RESUMO
The steps governing healing with or without fibrosis within the same microenvironment are unclear. After acute kidney injury (AKI), injured proximal tubular epithelial cells activate SOX9 for self-restoration. Using a multimodal approach for a head-to-head comparison of injury-induced SOX9 lineages, we identified a dynamic SOX9 switch in repairing epithelia. Lineages that regenerated epithelia silenced SOX9 and healed without fibrosis (SOX9on-off). By contrast, lineages with unrestored apicobasal polarity maintained SOX9 activity in sustained efforts to regenerate, which were identified as a SOX9on-on Cadherin6pos cell state. These reprogrammed cells generated substantial single-cell WNT activity to provoke a fibroproliferative response in adjacent fibroblasts, driving AKI to chronic kidney disease. Transplanted human kidneys displayed similar SOX9/CDH6/WNT2B responses. Thus, we have uncovered a sensor of epithelial repair status, the activity of which determines regeneration with or without fibrosis.
Assuntos
Injúria Renal Aguda , Túbulos Renais Proximais , Rim , Insuficiência Renal Crônica , Fatores de Transcrição SOX9 , Animais , Humanos , Camundongos , Injúria Renal Aguda/genética , Injúria Renal Aguda/patologia , Células Epiteliais , Fibrose , Rim/patologia , Regeneração , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/patologia , Fatores de Transcrição SOX9/genética , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/metabolismoRESUMO
The maintenance of cellular function relies on the close regulation of adenosine triphosphate (ATP) synthesis and hydrolysis. ATP hydrolysis by mitochondrial ATP Synthase (CV) is induced by loss of proton motive force and inhibited by the mitochondrial protein ATPase inhibitor (ATPIF1). The extent of CV hydrolytic activity and its impact on cellular energetics remains unknown due to the lack of selective hydrolysis inhibitors of CV. We find that CV hydrolytic activity takes place in coupled intact mitochondria and is increased by respiratory chain defects. We identified (+)-Epicatechin as a selective inhibitor of ATP hydrolysis that binds CV while preventing the binding of ATPIF1. In cells with Complex-III deficiency, we show that inhibition of CV hydrolytic activity by (+)-Epichatechin is sufficient to restore ATP content without restoring respiratory function. Inhibition of CV-ATP hydrolysis in a mouse model of Duchenne Muscular Dystrophy is sufficient to improve muscle force without any increase in mitochondrial content. We conclude that the impact of compromised mitochondrial respiration can be lessened using hydrolysis-selective inhibitors of CV.
Assuntos
Trifosfato de Adenosina , Mitocôndrias , Camundongos , Animais , Trifosfato de Adenosina/metabolismo , Mitocôndrias/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Proteínas/metabolismo , Homeostase , HidróliseRESUMO
This protocol focuses on the cloning and stable integration of sequences of interest by the use of a mosaic analysis with dual recombinases (MADR) plasmid that includes fusion proteins or independent proteins under the control of 2A peptide or IRES elements. Additionally, we describe how to generate a neural stem cell culture from Gt(ROSA)26Sortm4(ACTB-tdTomato, EGFP)Luo/J mice, and validate the MADR plasmids in vitro and in vivo by neonatal mouse brain electroporation. This protocol can be generalized to analyze any transgenic element using MADR technology. For complete details on the use and execution of this protocol, please refer to Kim et al. (2019).
Assuntos
Engenharia Genética/métodos , Vetores Genéticos/química , Recombinases/genética , Animais , Animais Geneticamente Modificados , Eletroporação , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Proteínas Luminescentes/genética , Camundongos , Plasmídeos/genética , Recombinases/metabolismo , Transfecção , Proteína Vermelha FluorescenteRESUMO
In situ transgenesis methods such as viruses and electroporation can rapidly create somatic transgenic mice but lack control over copy number, zygosity, and locus specificity. Here we establish mosaic analysis by dual recombinase-mediated cassette exchange (MADR), which permits stable labeling of mutant cells expressing transgenic elements from precisely defined chromosomal loci. We provide a toolkit of MADR elements for combination labeling, inducible and reversible transgene manipulation, VCre recombinase expression, and transgenesis of human cells. Further, we demonstrate the versatility of MADR by creating glioma models with mixed reporter-identified zygosity or with "personalized" driver mutations from pediatric glioma. MADR is extensible to thousands of existing mouse lines, providing a flexible platform to democratize the generation of somatic mosaic mice. VIDEO ABSTRACT.