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1.
J Vis Exp ; (141)2018 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-30507918

RESUMO

The regulation of LDL cholesterol uptake through LDLR-mediated endocytosis is an important area of study in various major pathologies including metabolic disorder, cardiovascular disease, and kidney disease. Currently, there is no available method to assess LDL uptake while simultaneously monitoring for health of the cells. The current study presents a protocol, using a live cell imaging analysis system, to acquire serial measurements of LDL influx with concurrent monitoring for cell health. This novel technique is tested in three human cell lines (hepatic, renal tubular epithelial, and coronary artery endothelial cells) over a four-hour time course. Moreover, the sensitivity of this technique is validated with well-known LDL uptake inhibitors, Dynasore and recombinant PCSK9 protein, as well as by an LDL uptake promoter, Simvastatin. Taken together, this method provides a medium-to-high throughput platform for simultaneously screening pharmacological activity as well as monitoring of cell morphology, hence cytotoxicity of compounds regulating LDL influx. The analysis can be used with different imaging systems and analytical software.


Assuntos
Membrana Celular/metabolismo , LDL-Colesterol/metabolismo , Imagem com Lapso de Tempo/métodos , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Linhagem Celular , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , LDL-Colesterol/agonistas , LDL-Colesterol/análise , Humanos , Hipolipemiantes/farmacologia , Pró-Proteína Convertase 9/análise , Pró-Proteína Convertase 9/metabolismo , Receptores de LDL/análise , Receptores de LDL/metabolismo , Serina Endopeptidases/análise , Serina Endopeptidases/metabolismo , Sinvastatina/farmacologia
2.
JCI Insight ; 3(6)2018 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-29563333

RESUMO

Alport syndrome is a rare hereditary renal disorder with no etiologic therapy. We found that osteopontin (OPN) is highly expressed in the renal tubules of the Alport mouse and plays a causative pathological role. OPN genetic deletion ameliorated albuminuria, hypertension, tubulointerstitial proliferation, renal apoptosis, and hearing and visual deficits in the Alport mouse. In Alport renal tubules we found extensive cholesterol accumulation and increased protein expression of dynamin-3 (DNM3) and LDL receptor (LDLR) in addition to dysmorphic mitochondria with defective bioenergetics. Increased pathological cholesterol influx was confirmed by a remarkably increased uptake of injected DiI-LDL cholesterol by Alport renal tubules, and by the improved lifespan of the Alport mice when crossed with the Ldlr-/- mice with defective cholesterol influx. Moreover, OPN-deficient Alport mice demonstrated significant reduction of DNM3 and LDLR expression. In human renal epithelial cells, overexpressing DNM3 resulted in elevated LDLR protein expression and defective mitochondrial respiration. Our results suggest a potentially new pathway in Alport pathology where tubular OPN causes DNM3- and LDLR-mediated enhanced cholesterol influx and impaired mitochondrial respiration.


Assuntos
Autoantígenos/metabolismo , Colágeno Tipo IV/metabolismo , Túbulos Renais/metabolismo , Mitocôndrias/metabolismo , Nefrite Hereditária/metabolismo , Osteopontina/metabolismo , Albuminúria/metabolismo , Animais , Apoptose , Autoantígenos/genética , Pressão Sanguínea , Colesterol/metabolismo , Colágeno/metabolismo , Colágeno Tipo IV/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Dinamina III/metabolismo , Células Epiteliais/metabolismo , Ácidos Graxos/metabolismo , Deleção de Genes , Testes Auditivos , Humanos , Hipertensão/metabolismo , Rim/metabolismo , Rim/patologia , Túbulos Renais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nefrite Hereditária/genética , Osteopontina/genética , Receptores de LDL , Smegmamorpha/metabolismo , Transcriptoma , Triglicerídeos/análise
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