RESUMO
Fibrosis is an exaggerated form of tissue repair that occurs with serious damage or repetitive injury and ultimately leads to organ failure due to the excessive scarring. Increased calcium ion entry through the TRPC6 channel has been associated with the pathogenesis of heart and glomerular diseases, but its role in renal interstitial fibrosis is unknown. We studied this by deletion of Trpc6 in mice and found it decreased unilateral ureteral obstruction-induced interstitial fibrosis and blunted increased mRNA expression of fibrosis-related genes in the ureteral obstructed kidney relative to that in the kidney of wild-type mice. Administration of BTP2, a pyrazol derivative known to inhibit function of several TRPC channels, also ameliorated obstruction-induced renal fibrosis and gene expression in wild-type mice. BTP2 inhibited carbachol-activated TRPC3 and TRPC6 channel activities in HEK293 cells. Ureteral obstruction caused over a 10-fold increase in mRNA expression for TRPC3 as well as TRPC6 in the kidneys of obstructed relative to the sham-operated mice. The magnitude of protection against obstruction-induced fibrosis in Trpc3 and Trpc6 double knockout mice was not different from that in Trpc6 knockout mice. Klotho, a membrane and soluble protein predominantly produced in the kidney, is known to confer protection against renal fibrosis. Administration of soluble klotho significantly reduced obstruction-induced renal fibrosis in wild-type mice, but not in Trpc6 knockout mice, indicating that klotho and TRPC6 inhibition act in the same pathway to protect against obstruction-induced renal fibrosis. Thus klotho and TRPC6 may be pharmacologic targets for treating renal fibrosis.
Assuntos
Anilidas/farmacologia , Glucuronidase/farmacologia , Nefropatias/prevenção & controle , Rim/efeitos dos fármacos , Canais de Cátion TRPC/antagonistas & inibidores , Tiadiazóis/farmacologia , Obstrução Ureteral/tratamento farmacológico , Agentes Urológicos/farmacologia , Animais , Citoproteção , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Fibrose , Regulação da Expressão Gênica , Predisposição Genética para Doença , Células HEK293 , Humanos , Rim/metabolismo , Rim/patologia , Nefropatias/genética , Nefropatias/metabolismo , Nefropatias/patologia , Proteínas Klotho , Masculino , Camundongos da Linhagem 129 , Camundongos Knockout , Fenótipo , RNA Mensageiro , Transdução de Sinais/efeitos dos fármacos , Canais de Cátion TRPC/deficiência , Canais de Cátion TRPC/genética , Canais de Cátion TRPC/metabolismo , Canal de Cátion TRPC6 , Transfecção , Obstrução Ureteral/genética , Obstrução Ureteral/metabolismo , Obstrução Ureteral/patologiaRESUMO
Filamentous inclusions of the microtubule-associated protein, tau, define a variety of neurodegenerative diseases known as tauopathies, including Alzheimer's disease (AD). To better understand the role of tau-mediated effects on pathophysiology and global central nervous system function, we extensively characterized gene expression, pathology and behavior of the rTg4510 mouse model, which overexpresses a mutant form of human tau that causes Frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). We found that the most predominantly altered gene expression pathways in rTg4510 mice were in inflammatory processes. These results closely matched the causal immune function and microglial gene-regulatory network recently identified in AD. We identified additional gene expression changes by laser microdissecting specific regions of the hippocampus, which highlighted alterations in neuronal network activity. Expression of inflammatory genes and markers of neuronal activity changed as a function of age in rTg4510 mice and coincided with behavioral deficits. Inflammatory changes were tau-dependent, as they were reversed by suppression of the tau transgene. Our results suggest that the alterations in microglial phenotypes that appear to contribute to the pathogenesis of Alzheimer's disease may be driven by tau dysfunction, in addition to the direct effects of beta-amyloid.
Assuntos
Doença de Alzheimer/genética , Expressão Gênica/genética , Redes Reguladoras de Genes/genética , Inflamação/genética , Proteínas tau/genética , Animais , Cromossomos Humanos Par 17/genética , Modelos Animais de Doenças , Feminino , Demência Frontotemporal/genética , Hipocampo/metabolismo , Humanos , Camundongos , Microglia/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Doenças Neurodegenerativas/genética , Neurônios/metabolismo , Transtornos Parkinsonianos/genéticaRESUMO
Embryonic stem (ES) cells offer unprecedented opportunities for in vitro drug discovery and safety assessment of compounds. Cardiomyocytes derived from ES cells enable development of predictive cardiotoxicity models to increase the safety of novel drugs. Heterogeneity of differentiated ES cells limits the development of reliable in vitro models for compound screening. We report an innovative and robust approach to isolate ES-derived cardiomyocytes using laser microdissection and pressure catapulting (LMPC). LMPC cells were readily applied onto 96-well format in vitro pharmacology assays. The expression of developmental and functional cardiac markers, Nkx 2.5, MLC2V, GATA-4, Connexin 43, Connexin 45, Serca-2a, cardiac alpha actin, and phospholamban, among others, was confirmed in LMPC ES-derived cardiomyocytes. Functional assays exhibited cardiac-like response to increased extracellular calcium (5.4 mM extracellular Ca2+) and L-type calcium channel antagonist (1 microM nifedipine). In conclusion, laser microdissection and pressure catapulting is a robust technology to isolate homogeneous ES-derived cell types from heterogeneous populations applicable to assay development.