RESUMO
Patients with CKD have an increased risk of cardiovascular mortality from arrhythmias and sudden cardiac death. We used a rat model of CKD (Cy/+) to study potential mechanisms of increased ventricular arrhythmias. Rats with CKD showed normal ejection fraction but hypertrophic myocardium. Premature ventricular complexes occurred more frequently in CKD rats than normal rats (42% versus 11%, P=0.18). By optical mapping techniques, action potential duration (APD) at 80% of repolarization was longer in CKD rats (78±4ms) than normal rats (63±3 ms, P<0.05) at a 200-ms pacing cycle length. Calcium transient (CaT) duration was comparable. Pacing cycle length thresholds to induce CaT alternans or APD alternans were longer in CKD rats than normal rats (100±7 versus 80±3 ms and 93±6 versus 76±4 ms for CaT and APD alternans, respectively, P<0.05), suggesting increased vulnerability to ventricular arrhythmia. Ventricular fibrillation was induced in 9 of 12 CKD rats and 2 of 9 normal rats (P<0.05); early afterdepolarization occurred in two CKD rats but not normal rats. The mRNA levels of TGF-ß, microRNA-21, and sodium calcium-exchanger type 1 were upregulated, whereas the levels of microRNA-29, L-type calcium channel, sarco/endoplasmic reticulum calcium-ATPase type 2a, Kv1.4, and Kv4.3 were downregulated in CKD rats. Cardiac fibrosis was mild and not different between groups. We conclude that cardiac ion channel and calcium handling are abnormal in CKD rats, leading to increased vulnerability to early afterdepolarization, triggered activity, and ventricular arrhythmias.
Assuntos
Arritmias Cardíacas/fisiopatologia , Regulação da Expressão Gênica , Insuficiência Renal Crônica/fisiopatologia , Potenciais de Ação , Animais , Cálcio/metabolismo , Sinalização do Cálcio , Modelos Animais de Doenças , Ecocardiografia , Eletrofisiologia , Fibrose/patologia , Frequência Cardíaca , Masculino , MicroRNAs/metabolismo , Miocárdio/patologia , RNA Mensageiro/metabolismo , Ratos , Fator de Crescimento Transformador beta/metabolismo , Fibrilação Ventricular/patologiaRESUMO
Meckel-Gruber syndrome is a severe autosomal, recessively inherited disorder characterized by bilateral renal cystic dysplasia, developmental defects of the central nervous system (most commonly occipital encephalocele), hepatic ductal dysplasia and cysts and polydactyly. MKS is genetically heterogeneous, with three loci mapped: MKS1, 17q21-24 (ref. 4); MKS2, 11q13 (ref. 5) and MKS3 (ref. 6). We have refined MKS3 mapping to a 12.67-Mb interval (8q21.13-q22.1) that is syntenic to the Wpk locus in rat, which is a model with polycystic kidney disease, agenesis of the corpus callosum and hydrocephalus. Positional cloning of the Wpk gene suggested a MKS3 candidate gene, TMEM67, for which we identified pathogenic mutations for five MKS3-linked consanguineous families. MKS3 is a previously uncharacterized, evolutionarily conserved gene that is expressed at moderate levels in fetal brain, liver and kidney but has widespread, low levels of expression. It encodes a 995-amino acid seven-transmembrane receptor protein of unknown function that we have called meckelin.
Assuntos
Anormalidades Múltiplas/genética , Mutação/genética , Proteínas/genética , Ratos Mutantes/genética , Animais , Sequência de Bases , Análise Mutacional de DNA , Modelos Animais de Doenças , Éxons/genética , Feminino , Marcadores Genéticos , Haplótipos , Humanos , Íntrons/genética , Masculino , Proteínas de Membrana , Dados de Sequência Molecular , Defeitos do Tubo Neural/genética , Linhagem , Mapeamento Físico do Cromossomo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , SíndromeRESUMO
Dichloroacetate (DCA) is a toxicant by-product from the chlorination disinfection process for municipal water. The levels would not affect people with normal renal and liver function. However, people with impaired renal or liver function may have an increased susceptibility to DCA toxicity as those are the organs affected by DCA. People (and rodents) with polycystic kidney disease (PKD) are polyuric, drink more fluids, and have both renal and liver pathology. In PKD, renal tubules and biliary epithelial cells proliferate to form cysts, which can eventually cause renal and/or liver dysfunction. Therefore, PKD may be a predisposing condition with an increased sensitivity to DCA toxicity. PCK rats are an orthologous model of human autosomal recessive PKD and were treated with 75 mg/l DCA in their drinking water. Male and female PCK and male Sprague-Dawley rats were treated from 4 to 8 wk of age, after which the severity of the renal and liver pathology induced by DCA were assessed. Only male PCK rats were adversely affected by DCA treatment, with an increase in the severity of renal cystic disease evinced by an increase in cystic enlargement and proteinuria. In conclusion, the chlorination byproduct DCA may adversely affect those with a preexisting renal disease, especially those who are polydipsic, like those with PKD.
Assuntos
Ácido Dicloroacético/toxicidade , Água Potável/efeitos adversos , Rim Policístico Autossômico Recessivo/induzido quimicamente , Animais , Feminino , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Despite intense investigation, mechanisms that facilitate the emergence of the pre-eclampsia phenotype in women are still unknown. Placental hypoxia, hypertension, proteinuria and oedema are the principal clinical features of this disease. It is speculated that hypoxia-driven disruption of the angiogenic balance involving vascular endothelial growth factor (VEGF)/placenta-derived growth factor (PLGF) and soluble Fms-like tyrosine kinase-1 (sFLT-1, the soluble form of VEGF receptor 1) might contribute to some of the maternal symptoms of pre-eclampsia. However, pre-eclampsia does not develop in all women with high sFLT-1 or low PLGF levels, and it also occurs in some women with low sFLT-1 and high PLGF levels. Moreover, recent experiments strongly suggest that several soluble factors affecting the vasculature are probably elevated because of placental hypoxia in the pre-eclamptic women, indicating that upstream molecular defect(s) may contribute to pre-eclampsia. Here we show that pregnant mice deficient in catechol-O-methyltransferase (COMT) show a pre-eclampsia-like phenotype resulting from an absence of 2-methoxyoestradiol (2-ME), a natural metabolite of oestradiol that is elevated during the third trimester of normal human pregnancy. 2-ME ameliorates all pre-eclampsia-like features without toxicity in the Comt(-/-) pregnant mice and suppresses placental hypoxia, hypoxia-inducible factor-1alpha expression and sFLT-1 elevation. The levels of COMT and 2-ME are significantly lower in women with severe pre-eclampsia. Our studies identify a genetic mouse model for pre-eclampsia and suggest that 2-ME may have utility as a plasma and urine diagnostic marker for this disease, and may also serve as a therapeutic supplement to prevent or treat this disorder.
Assuntos
Catecol O-Metiltransferase/deficiência , Estradiol/análogos & derivados , Estradiol/deficiência , Pré-Eclâmpsia/metabolismo , 2-Metoxiestradiol , Albuminas/análise , Animais , Pressão Sanguínea , Catecol O-Metiltransferase/análise , Catecol O-Metiltransferase/genética , Creatinina/urina , Modelos Animais de Doenças , Estradiol/sangue , Estradiol/urina , Feminino , Humanos , Hipertensão , Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Células Matadoras Naturais/imunologia , Tamanho da Ninhada de Vivíparos , Masculino , Camundongos , Placenta/enzimologia , Placenta/patologia , Pré-Eclâmpsia/sangue , Pré-Eclâmpsia/enzimologia , Pré-Eclâmpsia/urina , Gravidez , Proteinúria , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/sangueRESUMO
The morphology of the nephrons of the coelacanth Latimeria chalumnae was investigated by electron microscopy. Each nephron is composed of a large renal corpuscle with well vascularized glomerulus, ciliated neck segment, proximal tubule divided into first and second proximal segments, ciliated intermediate segment, distal tubule, collecting tubule, and duct. The podocytes of visceral epithelium contain large bi-lobed nuclei and their surface membranes pinch off vesicles into the cytoplasm. The processes of the podocytes give rise to pedicels that enclose narrow filtration slits. The endothelium of glomerular capillaries is attenuated and fenestrated. The short cytoplasmic processes of mesangial cells do not penetrate deeply into the sub-endothelial lamina. The glomerular basement membrane is about 286 nm in thickness. The pedicels also arise from podocyte cell bodies, and are connected by diaphragms and enclose slits, which open into narrow urinary spaces between podocytes. The cuboidal cells of the short neck segment display cilia with a characteristic pattern of 9+2 microtubules. The first proximal tubule segment differs from the second proximal segment in having densely packed microvilli, prominent endocytotic-lysosomal apparatus, and numerous basal membrane infoldings associated with mitochondria. The lateral cell membranes like those of other segments are straight and joined by desmosomes and apical adhering and tight junctions. The distal tubules display few short luminal microvilli and numerous basal mitochondria. The distal tubule, collecting tubule and duct are devoid of intercalated cells. The ultrastructure of the L chalumnae nephrons correlates well with their osmoregulatory function and resembles that of freshwater rainbow trout.
Assuntos
Peixes/anatomia & histologia , Rim/anatomia & histologia , Animais , FemininoRESUMO
Hypoxia is associated with tissue injury and fibrosis but its functional role in fibroblast activation and tissue repair/regeneration is unknown. Using kidney injury as a model system, we demonstrate that injured epithelial cells produce an increased number of exosomes with defined genetic information to activate fibroblasts. Exosomes released by injured epithelial cells promote proliferation, α-smooth muscle actin expression, F-actin expression, and type I collagen production in fibroblasts. Fibroblast activation is dependent on exosomes delivering TGF-ß1 mRNA among other yet to be identified moieties. This study suggests that TGF-ß1 mRNA transported by exosomes constitutes a rapid response to initiate tissue repair/regenerative responses and activation of fibroblasts when resident parenchyma is injured. The results also inform potential utility of exosome-targeted therapies to control tissue fibrosis.
Assuntos
Rim/lesões , Rim/fisiopatologia , Regeneração/fisiologia , Fator de Crescimento Transformador beta1/fisiologia , Animais , Hipóxia Celular/fisiologia , Células Cultivadas , Células Epiteliais/fisiologia , Exossomos/fisiologia , Fibroblastos/fisiologia , Fibrose , Humanos , Rim/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Células NIH 3T3 , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regeneração/genética , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Fator de Crescimento Transformador beta1/genéticaRESUMO
Mutations in inversin cause nephronophthisis type II, an autosomal recessive form of polycystic kidney disease associated with situs inversus, dilatation, and kidney cyst formation. Since cyst formation may represent a planar polarity defect, we investigated whether inversin plays a role in cell division. In developing nephrons from inv-/- mouse embryos we observed heterogeneity of nuclear size, increased cell membrane perimeters, cells with double cilia, and increased frequency of binuclear cells. Depletion of inversin by siRNA in cultured mammalian cells leads to an increase in bi- or multinucleated cells. While spindle assembly, contractile ring formation, or furrow ingression appears normal in the absence of inversin, mitotic cell rounding and the underlying rearrangement of the cortical actin cytoskeleton are perturbed. We find that inversin loss causes extensive filopodia formation in both interphase and mitotic cells. These cells also fail to round up in metaphase. The resultant spindle positioning defects lead to asymmetric division plane formation and cell division. In a cell motility assay, fibroblasts isolated from inv-/- mouse embryos migrate at half the speed of wild-type fibroblasts. Together these data suggest that inversin is a regulator of cortical actin required for cell rounding and spindle positioning during mitosis. Furthermore, cell division defects resulting from improper spindle position and perturbed actin organization contribute to altered nephron morphogenesis in the absence of inversin.
Assuntos
Actinas/fisiologia , Córtex Renal/citologia , Mitose/fisiologia , Fatores de Transcrição/metabolismo , Animais , Ensaios de Migração Celular , Células HEK293 , Células HeLa , Humanos , Córtex Renal/embriologia , Camundongos , Camundongos Knockout , Microscopia Confocal , Fatores de Transcrição/genéticaRESUMO
Gene therapy has been proposed as a novel alternative to treat kidney disease. This goal has been hindered by the inability to reliably deliver transgenes to target cells throughout the kidney, while minimizing injury. Since hydrodynamic forces have previously shown promising results, we optimized this approach and designed a method that utilizes retrograde renal vein injections to facilitate transgene expression in rat kidneys. We show, using intravital fluorescence two-photon microscopy, that fluorescent albumin and dextrans injected into the renal vein under defined conditions of hydrodynamic pressure distribute broadly throughout the kidney in live animals. We found injection parameters that result in no kidney injury as determined by intravital microscopy, histology, and serum creatinine measurements. Plasmids, baculovirus, and adenovirus vectors, designed to express EGFP, EGFP-actin, EGFP-occludin, EGFP-tubulin, tdTomato-H2B, or RFP-actin fusion proteins, were introduced into live kidneys in a similar fashion. Gene expression was then observed in live and ex vivo kidneys using two-photon imaging and confocal laser scanning microscopy. We recorded widespread fluorescent protein expression lasting more than 1 mo after introduction of transgenes. Plasmid and adenovirus vectors provided gene transfer efficiencies ranging from 50 to 90%, compared with 10-50% using baculovirus. Using plasmids and adenovirus, fluorescent protein expression was observed 1) in proximal and distal tubule epithelial cells; 2) within glomeruli; and 3) within the peritubular interstitium. In isolated kidneys, fluorescent protein expression was observed from the cortex to the papilla. These results provide a robust approach for gene delivery and the study of protein function in live mammal kidneys.
Assuntos
Adenoviridae/genética , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Rim/metabolismo , Plasmídeos/genética , Transgenes/genética , Actinas/genética , Actinas/metabolismo , Animais , Feminino , Terapia Genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hidrodinâmica , Rim/citologia , Masculino , Microscopia Confocal , Ocludina/genética , Ocludina/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMO
BACKGROUND/AIMS: Patients with chronic kidney disease (CKD) have a high prevalence of periodontal disease that may predispose to tooth loss and inflammation. The goal of this study was to test the hypotheses that a genetic rat model of progressive CKD would exhibit altered oral bone properties and that treatment with either bisphosphonates or calcium could attenuate these adverse changes. METHODS: At 25 weeks of age, rats were treated with zoledronate (ZOL), calcium gluconate, or their combination for 5 or 10 weeks. Mandible bone properties were assessed using micro-computed tomography to determine bone volume (BV/TV) and cementum-enamel junction to alveolar crest distance (CEJ-AC). RESULTS: Untreated CKD animals had significantly lower BV/TV at both 30 (-5%) and 35 (-14%) weeks of age and higher CEJ-AC (+27 and 29%) compared to normal animals. CKD animals had a significantly higher parathyroid hormone (PTH) compared to normal animals, yet similar levels of C-reactive protein (CRP). ZOL treatment normalized BV/TV over the first 5 weeks but this benefit was lost by 10 weeks. Calcium treatment, alone or in combination with ZOL, was effective in normalizing BV/TV at both time points. Neither ZOL nor calcium was able to correct the higher CEJ-AC caused by CKD. Calcium, but not ZOL, significantly reduced serum PTH, while neither treatment affected CRP. CONCLUSIONS: (i) This progressive animal model of CKD shows a clear mandibular skeletal phenotype consistent with periodontitis, (ii) the periodontitis is not associated with systemic inflammation as measured by CRP, and (iii) reducing PTH has positive effects on the mandible phenotype.
Assuntos
Doenças Ósseas/complicações , Doenças Ósseas/tratamento farmacológico , Cálcio/uso terapêutico , Difosfonatos/uso terapêutico , Imidazóis/uso terapêutico , Nefropatias/complicações , Mandíbula/efeitos dos fármacos , Animais , Doenças Ósseas/diagnóstico por imagem , Proteína C-Reativa/química , Cemento Dentário/química , Esmalte Dentário/química , Modelos Animais de Doenças , Heterozigoto , Inflamação , Nefropatias/diagnóstico por imagem , Masculino , Mandíbula/diagnóstico por imagem , Mandíbula/patologia , Hormônio Paratireóideo/metabolismo , Periodontite/tratamento farmacológico , Fenótipo , Ratos , Tomografia Computadorizada por Raios X , Microtomografia por Raio-X , Ácido ZoledrônicoRESUMO
BACKGROUND: Transglutaminase 2 (TGM2) is a calcium-dependent enzyme that can cross-link nearly all extracellular matrix (ECM) proteins and can facilitate cell-ECM interaction through integrins. Given the importance of the ECM in vascular calcification we tested the hypothesis that increased TGM2 activity may accelerate vascular calcification in chronic kidney disease (CKD). METHODS: We utilized thoracic aortas and vascular smooth muscle cells (VSMC) from the Cy/+ rat, a model of progressive CKD that develops arterial calcification on a normal phosphorus diet, compared to normal rats. RESULTS: VSMC isolated from CKD rats had increased expression and activity of TGM2 compared to cells from normal rats. The increased calcification and expression of alkaline phosphatase activity observed in VSMC from CKD rats compared to normal was inhibited in a dose-dependent manner with the TGM inhibitors cystamine and Z006. Matrix vesicles (MV) from CKD rat VSMC also had increased TGM2 expression and the calcification of MV on type I collagen could be inhibited with cystamine and accelerated by exogenous cross-linking of fibronectin or type I collagen with TGM2. Finally, the calcification of aorta rings from CKD rats in ex vivo cultures was inhibited with TGM2 inhibitor. CONCLUSION: These data demonstrate a role of TGM2 in the pathogenesis of vascular calcification in CKD through enhancement of MV-ECM calcification.
Assuntos
Exossomos/fisiologia , Matriz Extracelular/enzimologia , Proteínas de Ligação ao GTP/metabolismo , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/enzimologia , Insuficiência Renal Crônica/enzimologia , Transglutaminases/metabolismo , Calcificação Vascular/enzimologia , Animais , Aorta/enzimologia , Estudos de Casos e Controles , Células Cultivadas , Modelos Animais de Doenças , Proteínas da Matriz Extracelular/metabolismo , Proteínas de Ligação ao GTP/antagonistas & inibidores , Proteínas de Ligação ao GTP/fisiologia , Técnicas In Vitro , Proteína 2 Glutamina gama-Glutamiltransferase , Ratos , Transglutaminases/antagonistas & inibidores , Transglutaminases/fisiologiaRESUMO
Approximately 60,000 patients in the United States are waiting for a kidney transplant due to genetic, immunologic and environmentally caused kidney failure. Adult human renal stem cells could offer opportunities for autologous transplant and repair of damaged organs. Current data suggest that there are multiple progenitor types in the kidney with distinct localizations. In the present study, we characterize cells derived from human kidney papilla and show their capacity for tubulogenesis. In situ, nestin(+) and CD133/1(+) cells were found extensively intercalated between tubular epithelia in the loops of Henle of renal papilla, but not of the cortex. Populations of primary cells from the renal cortex and renal papilla were isolated by enzymatic digestion from human kidneys unsuited for transplant and immuno-enriched for CD133/1(+) cells. Isolated CD133/1(+) papillary cells were positive for nestin, as well as several human embryonic stem cell markers (SSEA4, Nanog, SOX2, and OCT4/POU5F1) and could be triggered to adopt tubular epithelial and neuronal-like phenotypes. Isolated papillary cells exhibited morphologic plasticity upon modulation of culture conditions and inhibition of asymmetric cell division. Labeled papillary cells readily associated with cortical tubular epithelia in co-culture and 3-dimensional collagen gel cultures. Heterologous organ culture demonstrated that CD133/1(+) progenitors from the papilla and cortex became integrated into developing kidney tubules. Tubular epithelia did not participate in tubulogenesis. Human renal papilla harbor cells with the hallmarks of adult kidney stem/progenitor cells that can be amplified and phenotypically modulated in culture while retaining the capacity to form new kidney tubules. This article is part of a Special Issue entitled: Polycystic Kidney Disease.
Assuntos
Células-Tronco Adultas/citologia , Células-Tronco Adultas/imunologia , Antígenos CD/metabolismo , Glicoproteínas/metabolismo , Medula Renal/citologia , Túbulos Renais/citologia , Túbulos Renais/crescimento & desenvolvimento , Peptídeos/metabolismo , Antígeno AC133 , Células-Tronco Adultas/transplante , Animais , Diferenciação Celular , Separação Celular , Técnicas de Cocultura , Ensaio de Unidades Formadoras de Colônias , Humanos , Camundongos , Técnicas de Cultura de Órgãos , Rim Policístico Autossômico Dominante/terapiaRESUMO
Male gender is a risk factor for progression of polycystic kidney disease (PKD). 17ß-Estradiol (E2) protects experimentally, but clinical use is limited by adverse effects. Novel E2 metabolites provide many benefits of E2 without stimulating the estrogen receptor, and thus may be safer. We hypothesized that E2 metabolites are protective in a model of PKD. Studies were performed in male control Han:SPRD rats, and in cystic males treated with orchiectomy, 2-methoxyestradiol, 2-hydroxyestradiol (2-OHE), or vehicle, from age 3 to 12 wk. Cystic rats exhibited renal functional impairment (â¼50% decrease in glomerular filtration and renal plasma flow rates, P < 0.05) and substantial cyst development (20.5 ± 2.0% of cortex area). 2-OHE was the most effective in limiting cysts (6.0 ± 0.7% of cortex area, P < 0.05 vs. vehicle-treated cystic rats) and preserving function, in association with suppression of proliferation, apoptosis, and angiogenesis markers. Downregulation of p21 expression and increased expression of Akt, the mammalian target of rapamycin (mTOR), and some of its downstream effectors were significantly reversed by 2-OHE. Thus, 2-OHE limits disease progression in a cystic rodent model. Mechanisms include reduced renal cell proliferation, apoptosis, and angiogenesis. These effects may be mediated, at least in part, by preservation of p21 and suppression of Akt and mTOR. Estradiol metabolites may represent a novel, safe intervention to slow progression of PKD.
Assuntos
Estradiol/análogos & derivados , Rim/efeitos dos fármacos , Doenças Renais Policísticas/tratamento farmacológico , 2-Metoxiestradiol , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Progressão da Doença , Estradiol/farmacologia , Estradiol/uso terapêutico , Rim/metabolismo , Rim/patologia , Masculino , Orquiectomia , Doenças Renais Policísticas/metabolismo , Doenças Renais Policísticas/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Resultado do TratamentoRESUMO
The network organization of type IV collagen consisting of α3, α4, and α5 chains in the glomerular basement membrane (GBM) is speculated to involve interactions of the triple helical and NC1 domain of individual α-chains, but in vivo evidence is lacking. To specifically address the contribution of the NC1 domain in the GBM collagen network organization, we generated a mouse with specific loss of α3NC1 domain while keeping the triple helical α3 chain intact by connecting it to the human α5NC1 domain. The absence of α3NC1 domain leads to the complete loss of the α4 chain. The α3 collagenous domain is incapable of incorporating the α5 chain, resulting in the impaired organization of the α3α4α5 chain-containing network. Although the α5 chain can assemble with the α1, α2, and α6 chains, such assembly is incapable of functionally replacing the α3α4α5 protomer. This novel approach to explore the assembly type IV collagen in vivo offers novel insights in the specific role of the NC1 domain in the assembly and function of GBM during health and disease.
Assuntos
Colágeno Tipo IV/química , Colágeno/química , Albuminas/metabolismo , Sequência de Aminoácidos , Animais , Creatina/urina , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Genótipo , Humanos , Rim/metabolismo , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Nefrite Hereditária/genética , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , Homologia de Sequência de AminoácidosRESUMO
The development and progression of renal cysts appears to be driven by reduced cellular calcium and increased cyclic adenosine monophosphate (cAMP) from G-protein-coupled receptors. To test whether treatment with a calcimimetic that stimulates the G-protein-coupled calcium-sensing receptor might normalize cystic epithelial cell intracellular calcium and cAMP, thereby inhibiting cyst progression, we used pcy mice. These animals develop cysts principally in the collecting duct, as do humans with nephronophthisis (NPHP). We administered the calcimimetic R-568 mixed in their food at early or late stages in the pathogenesis of cyst formation. The treatment reduced cyst enlargement, and the early treatment inhibited development of renal fibrosis. Although the effect of later treatment was more modest, both stages of the disease responded positively to treatment. Additionally, R-568 decreased total kidney cAMP in the pcy mice and, in vitro, decreased cAMP levels and cell proliferation, while increasing intracellular calcium in immortalized human autosomal recessive polycystic kidney disease renal epithelial cells. The latter two effects were unique to R-568 and not replicated by raising extracellular calcium. Thus, treating pcy mice with R-568 was effective in reducing cyst progression in this rodent model of NPHP. Direct studies will be needed to determine whether these results can be applied to the human disease.
Assuntos
Compostos de Anilina/farmacologia , Calcimiméticos/farmacologia , Doenças Renais Císticas/patologia , Doenças Renais Císticas/prevenção & controle , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , AMP Cíclico/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Doenças Renais Císticas/genética , Doenças Renais Císticas/metabolismo , Cinesinas/genética , Masculino , Camundongos , Camundongos Mutantes , Mutação de Sentido Incorreto , Fenetilaminas , Rim Policístico Autossômico Recessivo/tratamento farmacológico , Rim Policístico Autossômico Recessivo/metabolismo , Rim Policístico Autossômico Recessivo/patologia , Propilaminas , Receptores de Detecção de Cálcio/agonistasRESUMO
Meckel syndrome (MKS) is a lethal disorder characterized by renal cystic dysplasia, encephalocele, polydactyly and biliary dysgenesis. It is highly genetically heterogeneous with nine different genes implicated in this disorder. MKS is thought to be a ciliopathy because of the range of phenotypes and localization of some of the implicated proteins. However, limited data are available about the phenotypes associated with MKS1 and MKS3, and the published ciliary data are conflicting. Analysis of the wpk rat model of MKS3 revealed functional defects of the connecting cilium in the eye that resulted in lack of formation of the outer segment, whereas infertile wpk males developed spermatids with very short flagella that did not extend beyond the cell body. In wpk renal collecting duct cysts, cilia were generally longer than normal, with additional evidence of cells with multiple primary cilia and centrosome over-duplication. Kidney tissue and cells from MKS1 and MKS3 patients showed defects in centrosome and cilia number, including multi-ciliated respiratory-like epithelia, and longer cilia. Stable shRNA knockdown of Mks1 and Mks3 in IMCD3 cells induced multi-ciliated and multi-centrosomal phenotypes. These studies demonstrate that MKS1 and MKS3 are ciliopathies, with new cilia-related eye and sperm phenotypes defined. MKS1 and MKS3 functions are required for ciliary structure and function, including a role in regulating length and appropriate number through modulating centrosome duplication.
Assuntos
Anormalidades Múltiplas/genética , Centrossomo/metabolismo , Cílios/metabolismo , Proteínas de Membrana/metabolismo , Mutação , Proteínas/metabolismo , Anormalidades Múltiplas/metabolismo , Anormalidades Múltiplas/patologia , Animais , Cílios/genética , Feminino , Humanos , Proteínas de Membrana/genética , Proteínas/genética , Ratos , Ratos WistarRESUMO
Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the slow growth of multiple fluid-filled cysts predominately in the kidney tubules and liver bile ducts. Elucidation of mechanisms that control cyst growth will provide the basis for rational therapeutic intervention. We used electrophysiological methods to identify lysophosphatidic acid (LPA) as a component of cyst fluid and serum that stimulates secretory Cl- transport in the epithelial cell type that lines renal cysts. LPA effects are manifested through receptors located on the basolateral membrane of the epithelial cells resulting in stimulation of channel activity in the apical membrane. Concentrations of LPA measured in human ADPKD cyst fluid and in normal serum are sufficient to maximally stimulate ion transport. Thus, cyst fluid seepage and/or leakage of vascular LPA into the interstitial space are capable of stimulating epithelial cell secretion resulting in cyst enlargement. These observations are particularly relevant to the rapid decline in renal function in late-stage disease and to the "third hit" hypothesis that renal injury exacerbates cyst growth.
Assuntos
Lisofosfolipídeos/farmacologia , Rim Policístico Autossômico Dominante/metabolismo , Amilorida/farmacologia , Linhagem Celular , Cloretos/metabolismo , Líquido Cístico/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Transporte de Íons , Lisofosfolipídeos/análise , Rim Policístico Autossômico Dominante/patologia , Receptores de Ácidos Lisofosfatídicos/metabolismo , Bloqueadores dos Canais de Sódio/farmacologiaRESUMO
BACKGROUND: The objective of the current study was to determine if altered regulation of matrix metalloproteinases (MMPs) may predispose to extracellular matrix degradation, facilitating arterial calcification in chronic kidney disease (CKD) using a progressive model of CKD-MBD, the Cy/+ rat. METHODS: Sera were collected from normal or CKD rats at various times and MMP-2 and MMP-9 levels determined by ELISA or zymography. Aorta tissue was harvested at sacrifice for RT-PCR and immunostaining. Calcification of aorta rings was assessed with MMP inhibitors. RESULTS: There was an increase in MMP-2, MMP-9, TIMP-1, and RUNX-2 expression in the aorta with progressive CKD, and increased MMP-2 activity in the serum. Immunostaining revealed increased expression of MMP-2 and MMP-9 in areas of aorta calcification. There was also an upregulation of MMP-2 and MMP-9 in vascular smooth muscle cells (VSMC) from CKD rats. MMP inhibitors decreased calcification of aorta rings from normal and CKD rats. High phosphorus increased MMP-2 and MMP-9 expressions in VSMC from normal rats but not from CKD rats. CONCLUSION: MMP-2 and MMP-9 expression and activity are increased with progressive CKD, and blockade of MMP activity can inhibit arterial calcification. These data suggest degradation of the extracellular matrix is a critical step in the pathogenesis of arterial calcification in CKD.
Assuntos
Nefropatias/sangue , Nefropatias/complicações , Metaloproteinase 2 da Matriz/fisiologia , Metaloproteinase 9 da Matriz/fisiologia , Calcificação Vascular/etiologia , Animais , Doença Crônica , Metaloproteinase 2 da Matriz/sangue , Metaloproteinase 9 da Matriz/sangue , Ratos , Regulação para CimaRESUMO
BACKGROUND: Meckel syndrome (MKS) is a fatal autosomal recessive condition with prominent renal cystic pathology. Renal protein misexpression was evaluated in the Wpk rat model of human MKS3 gene disease to identify biomarkers for the staging of renal cystic progression. METHODS: Misexpressed proteins were compared between early and late stages of MKS renal cystic disease using proteomic analysis (two-dimensional gel electrophoresis with LC-MS/MS identification) followed by Western blot analysis. RESULTS: A proteomic analysis identified 76 proteins with statistically different, normalized abundance in at least one group. Subsequently, Western blot was used to confirm differential expression in several of these and polycystic kidney disease (PKD)-associated proteins. Galectin-1 and vimentin were identified as overexpressed proteins, which have been previously found in the jck mouse model of nephronophthisis 9. Ciliopathic PKD proteins, polycystins 1 & 2, and fibrocystin were also differentially expressed in Wpk kidney. CONCLUSION: In the Wpk rat, misexpressed proteins were identified that were also implicated in other forms of cystic disease. Numerous proteins were either over- or underexpressed in late-stage disease. Differences in protein expression may serve as biomarkers of cystic disease and its progression.
Assuntos
Rim/metabolismo , Proteoma/metabolismo , Animais , Western Blotting , Transtornos da Motilidade Ciliar/genética , Transtornos da Motilidade Ciliar/metabolismo , Modelos Animais de Doenças , Eletroforese em Gel Bidimensional , Encefalocele/genética , Encefalocele/metabolismo , Proteína 3 Ligante de Ácido Graxo , Proteínas de Ligação a Ácido Graxo/metabolismo , Feminino , Galectinas/metabolismo , Humanos , Laminina/metabolismo , Masculino , Camundongos , Doenças Renais Policísticas/genética , Doenças Renais Policísticas/metabolismo , Proteômica , Ratos , Ratos Mutantes , Receptores de Superfície Celular/metabolismo , Canais de Cátion TRPP/metabolismo , Espectrometria de Massas em Tandem , Vimentina/metabolismoRESUMO
The polycystic kidney diseases (PKDs) are a group of genetic disorders causing significant renal failure and death in children and adults. There are no effective treatments. Two childhood forms, autosomal recessive PKD (ARPKD) and nephronophthisis (NPH), are characterized by collecting-duct cysts. We used animal models orthologous to the human disorders to test whether a vasopressin V2 receptor (VPV2R) antagonist, OPC31260, would be effective against early or established disease. Adenosine-3',5'-cyclic monophosphate (cAMP) has a major role in cystogenesis, and the VPV2R is the major cAMP agonist in the collecting duct. OPC31260 administration lowered renal cAMP, inhibited disease development and either halted progression or caused regression of established disease. These results indicate that OPC31260 may be an effective treatment for these disorders and that clinical trials should be considered.
Assuntos
Antagonistas dos Receptores de Hormônios Antidiuréticos , Benzazepinas/uso terapêutico , Doenças Renais Policísticas/tratamento farmacológico , Doenças Renais Policísticas/metabolismo , Receptores de Vasopressinas/metabolismo , Animais , Aquaporina 2 , Aquaporina 6 , Aquaporinas/genética , Aquaporinas/metabolismo , Benzazepinas/metabolismo , AMP Cíclico/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Humanos , Rim/metabolismo , Rim/patologia , Camundongos , Doenças Renais Policísticas/fisiopatologia , Proteínas/genética , Proteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Canais de Cátion TRPPRESUMO
Autosomal dominant polycystic kidney disease (ADPKD) is a leading cause of end-stage renal disease. The vasopressin V2 receptor (VPV2R) antagonist OPC31260 has been effective in two animal models of PKD with pathologies that are probably related. Here we show, in a mouse model of ADPKD (Pkd2(-/tm1Som)), a similar cellular phenotype and response to OPC31260 treatment, with reduction of renal cyclic AMP (cAMP) levels, prevention of renal enlargement, marked inhibition of cystogenesis and protection of renal function.