PMID- 24179166
OWN - NLM
STAT- MEDLINE
DCOM- 20140414
LR  - 20231213
IS  - 1533-3450 (Electronic)
IS  - 1046-6673 (Print)
IS  - 1046-6673 (Linking)
VI  - 25
IP  - 2
DP  - 2014 Feb
TI  - New autophagy reporter mice reveal dynamics of proximal tubular autophagy.
PG  - 305-15
LID - 10.1681/ASN.2013040374 [doi]
AB  - 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.
FAU - Li, Ling
AU  - Li L
AD  - Department of Pediatrics, Columbia University College of Physicians and Surgeons, 
      New York, New York; and.
FAU - Wang, Zhao V
AU  - Wang ZV
FAU - Hill, Joseph A
AU  - Hill JA
FAU - Lin, Fangming
AU  - Lin F
LA  - eng
GR  - U01 HL100401/HL/NHLBI NIH HHS/United States
GR  - R01 DK083411/DK/NIDDK NIH HHS/United States
GR  - HL-100401/HL/NHLBI NIH HHS/United States
GR  - R01 HL080144/HL/NHLBI NIH HHS/United States
GR  - HL-0980842/HL/NHLBI NIH HHS/United States
GR  - HL-080144/HL/NHLBI NIH HHS/United States
GR  - R01DK083411/DK/NIDDK NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Research Support, Non-U.S. Gov't
DEP - 20131031
PL  - United States
TA  - J Am Soc Nephrol
JT  - Journal of the American Society of Nephrology : JASN
JID - 9013836
RN  - 0 (Culture Media)
RN  - 0 (Luminescent Proteins)
RN  - 0 (Map1lc3b protein, mouse)
RN  - 0 (Microtubule-Associated Proteins)
RN  - 0 (Multiprotein Complexes)
RN  - 0 (Recombinant Fusion Proteins)
RN  - 0 (enhanced green fluorescent protein)
RN  - 147336-22-9 (Green Fluorescent Proteins)
RN  - 886U3H6UFF (Chloroquine)
RN  - EC 2.7.1.1 (mTOR protein, mouse)
RN  - EC 2.7.11.1 (Mechanistic Target of Rapamycin Complex 1)
RN  - EC 2.7.11.1 (TOR Serine-Threonine Kinases)
RN  - W36ZG6FT64 (Sirolimus)
SB  - IM
MH  - Acute Kidney Injury/*pathology
MH  - Animals
MH  - Autophagy/genetics/*physiology
MH  - Cells, Cultured
MH  - Chloroquine/pharmacology
MH  - Culture Media/pharmacology
MH  - Epithelial Cells/chemistry/pathology
MH  - Female
MH  - *Genes, Reporter
MH  - Green Fluorescent Proteins/analysis/genetics
MH  - Hydrogen-Ion Concentration
MH  - Kidney Tubules, Proximal/*pathology/physiology
MH  - Luminescent Proteins/analysis/genetics
MH  - Mechanistic Target of Rapamycin Complex 1
MH  - Mice
MH  - Mice, Transgenic
MH  - Microtubule-Associated Proteins/genetics
MH  - Multiprotein Complexes/antagonists & inhibitors/physiology
MH  - Phagosomes/chemistry/physiology
MH  - Recombinant Fusion Proteins/metabolism
MH  - Regeneration/physiology
MH  - Reperfusion Injury/*pathology
MH  - Sirolimus/pharmacology
MH  - TOR Serine-Threonine Kinases/antagonists & inhibitors/physiology
MH  - Vacuoles/chemistry/physiology
MH  - Red Fluorescent Protein
PMC - PMC3904563
EDAT- 2013/11/02 06:00
MHDA- 2014/04/15 06:00
PMCR- 2015/02/01
CRDT- 2013/11/02 06:00
PHST- 2013/11/02 06:00 [entrez]
PHST- 2013/11/02 06:00 [pubmed]
PHST- 2014/04/15 06:00 [medline]
PHST- 2015/02/01 00:00 [pmc-release]
AID - ASN.2013040374 [pii]
AID - 2013040374 [pii]
AID - 10.1681/ASN.2013040374 [doi]
PST - ppublish
SO  - J Am Soc Nephrol. 2014 Feb;25(2):305-15. doi: 10.1681/ASN.2013040374. Epub 2013 
      Oct 31.