation of extracellular matrices may inhibit the angiotensin II-stimulated proliferation of human mesangial cells (HMCs). The process is regulated by p44/42 MAP kinase activity through the mediation of paxillin and GTPase activating proteins. In this report, we investigated the effect of integrin molecules on the angiotensin II-induced p44/42 MAP kinase activation in non-adherent HMCs. The results demonstrated that incubation of cells with both antibody to integrin beta(1) chain (K20) and GRGDS peptide induced integrin clustering, paxillin aggregation, and marked suppression of angiotensin II-induced p44/42 MAP kinase activation. On the other hand, incubation of cells with K20 alone induced integrin clustering without paxillin aggregation and the suppressive effect on angiotensin II-stimulated p44/42 MAP kinase activity. Our results strongly suggest the pivotal role of integrins in the inhibitory effect of focal adhesion on p44/42 MAP kinase activity, the checking system against angiotensin II-induced MAP kinase overactivation.
Asunto(s)
Angiotensina II/farmacología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Adhesión Celular/fisiología , Mesangio Glomerular/enzimología , Integrinas/fisiología , Proteínas Quinasas Activadas por Mitógenos , Anticuerpos/farmacología , Antígenos CD/metabolismo , Moléculas de Adhesión Celular/metabolismo , Células Cultivadas , Proteínas del Citoesqueleto/metabolismo , Activación Enzimática/efectos de los fármacos , GTP Fosfohidrolasas/metabolismo , Humanos , Integrina alfa5 , Integrina beta1/inmunología , Integrina beta1/fisiología , Proteína Quinasa 3 Activada por Mitógenos , Paxillin , Fosfoproteínas/metabolismoRESUMEN
The overproduction of nitric oxide (NO) is reported in the diabetic kidney and considered to be involved in glomerular hyperfiltration. The precise mechanism of NO production in the diabetic kidney is, however, not known. In this report, we compare the localization of endothelial cell nitric oxide synthase (ecNOS) isoform expression in the kidney tissue of streptozotocin (STZ)-induced diabetic rats and 5/6 nephrectomized rats and clarify the pivotal role of ecNOS for the glomerular hyperfiltration in the early stages of diabetic nephropathy. In diabetic rats, the diameters of afferent arterioles, the glomerular volume, creatinine clearance, and urinary NO2/NO3 were increased after the induction of diabetes. Efferent arterioles were, however, not altered. Insulin or L-NAME treatment returned the diameters of afferent arterioles, glomerular volume, creatinine clearance, and urinary NO2/NO3 to normal. The expression of ecNOS in afferent arterioles and glomeruli of diabetic rats increased during the early stages of the disease, but was not altered in efferent arterioles. Treatment with either insulin or L-NAME decreased ecNOS expression in afferent arterioles and in glomeruli. In contrast, the ecNOS expression was upregulated in both afferent and efferent arterioles and in the glomeruli of 5/6 nephrectomized rats, where the dilatation of afferent and efferent arterioles and glomerular enlargement were observed. Treatment with L-NAME ameliorated the ecNOS expression and dilatation of arterioles. We conclude that enhanced NO synthesis by ecNOS in afferent arterioles and glomerular endothelial cells in response to the hyperglycaemic state could cause preferential dilatation of afferent arterioles, which ultimately induces glomerular enlargement and glomerular hyperfiltration.