Fig 1.
LPS Induces Autophagy in RTEC: Western Blot for LC3 in HPT1b cells incubated over increasing time with 1 microgram /mL LPS in the absence (A and B) or presence of 100nM Baf A1 (C and D). LPS-induced autophagy was confirmed by immunofluorescence staining of LC3 in HPT1b cells before (E) and after (F) 24-hour treatment with 1 microgram /mL LPS.
Fig 2.
LPS Induces Autophagy in RTEC in vivo: GFP-LC3 reporter mice demonstrate diffuse faint GFP fluorescence in RTEC of control-injected mice (A,B; 630X and 1000X, respectively). LPS injection induced marked increase in abundance of green fluorescent puncta in RTEC (C,D; 630X and 1000X, respectively). Western blot for LC3 demonstrates increased LC3-II accumulation in LPS-treated as compared to control injected mice (E).
Fig 3.
LPS-induced autophagy is TLR4 dependent: Western blot for LC3 and corresponding quantification in lysates from renal cortex of C57BL/10ScN mice 24hr after injection with 15mg/kg LPS or control sterile saline demonstrated no increased autophagy (A, C). Immunodetection of LC3 in primary RTEC isolated from C57BL/10ScN mice grown in the absence or presence of 1 microgram /mL LPS for 24 hours (B, D) revealed no differences in the abundance or distribution of LC3 isoforms.
Fig 4.
Proximal tubule-specific knockout of ATG7 increases severity of LPS-induced AKI: (A) Decreased ATG7 and LC3-II in cortical lysates from ATGKO PEPCK-Cre;Atg7fl/fl mice compared to control Atg7fl/fl mice. (B) Proximal tubule-specific deletion of ATG7 worsened LPS-induced AKI as reflected by higher BUN levels after 24 and 48 hours (* = p < 0.05 compared to controls at same time point). Administration of LPS induced only modest histological changes in control mice (C) with preserved architecture and only focal vacuolization (arrow), whereas ATG7KO kidneys (D) showed more diffuse indications of proximal tubular injury including substantial coarse vacuolization of proximal tubular epithelium (large arrow) with degenerating nuclei (small arrow).
Fig 5.
LPS exposure increases IL6 expression and STAT3 activation in ATG7KO RTEC cells.
qPCR analysis demonstrated increased LPS-induced IL-6 expression in cortex of ATG7KO kidneys compared to LPS injected controls (A) which was accompanied by (B) decreased LC3-II and increased STAT3 phosphorylation. (C) Densitometric analysis of the expression of phospho-STAT3 relative to total STAT3. (D)Western blot of FKT- and FKT+ cells indicated that the ablation of Atg7 impaired accumulation of the LC3-II isoform in response to LPS (D). At the same time, qPCR analyses showed that the LPS induction of IL-6 was enhanced in the ATG7 deficient FKT- cell line (E) and in RTEC isolated from wild type mice co-incubated in the absence or presence of 5mM 3MA (F).