title: New autophagy reporter mice reveal dynamics of proximal tubular autophagy.
abstract: The accumulation of autophagosomes in postischemic kidneys may be renoprotective, but whether this accumulation results from the induction of autophagy or from obstruction within the autophagic process is unknown. Utilizing the differential pH sensitivities of red fluorescent protein (RFP; pKa 4.5) and enhanced green fluorescent protein (EGFP; pKa 5.9), we generated CAG-RFP-EGFP-LC3 mice to distinguish early autophagic vacuoles from autolysosomes. In vitro and in vivo studies confirmed that in response to nutrient deprivation, renal epithelial cells in CAG-RFP-EGFP-LC3 mice produce autophagic vacuoles expressing RFP and EGFP puncta. EGFP fluorescence diminished substantially in the acidic environment of the autolysosomes, whereas bright RFP signals remained. Under normal conditions, nephrons expressed few EGFP and RFP puncta, but ischemia-reperfusion injury (IRI) led to dynamic changes in the proximal tubules, with increased numbers of RFP and EGFP puncta that peaked at 1 day after IRI. The number of EGFP puncta returned to control levels at 3 days after IRI, whereas the high levels of RFP puncta persisted, indicating autophagy initiation at day 1 and autophagosome clearance during renal recovery at day 3. Notably, proliferation decreased in cells containing RFP puncta, suggesting that autophagic cells are less likely to divide for tubular repair. Furthermore, 87% of proximal tubular cells with activated mechanistic target of rapamycin (mTOR), which prevents autophagy, contained no RFP puncta. Conversely, inhibition of mTOR complex 1 induced RFP and EGFP expression and decreased cell proliferation. In summary, our results highlight the dynamic regulation of autophagy in postischemic kidneys and suggest a role of mTOR in autophagy resolution during renal repair.
authors.affiliation: Department of Pediatrics, Columbia University College of Physicians and Surgeons, New York, New York; and.
authors.forename: Ling
authors.initials: L
authors.lastname: Li
authors.forename: Zhao V
authors.initials: ZV
authors.lastname: Wang
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Hill
authors.forename: Fangming
authors.initials: F
authors.lastname: Lin
mesh_terms: D058186:Acute Kidney Injury / Q000473:pathology*; D000818:Animals; D001343:Autophagy / Q000235:genetics / Q000502:physiology*; D002478:Cells, Cultured; D002738:Chloroquine / Q000494:pharmacology; D003470:Culture Media / Q000494:pharmacology; D004847:Epithelial Cells / Q000737:chemistry / Q000473:pathology; D005260:Female; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007687:Kidney Tubules, Proximal / Q000473:pathology* / Q000502:physiology; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D000076222:Mechanistic Target of Rapamycin Complex 1; D051379:Mice; D008822:Mice, Transgenic; D008869:Microtubule-Associated Proteins / Q000235:genetics; D046912:Multiprotein Complexes / Q000037:antagonists & inhibitors / Q000502:physiology; D010588:Phagosomes / Q000737:chemistry / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012038:Regeneration / Q000502:physiology; D015427:Reperfusion Injury / Q000473:pathology*; D020123:Sirolimus / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000502:physiology; D014617:Vacuoles / Q000737:chemistry / Q000502:physiology; D000097573:Red Fluorescent Protein
chemical_list: D002738:Chloroquine; D003470:Culture Media; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D000076222:Mechanistic Target of Rapamycin Complex 1; D008869:Microtubule-Associated Proteins; D046912:Multiprotein Complexes; D011993:Recombinant Fusion Proteins; D020123:Sirolimus; D058570:TOR Serine-Threonine Kinases; D000097573:Red Fluorescent Protein; C480860:Map1lc3b protein, mouse; C485184:enhanced green fluorescent protein; C546843:mTOR protein, mouse