RESUMO
We previously described early results of a nonchimeric operational tolerance protocol in human leukocyte antigen (HLA)-identical living donor renal transplants and now update these results. Recipients given alemtuzumab, tacrolimus/MPA with early sirolimus conversion were multiply infused with donor hematopoietic CD34(+) stem cells. Immunosuppression was withdrawn by 24 months. Twelve months later, operational tolerance was confirmed by rejection-free transplant biopsies. Five of the first eight enrollees were initially tolerant 1 year off immunosuppression. Biopsies of three others after total withdrawal showed Banff 1A acute cellular rejection without renal dysfunction. With longer follow-up including 5-year posttransplant biopsies, four of the five tolerant recipients remain without rejection while one developed Banff 1A without renal dysfunction. We now add seven new subjects (two operationally tolerant), and demonstrate time-dependent increases of circulating CD4(+) CD25(+++) CD127(-) FOXP3(+) Tregs versus losses of Tregs in nontolerant subjects (p < 0.001). Gene expression signatures, developed using global RNA expression profiling of sequential whole blood and protocol biopsy samples, were highly associative with operational tolerance as early as 1 year posttransplant. The blood signature was validated by an external Immune Tolerance Network data set. Our approach to nonchimeric operational HLA-identical tolerance reveals association with Treg immunophenotypes and serial gene expression profiles.
Assuntos
Biomarcadores/análise , Antígenos HLA/genética , Antígenos HLA/imunologia , Falência Renal Crônica/imunologia , Transplante de Rim , Quimeras de Transplante/imunologia , Tolerância ao Transplante/imunologia , Adulto , Idoso , Feminino , Seguimentos , Perfilação da Expressão Gênica , Genômica/métodos , Taxa de Filtração Glomerular , Sobrevivência de Enxerto , Histocompatibilidade , Humanos , Imunofenotipagem , Falência Renal Crônica/genética , Falência Renal Crônica/cirurgia , Testes de Função Renal , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Retrospectivos , Fatores de Risco , Quimeras de Transplante/genéticaRESUMO
Diabetic Nephropathy (DN) is believed to be a major microvascular complication of diabetes. The hallmark of DN includes deposition of Extracellular Matrix (ECM) proteins, such as, collagen, laminin and fibronectin in the mesangium and renal tubulo-interstitium of the glomerulus and basement membranes. Such an increased expression of ECM leads to glomerular and tubular basement membranes thickening and increase of mesangial matrix, ultimately resulting in glomerulosclerosis and tubulointerstitial fibrosis. The characteristic morphologic glomerular mesangial lesion has been described as Kimmelstiel-Wilson nodule, and the process at times is referred to as diabetic nodular glomerulosclerosis. Thus, the accumulation of ECM proteins plays a critical role in the development of DN. The relevant mechanism(s) involved in the increased ECM expression and their regulation in the kidney in diabetic state has been extensively investigated and documented in the literature. Nevertheless, there are certain other mechanisms that may yet be conclusively defined. Recent studies demonstrated that some of the new signaling pathways or molecules including, Notch, Wnt, mTOR, TLRs and small GTPase may play a pivotal role in the modulation of ECM regulation and expression in DN. Such modulation could be operational for instance Notch through Notch1/Jagged1 signaling, Wnt by Wnt/ß- catenin pathway and mTOR via PI3-K/Akt/mTOR signaling pathways. All these pathways may be critical in the modulation of ECM expression and tubulo-interstitial fibrosis. In addition, TLRs, mainly the TLR2 and TLR4, by TLR2- dependent and TGF-ß-dependent conduits, may modulate ECM expression and generate a fibrogenic response. Small GTPase like Rho, Ras and Rab family by targeting relevant genes may also influence the accumulation of ECM proteins and renal fibrosis in hyperglycemic states. This review summarizes the recent information about the role and mechanisms by which these molecules and signaling pathways regulate ECM synthesis and its expression in high glucose ambience in vitro and in vivo states. The understanding of such signaling pathways and the molecules that influence expression, secretion and amassing of ECM may aid in developing strategies for the amelioration of diabetic nephropathy.
Assuntos
Nefropatias Diabéticas/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Animais , Matriz Extracelular/metabolismo , Humanos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Receptores Notch/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Receptores Toll-Like/metabolismo , beta Catenina/metabolismoRESUMO
Matrix metalloproteinases (MMPs) are proteolytic enzymes belonging to the family of zinc-dependent endopeptidases that are capable of degrading almost all the proteinaceous components of the extracellular matrix (ECM). It is known that MMPs play a role in a number of renal diseases, such as, various forms of glomerulonephritis and tubular diseases, including some of the inherited kidney diseases. In this regard, ECM accumulation is considered to be a hallmark morphologic finding of diabetic nephropathy, which not only is related to the excessive synthesis of matrix proteins, but also to their decreased degradation by the MMPs. In recent years, increasing evidence suggest that there is a good correlation between the activity or expression of MMPs and progression of renal disease in patients with diabetic nephropathy and in various experimental animal models. In such a diabetic milieu, the expression of MMPs is modulated by high glucose, advanced glycation end products (AGEs), TGF-ß, reactive oxygen species (ROS), transcription factors and some of the microRNAs. In this review, we focused on the structure and functions of MMPs, and their role in the pathogenesis of diabetic nephropathy.
Assuntos
Nefropatias Diabéticas/metabolismo , Metaloproteinases da Matriz/metabolismo , Animais , Humanos , Metaloproteinases da Matriz/química , MicroRNAs/metabolismoRESUMO
Diabetic nephropathy is a leading cause of end-stage renal failure worldwide. Its morphologic characteristics include glomerular hypertrophy, basement membrane thickening, mesangial expansion, tubular atrophy, interstitial fibrosis and arteriolar thickening. All of these are part and parcel of microvascular complications of diabetes. A large body of evidence indicates that oxidative stress is the common denominator link for the major pathways involved in the development and progression of diabetic micro- as well as macro-vascular complications of diabetes. There are a number of macromolecules that have been implicated for increased generation of reactive oxygen species (ROS), such as, NAD(P)H oxidase, advanced glycation end products (AGE), defects in polyol pathway, uncoupled nitric oxide synthase (NOS) and mitochondrial respiratory chain via oxidative phosphorylation. Excess amounts of ROS modulate activation of protein kinase C, mitogen-activated protein kinases, and various cytokines and transcription factors which eventually cause increased expression of extracellular matrix (ECM) genes with progression to fibrosis and end stage renal disease. Activation of renin-angiotensin system (RAS) further worsens the renal injury induced by ROS in diabetic nephropathy. Buffering the generation of ROS may sound a promising therapeutic to ameliorate renal damage from diabetic nephropathy, however, various studies have demonstrated minimal reno-protection by these agents. Interruption in the RAS has yielded much better results in terms of reno-protection and progression of diabetic nephropathy. In this review various aspects of oxidative stress coupled with the damage induced by RAS are discussed with the anticipation to yield an impetus for designing new generation of specific antioxidants that are potentially more effective to reduce reno-vascular complications of diabetes.
Assuntos
Antioxidantes , Diabetes Mellitus/metabolismo , Diabetes Mellitus/fisiopatologia , Nefropatias Diabéticas , Falência Renal Crônica/metabolismo , Falência Renal Crônica/fisiopatologia , Rim/metabolismo , Rim/fisiopatologia , NADPH Oxidases/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Sistema Renina-Angiotensina , Transdução de Sinais/efeitos dos fármacos , Animais , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Nefropatias Diabéticas/tratamento farmacológico , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/fisiopatologia , Humanos , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Coelhos , Ratos , Sistema Renina-Angiotensina/efeitos dos fármacosRESUMO
Serial changes in glomerular capillary loop gene expression were used to uncover mechanisms contributing to primary glomerular disease in rat models of passive Heymann nephritis and puromycin nephrosis. Before the onset of proteinuria, podocyte protein-tyrosine phosphatase (GLEPP1) expression was transiently decreased in the nephrosis model, whereas the immune costimulatory molecule B7.1 was stimulated in both models. To relate these changes to the development of proteinuria, the time of onset and intensity of proteinuria were altered. When the models were induced simultaneously, proteinuria and anasarca occurred earlier with the collapse of glomerular capillary loops. Upregulation of B7.1 with the downregulation of GLEPP1, Wilms' tumor gene (WT1), megalin, and vascular endothelial growth factor started early and persisted through the course of disease. In the puromycin and the combined models, changes in GLEPP1 expression were corticosteroid-sensitive, whereas B7.1, WT1, vascular endothelial growth factor, and most slit diaphragm genes involved later in the combined model, except podocin, were corticosteroid-resistant. There was a very early increase in the nuclear expression of podocyte transcription factors ZHX2 and ZHX1 that may be linked to the changes in gene expression in the combined proteinuric model. Our studies suggest that an early and persistent change in mostly steroid-resistant glomerular gene expression is the hallmark of severe and progressive glomerular disease.
Assuntos
Regulação da Expressão Gênica/fisiologia , Glomerulonefrite/genética , Glomerulonefrite/fisiopatologia , Glomérulos Renais/metabolismo , Animais , Antígeno B7-1/genética , Antígeno B7-1/metabolismo , Modelos Animais de Doenças , Glomerulonefrite/patologia , Glomerulonefrite Membranosa/genética , Glomerulonefrite Membranosa/patologia , Glomerulonefrite Membranosa/fisiopatologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Glomérulos Renais/patologia , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Nefrose/genética , Nefrose/patologia , Nefrose/fisiopatologia , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/metabolismo , Proteinúria/genética , Proteinúria/patologia , Proteinúria/fisiopatologia , Ratos , Ratos Wistar , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas WT1/genética , Proteínas WT1/metabolismoRESUMO
BACKGROUND: Diabetic nephropathy accounts for over 30% of the end-stage renal disease (ESRD). A number of defined mechanisms and molecules that are involved in its pathogenesis are known, while others remain to be identified. METHODS: Suppression subtraction hybridization (SSH)-polymerase chain reaction (PCR) was employed to search for new genes that may be relevant to the pathogenesis of diabetic nephropathy during embryonic development, the time when the kidney is most susceptible to various forms of stress. A diabetic state was induced in pregnant mice at day-13 of gestation by administration of streptozotocin. The kidneys of newborn mice with blood glucose level> 200 mg/dL were harvested, mRNA isolated and subjected to SSH-PCR. Several differentially expressed cDNA fragments with up-regulated expression were isolated. One of the cDNA fragments had homology with human Ras-like guanine 5'-triphosphate (GTPase), Rap1b gene. By utilizing the lambdaZAP II mouse cDNA library and SMART RACE amplification, a full-length Rap1b cDNA was isolated. A recombinant protein was generated in pET15b bacterial expression system. An anti-Rap1b antibody was raised in rabbits by immunizing them with the fusion protein, and its specificity was confirmed by Western blot analysis. RESULTS: Rap1b cDNA had an open reading frame of 552 bp with a predicted putative protein size of approximately 21 kD. In vitro translation verified the authentication of the Rap1b cDNA clone. Northern blot analyses revealed a single approximately 2.3 kb Rap1b mRNA transcript. Its expression was up-regulated in several tissues, including the kidney of newborn diabetic mice. The degree of up-regulation of Rap1b mRNA expression was proportional to the blood glucose levels. Western blot analyses confirmed the hyperglycemia-induced up-regulation of the Rap1b expression. In situ hybridization and immunofluorescence studies revealed that Rap1b was expressed in the inner medullary collecting tubules. During hyperglycemia, its expression was accentuated and extended into the outer medullary and cortical collecting tubules. Similar up-regulation of Rap1b was observed when embryonic kidneys, harvested at day-13 of gestation, were exposed to high glucose ambience. CONCLUSION: The data suggest that Rap1b, a GTP-binding protein that plays a critical role in various signaling intracellular events, is another molecule that may be relevant to the pathobiology of diabetic nephropathy.
Assuntos
Proteínas rap de Ligação ao GTP/metabolismo , Sequência de Aminoácidos/genética , Animais , Animais Recém-Nascidos/fisiologia , Sequência de Bases/genética , DNA Complementar/genética , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/fisiopatologia , Embrião de Mamíferos/metabolismo , Expressão Gênica , Glucose/fisiologia , Hiperglicemia/genética , Hiperglicemia/metabolismo , Rim/fisiopatologia , Camundongos , Camundongos Endogâmicos ICR , Dados de Sequência Molecular , Hibridização de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase , Coelhos , Técnica de Subtração , Regulação para Cima , Proteínas rap de Ligação ao GTP/genéticaRESUMO
Aldo-keto reductases (AKRs) are a family of monomeric oxido-reductases with molecular weight ranging from 35-40 kDa and currently includes upwards of 60 members. They are expressed in a wide variety of tissues, where they catalyze the NADPH-dependent reduction of various aliphatic and aromatic aldehydes and ketones. The functions of most of the family members are not well defined. But two members, aldehyde reductase (AKRIA) and aldose reductase (AKRIB), have been extensively studied. The latter has received the most attention since being relevant to the complications of diabetes mellitus. It is up-regulated during hyperglycemia, and at the same time there is an increased activity of the sorbitol pathway and non-enzymatic glycation of proteins with ensuing damage in various tissues. It is developmentally regulated in the ocular lens, and is believed to modulate lens fiber morphogenesis during fetal life. Unlike the other AKR family members that are ubiquitously expressed, recently a renal-specific oxio-reductase has been described that is expressed exclusively in the proximal tubules. Although, it has no homology with other AKR members, it binds to NADPH with high affinity and is up-regulated in streptozotocin-induced diabetes in mice. It is also developmentally regulated and seems to selectively modulate renal tubulogenesis during embryonic life.
Assuntos
Oxirredutases do Álcool/metabolismo , Nefropatias Diabéticas/enzimologia , Rim/enzimologia , Aldeído Redutase/metabolismo , Aldo-Ceto Redutases , Animais , Humanos , Rim/embriologiaRESUMO
Glucose is the main source or energy for the mammalian cells and its entry is mediated via various transporters. About 7 facilitative (GULT-1 to -7) and 2 concentrative glucose transporters (SGLT-1 and -2) have been identified. The facilitative glucose transporters allow the glucose entry into the cell interior due to the concentration gradient and the latter via the Na+-dependent electrochemical gradient. They have similar structural motifs with 12-14 putative transmembrane domains with a predicted protein size varying from 50 to 76kDa. Some of the facilitative glucose transporters (GLUT-1, -2, -4 and -5) and both the sodium glucose co-transporters (SGLT-1 and -2) are expressed in the kidney. The transporters that are involved in the major transport of glucose in the kidney include GLUT-2 and SGLT-2. They are of high capacity and low affinity type and are expressed in the S1 segment of the proximal tubule. All the transporters expressed in the kidney are developmentally regulated. The mRNA expression of renal GLUTs is variable during the fetal and postnatal periods. On the other hand the mRNA of SGLTs increases steadily from the fetal period to maturity along with the increase in their functional activity, i.e., glucose uptake. Recent studies indicate that the SGLTs are believed to selectively regulate tubulogenesis since they are expressed in the metanephric tubules very early in the embryonic life in mammals.
Assuntos
Rim/embriologia , Rim/metabolismo , Proteínas de Transporte de Monossacarídeos/fisiologia , Animais , Humanos , Mamíferos , Proteínas de Transporte de Monossacarídeos/metabolismoRESUMO
It currently is thought that human immunodeficiency virus-associated nephropathy (HIVAN) occurs late in the course of HIV infection. Although HIVAN may be the presenting manifestation of acquired immunodeficiency syndrome (AIDS), it usually occurs after a prolonged period of viral infection often associated with high levels of HIV viremia. The patient described here developed HIVAN as a manifestation of acute retroviral syndrome. A 41-year-old black man presented with nephrotic range proteinuria, renal insufficiency, and acute gastrointestinal and pulmonary symptoms. He recently had been treated for primary syphilis. Two HIV serologic tests, performed 3 months apart, were negative. Renal biopsy was consistent with HIVAN. After the biopsy, the patient was discovered to have more than 700,000 viral copies per mL in his blood. CD4(+) count was greater than 500/mm(3). Six weeks later, enzyme-linked immunosorbent assay and Western blot analyses for HIV antibody became positive. HIVAN can occur early in the course of HIV infection, even during acute infection before seroconversion, and prolonged exposure to virus is not necessary for this renal involvement to occur in the susceptible host.
Assuntos
Nefropatia Associada a AIDS/patologia , Soronegatividade para HIV , Rim/patologia , Adulto , Biópsia , Soropositividade para HIV/diagnóstico , Humanos , Rim/virologia , Masculino , Insuficiência Renal/patologia , Insuficiência Renal/virologia , Viremia/complicaçõesRESUMO
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.
Assuntos
Nefropatias Diabéticas/genética , Perfilação da Expressão Gênica , Glomerulosclerose Segmentar e Focal/genética , Rim/fisiopatologia , Animais , Diabetes Mellitus Experimental , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Expressão Gênica , Glomerulosclerose Segmentar e Focal/metabolismo , Glomerulosclerose Segmentar e Focal/patologia , Rim/patologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Nefrectomia , Análise de Sequência com Séries de Oligonucleotídeos , Valores de ReferênciaRESUMO
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.
Assuntos
Carboxipeptidases/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Túbulos Renais Coletores/enzimologia , Rim/enzimologia , Glicoproteínas de Membrana/genética , Sequência de Aminoácidos , Enzima de Conversão de Angiotensina 2 , Animais , Sequência de Bases , Carboxipeptidases/química , Carboxipeptidases/metabolismo , Membrana Celular/enzimologia , Citosol/enzimologia , Embrião de Mamíferos , Desenvolvimento Embrionário e Fetal , Humanos , Rim/embriologia , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos , Peptidil Dipeptidase A , Sinais Direcionadores de Proteínas/genética , RNA Mensageiro/genética , Ratos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transcrição GênicaRESUMO
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.
Assuntos
Adjuvantes Imunológicos/uso terapêutico , Anticorpos/imunologia , Antígenos de Diferenciação/uso terapêutico , Galectinas , Hemaglutininas/uso terapêutico , Soros Imunes/imunologia , Lectinas/uso terapêutico , Nefrite/tratamento farmacológico , Nefrite/imunologia , Adjuvantes Imunológicos/farmacocinética , Animais , Antígenos de Diferenciação/metabolismo , Apoptose/efeitos dos fármacos , Autoanticorpos , Dexametasona/uso terapêutico , Feminino , Galectina 1 , Galectina 3 , Glucocorticoides/uso terapêutico , Hemaglutininas/metabolismo , Rim/metabolismo , Glomérulos Renais/patologia , Lectinas/metabolismo , Nefrite/patologia , Nefrite/fisiopatologia , Nefrite/urina , Proteinúria/urina , Ratos , Ratos Endogâmicos WKY , Baço/fisiopatologia , Linfócitos T/fisiologiaRESUMO
Expression and role of sodium glucose cotransporter (SGLT-1) in tubulogenesis were investigated during renal development. A mouse SGLT-1 cDNA was cloned, and it had substantial homology with human and rat forms. Four mRNA transcripts were detected, which differed in size from other species. SGLT-1 transcripts were detected at day 13 of gestation, and their expression increased during later stages extending into the postnatal period. A high mRNA and protein expression of SGLT-1 was seen in tubular segments of the inner cortex and outer medulla at day 16, and it was developmentally regulated. Treatment with SGLT-1 antisense selectively decreased the population of tubules in the metanephric explants. Expression of glomerular mRNA and WGA binding were unchanged. SGLT-1 activity, as measured by [(14)C]methyl-alpha-D-glucopyranoside uptake, increased during gestation in the tubular segments where it is expressed. Glucose uptake was inhibited by the treatment with SGLT-1 antisense and D-galactose. The data suggest that SGLT-1 exhibits a restricted spatiotemporal expression with functional activity confined to the corresponding tubular segments, and it selectively maintains renal tubulogenesis during development.
Assuntos
Túbulos Renais/embriologia , Glicoproteínas de Membrana/fisiologia , Proteínas de Transporte de Monossacarídeos/fisiologia , Sequência de Aminoácidos/genética , Animais , Northern Blotting , Southern Blotting , Clonagem Molecular , DNA Complementar/genética , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/fisiologia , Desenvolvimento Embrionário e Fetal/fisiologia , Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Dados de Sequência Molecular , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , Oligonucleotídeos Antissenso/farmacologia , RNA Mensageiro/metabolismo , Ratos , Homologia de Sequência de Aminoácidos , Transportador 1 de Glucose-SódioRESUMO
Aldose reductase (ALR2), a NADPH-dependent aldo-keto reductase (AKR), is widely distributed in mammalian tissues and has been implicated in complications of diabetes, including diabetic nephropathy. To identify a renal-specific reductase belonging to the AKR family, representational difference analyses of cDNA from diabetic mouse kidney were performed. A full-length cDNA with an ORF of 855 nt and yielding a approximately 1.5-kb mRNA transcript was isolated from a mouse kidney library. Human and rat homologues also were isolated, and they had approximately 91% and approximately 97% amino acid identity with mouse protein. In vitro translation of the cDNA yielded a protein product of approximately 33 kDa. Northern and Western blot analyses, using the cDNA and antirecombinant protein antibody, revealed its expression exclusively confined to the kidney. Like ALR2, the expression was up-regulated in diabetic kidneys. Its mRNA and protein expression was restricted to renal proximal tubules. The gene neither codistributed with Tamm-Horsfall protein nor aquaporin-2. The deduced protein sequence revealed an AKR-3 motif located near the N terminus, unlike the other AKR family members where it is confined to the C terminus. Fluorescence quenching and reactive blue agarose chromatography studies revealed that it binds to NADPH with high affinity (K(dNADPH) = 66.9 +/- 2.3 nM). This binding domain is a tetrapeptide (Met-Ala-Lys-Ser) located within the AKR-3 motif that is similar to the other AKR members. The identified protein is designated as RSOR because it is renal-specific with properties of an oxido-reductase, and like ALR2 it may be relevant in the renal complications of diabetes mellitus.
Assuntos
Aldeído Redutase/genética , Diabetes Mellitus Experimental/enzimologia , Rim/enzimologia , Aldeído Redutase/metabolismo , Animais , Animais Recém-Nascidos , Sequência de Bases , DNA Complementar , Diabetes Mellitus Experimental/genética , Biblioteca Gênica , Humanos , Hiperglicemia/enzimologia , Camundongos , Camundongos Endogâmicos ICR , Dados de Sequência Molecular , Fases de Leitura Aberta , Biossíntese de Proteínas , RNA Mensageiro/genética , Ratos , Transcrição GênicaRESUMO
We previously isolated and identified steroid receptor coactivator-1 (SRC-1) and peroxisome proliferator-activated receptor (PPAR)-binding protein (PBP/PPARBP) as coactivators for PPAR, using the ligand-binding domain of PPARgamma as bait in a yeast two-hybrid screening. As part of our continuing effort to identify cofactors that influence the transcriptional activity of PPARs, we now report the isolation of a novel coactivator from mouse, designated PRIP (peroxisome proliferator-activated receptor interacting protein), a nuclear protein with 2068 amino acids and encoded by 13 exons. Northern analysis showed that PRIP mRNA is ubiquitously expressed in many tissues of adult mice. PRIP contains two LXXLL signature motifs. The amino-terminal LXXLL motif (amino acid position 892 to 896) of PRIP was found to be necessary for nuclear receptor interaction, but the second LXXLL motif (amino acid position 1496 to 1500) appeared unable to bind PPARgamma. Deletion of the last 12 amino acids from the carboxyl terminus of PPARgamma resulted in the abolition of the interaction between PRIP and PPARgamma. PRIP also binds to PPARalpha, RARalpha, RXRalpha, ER, and TRbeta1, and this binding is increased in the presence of specific ligands. PRIP acts as a strong coactivator for PPARgamma in the yeast and also potentiates the transcriptional activities of PPARgamma and RXRalpha in mammalian cells. A truncated form of PRIP (amino acids 786-1132) acts as a dominant-negative repressor, suggesting that PRIP is a genuine coactivator.
Assuntos
Proteínas de Transporte/genética , Proteínas Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/metabolismo , Clonagem Molecular , Subunidade 1 do Complexo Mediador , Camundongos , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/isolamento & purificação , Proteínas Nucleares/metabolismoRESUMO
Extracellular matrix (ECM) proteins, their integrin receptors, and matrix metalloproteinases (MMPs), the ECM-degrading enzymes, are believed to be involved in various biological processes, including embryogenesis. In the present study, we investigated the role of membrane type MMP, MT-1-MMP, an activator pro-MMP-2, in metanephric development. Also, its relationship with MMP-2 and its inhibitor, TIMP-2, was studied. Since mRNAs of MT-1-MMP and MMP-2 are respectively expressed in the ureteric bud epithelia and mesenchyme, they are ideally suited for juxtacrine/paracrine interactions during renal development. Northern blot analyses revealed a single approximately 4.5-kb mRNA transcript of MT-1-MMP, and its expression was developmentally regulated. Inclusion of MT-1-MMP antisense oligodeoxynucleotide (ODN) in the culture media induced dysmorphogenetic changes in the embryonic metanephros. MMP-2 antisense ODN also induced similar changes, but they were relatively less; on the other hand TIMP-2 antisense ODN induced a mild increase in the size of explants. Concomitant exposure of MT-1-MMP and MMP-2 antisense ODNs induced profound alterations in the metanephroi. Treatment of TIMP-2 antisense ODN to metanephroi exposed to MT-1-MMP/MMP-2 antisense notably restored the morphology of the explants. Specificity of the MT-1-MMP antisense ODN was reflected in the selective decrease in its mRNA and protein expression. The MT-1-MMP antisense ODN also resulted in a failure in the activation of pro-MMP-2 to MMP-2. These findings suggest that the trimacromolecular complex of MT-1-MMP:MMP-2:TIMP-2 modulates the organogenesis of the metanephros, conceivably by mediating paracrine/juxtacrine epithelial:mesenchymal interactions.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Rim/embriologia , Rim/crescimento & desenvolvimento , Metaloproteinase 2 da Matriz/genética , Metaloproteinases da Matriz/genética , Metaloendopeptidases , Transcrição Gênica , Envelhecimento , Animais , Desenvolvimento Embrionário e Fetal , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica , Idade Gestacional , Rim/enzimologia , Metaloproteinases da Matriz Associadas à Membrana , Camundongos , Camundongos Endogâmicos ICR , Morfogênese/efeitos dos fármacos , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Técnicas de Cultura de Órgãos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Inibidor Tecidual de Metaloproteinase-2/genéticaRESUMO
Tubulointerstitial nephritis antigen (TIN-ag) is an extracellular matrix protein and is expressed in the renal tubular basement membranes. Its role in metanephric development was investigated. TIN-ag cDNA, isolated from the newborn mouse library, had an ORF of 1,425 nucleotides, a putative signal sequence, and an ATP/GTP-binding site. The translated sequence had approximately 80% identity with rabbit TIN-ag. Among various tissues, TIN-ag mRNA was primarily expressed in the newborn kidney. In the embryonic metanephros, TIN-ag expression was confined to the distal convolution or pole of the S-shaped body, the segment of the nascent nephron that is the progenitor of renal tubules. Treatment with TIN-ag antisense oligodeoxynucleotide induced dysmorphogenesis of the embryonic metanephroi, malformation of the S-shaped body, and a decrease in the tubular population, whereas the glomeruli were unaffected. Treatment also led to a decrease of TIN-Ag mRNA, de novo synthesis of TIN-ag protein, and its antibody reactivity. The mRNA expression of glomerular epithelial protein 1 (a marker for renal podocytes), anti-heparan-sulfate-proteoglycan antibody reactivity, and wheat germ agglutinin lectin staining of the metanephros were unaffected. The anti-TIN-ag antibody treatment also caused deformation of the S-shaped body and a reduction in the tubular population, whereas the glomeruli were unchanged. The data suggest that the TIN-ag, unlike other basement membrane proteins, selectively regulates tubulogenesis, whereas glomerulogenesis is largely unaffected.
Assuntos
Moléculas de Adesão Celular/fisiologia , Proteínas da Matriz Extracelular/fisiologia , Glomérulos Renais/embriologia , Túbulos Renais/embriologia , Glicoproteínas de Membrana/fisiologia , Nefrite Intersticial/imunologia , Proteínas de Ligação a Telômeros , Sequência de Aminoácidos , Animais , Sequência de Bases , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/imunologia , DNA Complementar/análise , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/imunologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos ICR , Dados de Sequência Molecular , Oligonucleotídeos Antissenso/farmacologia , Proteínas Tirosina Fosfatases/genética , RNA Mensageiro/análise , Coelhos , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a ReceptoresRESUMO
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.
Assuntos
Testes Genéticos , Glomerulosclerose Segmentar e Focal/genética , Rim/fisiologia , Nefrectomia , Animais , Northern Blotting , Southern Blotting , Cicatriz/patologia , DNA Complementar/análise , Diabetes Mellitus Experimental/genética , Nefropatias Diabéticas/genética , Expressão Gênica/fisiologia , Hipertrofia , Rim/patologia , Rim/cirurgia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Reação em Cadeia da Polimerase , RNA Mensageiro/análiseRESUMO
Fibrillin-2 is an extracellular matrix protein. It is associated with elastic fibers in several tissues and is believed to serve as a ligand for alphavbeta3 integrin, the latter being a known morphogen. In this study, the role of fibrillin-2 in lung development was investigated. Also, rat fibrillin-2 cDNA was isolated and sequenced and its spatiotemporal expression determined. It had approximately 88% homology with human fibrillin-2 and had Ca(2+) binding epidermal growth factor-like domains, transforming growth factor-beta binding protein motifs, and two RGD binding sites. Northern blot analysis revealed an approximately 10-kb transcript, and fibrillin-2 expression was developmentally regulated, and it paralleled that of tropoelastin. At day 13 of gestation, fibrillin-2 was expressed in the mesenchyme and at the epithelial:mesenchymal interface. From day 13 to 19 of gestation, its expression intensified and was confined around the tracheobronchial airways, while it lessened during the postnatal period. Immunoprecipitation revealed an approximately 350-kDa band by SDS-PAGE. Treatment with fibrillin-2 antisense oligodeoxynucleotide induced dysmorphogenesis of the lung explants. They were smaller and had rudimentary lung bud branches, collapsed conducting airways, and loose expanded mesenchyme. Concomitantly, fibrillin-2 mRNA, antibody reactivity in the explants, and fibrillin-2-specific radioincorporation were reduced. Anti-alphav and -laminin antibody reactivity and their respective incorporated specific radioactivities were unaltered. These data indicate that fibrillin-2 modulates organogenesis of the lung in the context of epithelial:mesenchymal interactions. Conceivably, the collapse of the conducting airways may also be related to the perturbed biology of the fibrillin-2 interacting protein, i.e., elastin, the latter being critical for the normal biophysiology of the lungs.