Augmenting Autophagy to Treat Acute Kidney Injury during Endotoxemia in Mice

To determine that 1) an age-dependent loss of inducible autophagy underlies the failure to recover from AKI in older, adult animals during endotoxemia, and 2) pharmacologic induction of autophagy, even after established endotoxemia, is of therapeutic utility in facilitating renal recovery in aged mice.

C57Bl/6 mice of 8 (young) and 45 (adult) weeks of age.

These novel results demonstrate a role for autophagy in the context of LPS-induced AKI and support further investigation into like interventions that have potential to alter the natural history of disease.

Ethics Statement All experiments were performed in accordance with the National Institutes of Health guidelines under protocols approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh (protocol # 1006391B). All surgery was performed under halothane inhalational anesthesia, and all efforts were made to minimize suffering.

Autophagy was inhibited using in vivo RNAi of VPS34 as previously performed; this technique effectively and specifically inhibits the expression of the targeted protein of interest [9] , [10] . Mice were administered VPS34 or scrambled, non-target siRNA (6 mg/kg) by hydrodynamic tail vein injection delivered in (animal mass/10) mL lactated ringers as previously performed [9] . After 72 hours mice were randomly allocated to each experimental condition.

We performed cecal ligation and puncture (CLP) as previously described, using a single 21-gauge puncture, a model we have optimized to enable the evaluation of cellular/organ biology and physiology [11] . Sham animals underwent anesthesia and laparotomy with bowel manipulation. All mice received volume resuscitation with 0.9% saline (2 ml/kg SQ).

Total cellular lysate was extracted at 4°C in 500 µL of lysis buffer [11] . Protein concentration was determined using a bicinchoninic acid protein assay (Pierce, Rockford, IL).

Kidneys were flushed with PBS and then perfused with 2% paraformaldehyde. After 2 h fixation, samples were transferred to 30% sucrose for 24 h with a total of three sucrose changes. The samples were cryopreserved in liquid nitrogen cooled 2-methylbutane and stored at −80°C until sectioning. Cryopreserved tissues were sectioned to 6 mm thickness and incubated with 2% bovine serum albumin (BSA) in PBS for 1 h, followed by 5 washes with PBS+0.5% BSA (PBB). This was followed by an overnight incubation of samples with anti-LC3-I/II antibody in PBS+5% BSA (Novus, 5 ug/ml) in the presence of Triton X-100 at 0.1% at 4C. The slides were washed with 0.5% BSA, followed by a 1 hour incubation with a Cy3 secondary antibody (goat anti-rabbit, 1:1000, Jackson ImmunoResearch Laboratories) plus AlexaFluor-488 phallodin (1:250, Invitrogen). The slides were rinsed, and HOECHST dye (1 mg/100 ml bisbenzimide) was applied for 30 s. The slides were rinsed with PBS and coverslipped with gelvatol, a water-soluble mounting media. Slides were imaged with an Olympus Fluoview 1000 confocal scanning microscope (Olympus, Melville, NY). Imaging conditions were maintained at identical settings within each antibody-labeling experiment with original gating performed using the negative control. Quantification was performed using Metamorph to determine the mean fluorescent intensity (MFI) of punctate LC3b adjusted for actin MFI (Molecular Devices, Sunnyvale, CA).

Renal function was determined by assaying serum for blood urea nitrogen (BUN) and cystatin C, respectively using the DRI-CHEM 4000 Chemistry Analyzer (Heska, Loveland, CO) and an enzyme immunoassay kit (R&D, Minneapolis, MN). Cystatin C has emerged as a more precise marker of glomerular filtration rate and has been validated in both human and murine studies [17] , [18] .

As shown in Figure 1a , adult mice, by comparison to their younger counterparts, exhibited non-recovery of renal function after LPS administration. Both groups have similar BUN and cystatin concentrations at baseline and 18 hours after LPS, the latter indicating comparable susceptibility to AKI. By 48 hours, however, young mice demonstrated normalization of renal function, whereas adult mice exhibited persistent elevations in both BUN (25 vs. 133 mg/dL, p = 0.003) and cystatin (649 vs. 1302 ng/mL, p = 0.003). 10.1371/journal.pone.0069520.g001 Figure 1 Adult mice exhibit non-recovery of renal function and diminished autophagy during endotoxemia. (a) Time course plots for serum BUN and cystatin concentrations after LPS comparing adult and young mice (n = 8–11 mice per group per timepoint). (b) The effects of LPS on autophagy in the renal cortices of adult and young mice were assessed by immunoblot for LC3b (16KD). Representative blot is shown at 48 h timepoint after LPS from n = 4 experiments (8–9 mice per experiment). Corresponding densitometry compares total LC3b of older aged adult and younger animals exposed to LPS. (c) Immunofluorescent microscopy (20X) of renal cortex in adult and young mice harvested at 48 h after LPS. LC3 (Cy3, red), actin (Alexa488, green), nucleus (Hoechst, blue). Representative images of n = 4 experiments (8 mice per experiment). (d) Transmission electron microscopy (10 5 X) of renal cortex of young and adult mice 48 h after endotoxemia. Inset (5×10 5 X). Representative images of n = 4 experiments (8 mice per experiment). arrowheads, autophagosomes; arrows, autophagolysosomes. Data are means ± s.e.m.; rank sum test. The impairment in renal recovery observed in adult mice correlated with reduced renal autophagy as assessed by three independent methods: immunoblot, immunofluorescence, and electron microscopy (EM). Representative immunoblot and corresponding densitometry show significantly less LC3b expression in adult animals than in younger animals 48 hours after LPS (p<0.001) ( Fig. 1b ). Young animals, by contrast to adult animals, exhibited more immunofluorescence and a more pronounced punctate staining pattern for LC3 (MFI: 0.062 vs. 0.003, p = 0.03), the latter indicating the association of the conjugated form of LC3 (LC3b) with the autophagosomal membrane ( Fig. 1c ). EM illustrates numerous multi-membranous autophagosomes and autophagolysosomes in the renal cortex of younger animals, which are notably reduced in adult animals, which exhibit swollen, damaged mitochondria lacking prototypical architectural features ( Fig. 1d ): proportion of cytoplasmic area 0.06 vs. 0.029, p = 0.005. We did not observe notable differences in basal (i.e. without LPS) autophagy between young and adult animals ( Fig. 1b ; immunofluorescence and electron microscopy data not shown). We hypothesized that inhibiting autophagy would render young animals ‘adult’ and interfere with recovery from AKI. VPS34, an evolutionarily conserved class III phosphoinosititde 3-kinase, is an indispensible upstream regulator of autophagy [10] . We have previously used RNAi to inhibit the in vivo expression of VPS34 as means to regulate autophagy in vivo [9] , [10] . As shown in Figure 2a , by comparison to non-target, scrambled siRNA (RNAi NT ), VPS34 siRNA (RNAi VPS34 ) effectively inhibited the induced expression of VPS34 in renal tubular cells during endotoxemia ( Fig. 2a .), which correlated with attenuated autophagy, as evidenced by reduced punctate LC3 expression ( Fig. 2b ). Representative immunoblot shows significantly less LC3b expression in RNAi VPS34 by comparison to RNAi NT animals 48 hours after LPS ( Fig. 2c ). RNAi VPS34 mice, by comparison to RNAi NT mice, failed to recover from AKI, as characterized by persistently elevated BUN (29 vs. 133 mg/dL, p = 0.02) and cystatin (636 vs. 1367 ng/mL, p = 0.02) concentrations 48 hours after LPS. Thus, inhibiting autophagy in young mice produced a temporal pattern of AKI similar to that of aged, adult mice. 10.1371/journal.pone.0069520.g002 Figure 2 Inhibition of VPS34 attenuates autophagy and inhibits recovery of renal function in young mice during endotoxemia. Immunofluorescent microscopy (20X) of renal cortex harvested at 48 hours after LPS in young mice from 4 experimental groups: NT siRNA and saline control, VPS34 siRNA and saline control, NT siRNA and LPS, and VPS34 siRNA and LPS. (a) VPS34 (Cy3, red), actin (Alexa488, green), nucleus (Hoechst, blue). Representative images of n = 4 experiments (4 mice per experiment). (b) LC3 (Cy3, red), actin (Alexa488, green), nucleus (Hoechst, blue). Representative images of n = 4 experiments (4 mice per experiment). (c) Immunoblot analysis of renal cortex lysate detecting LC3b (16KD) in untreated mice (lanes 1 and 2) and mice treated with Non-target siRNA and LPS (lanes 3 and 4) or VPS34 siRNA and LPS (lanes 5 and 6). Representative blot is shown at 48 h timepoint after LPS from n = 2 experiments (6 mice per experimen