New autophagy reporter mice reveal dynamics of proximal tubular autophagy

Ling Li, Zhao Wang, Joseph A Hill, Fangming Lin

Research output: Contribution to journalArticle

53 Citations (Scopus)

Abstract

The accumulation of autophagosomes in postischemic kidneys may be renoprotective, but whether this accumulation results from the inductionof 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 todynamic changesin the proximaltubules, 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.

Original languageEnglish (US)
Pages (from-to)305-315
Number of pages11
JournalJournal of the American Society of Nephrology
Volume25
Issue number2
DOIs
StatePublished - Feb 2014

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Autophagy
Kidney
Reperfusion Injury
Sirolimus
Vacuoles
Nephrons
Fluorescence
Epithelial Cells
Cell Proliferation
Food
Autophagosomes

ASJC Scopus subject areas

  • Nephrology

Cite this

New autophagy reporter mice reveal dynamics of proximal tubular autophagy. / Li, Ling; Wang, Zhao; Hill, Joseph A; Lin, Fangming.

In: Journal of the American Society of Nephrology, Vol. 25, No. 2, 02.2014, p. 305-315.

Research output: Contribution to journalArticle

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