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. 2012 Mar;23(3):412-20.
doi: 10.1681/ASN.2011070690. Epub 2011 Dec 22.

Inhibition of MTOR disrupts autophagic flux in podocytes

Davide P Cinà  1 , Tuncer Onay, Aarti Paltoo, Chengjin Li, Yoshiro Maezawa, Javier De Arteaga, Andrea Jurisicova, Susan E Quaggin
Affiliations

Inhibition of MTOR disrupts autophagic flux in podocytes

Davide P Cinà et al. J Am Soc Nephrol. 2012 Mar.

Abstract

Inhibitors of the mammalian target of rapamycin (MTOR) belong to a family of drugs with potent immunosuppressive, antiangiogenic, and antiproliferative properties. De novo or worsening proteinuria can occur during treatment with these agents, but the mechanism by which this occurs is unknown. We generated and characterized mice carrying a podocyte-selective knockout of the Mtor gene. Although Mtor was dispensable in developing podocytes, these mice developed proteinuria at 3 weeks and end stage renal failure by 5 weeks after birth. Podocytes from these mice exhibited an accumulation of the autophagosome marker LC3 (rat microtubule-associated protein 1 light chain 3), autophagosomes, autophagolysosomal vesicles, and damaged mitochondria. Similarly, human podocytes treated with the MTOR inhibitor rapamycin accumulated autophagosomes and autophagolysosomes. Taken together, these results suggest that disruption of the autophagic pathway may play a role in the pathogenesis of proteinuria in patients treated with MTOR inhibitors.

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Figures

Figure 1.
Figure 1.
Generation of podocyte-selective knockout of the Mtor gene. (A) BAC recombineering construct. LoxP sites (triangles) are inserted around the first three exons of the Mtor gene. The neomycin selection cassette is used to select for positive embryonic stem cell clones, then removed by FLPe recombinase-mediated excision. Cre-mediated excision results in a null Mtor allele. (B) Southern blot analysis confirms correctly targeted alleles. (C) A null Mtor allele (Mtordel) is generated by breeding floxed mice to a pCaggs-Cre driver strain that deletes at the one cell stage (germline deletion). A podocyte-selective knockout is generated by breeding Mtorflox/del mice to a Podocin-Cre driver strain. (D) Table showing correlation between genotype and survival. As predicted, Mtordel/del (Mtor−/−) mice die in utero. (E) Southern blot analysis of renal cortical genomic DNA confirms the presence of a deleted Mtor allele in Mtor pod-KO mice. (F) Western blot analysis of isolated glomerular protein confirms downregulation of Mtor protein in the glomeruli from Mtor pod-KO mice compared with Mtorwt/flox mice. Each lane contains glomeruli pooled from four mice. Wt, wild-type allele; Tg, transgenic Mtorflox allele.
Figure 2.
Figure 2.
Disease course and glomerular histology in Mtor pod-KO mice. (A) Mtor pod-KO mice appear smaller than their control littermates at 3 weeks of age. (B) Mtor pod-KO mice are significantly growth restricted by 4 weeks of age (*P<0.01; **P<0.05). (C) Protein/creatinine ratios are significantly increased by 3 weeks of age in Mtor pod-KO mice compared with wild-type (*P<0.01; **P<0.05). (D) At 3 weeks of age, glomeruli look similar in mutants and controls although proteinaceous casts are visible in tubules (periodic acid–Schiff stain, magnification ×40). By 4 weeks of age, glomerular scarring and vacuolization of podocytes are evident. (E) By 5 weeks of age, glomeruli are end stage with widespread vacuolization of podocytes. (F) Electron micrographs from control and mutant glomeruli at 3 weeks of age show focal foot process effacement in mutants compared with controls. The endothelium and glomerular basement membrane appear largely intact. Scale bars represent 1 μm. (G) Electron micrographs of glomeruli from 2-week-old and 3-week-old Mtor pod-KO mice show frequent double-membraned cytoplasmic vesicles characteristic of autophagosomes. Scale bar in the top left panel represents 500 nm. All other scale bars represent 1 μm. (H) Isolated glomeruli from Mtor pod-KO mice show increased levels of LC3-II compared with controls. AV, autophagosomal vesicles; ALV, autophagolysosomal vesicles.
Figure 3.
Figure 3.
Model explaining the effect of MTOR inhibition in podocytes. (A) Physiologic level of MTOR activity inhibits autophagy (step 1), and maintains it at a basal level (step 2) in podocytes. MTOR reactivation allows autophagolysosomal reformation and the cycle of autophagy to complete itself. (B) MTOR inhibition (step 1) disrupts the autophagic pathway at two points. First, it relieves chronic suppression, resulting in activation and enhanced autophagy (step 2). However, MTOR inhibition will also lead to suppression of the reformation of lysosomes and autophagosomes, ultimately resulting in an accumulation of ALVs (step 3), damaged intracellular organelles such as mitochondria, and cell death. Lyso, lysosome; AV, autophagosomal vesicle; ALV, autophagolysosomal vesicle.
Figure 4.
Figure 4.
Chronic MTOR inhibition disrupts the autophagic pathway in podocytes in vitro. (A) Human podocytes stably transfected with LC3-GFP and treated with rapamycin exhibit increased fluorescent-tagged ringed structures characteristic of autophagosomes. The AVs colocalize with the lysosomal marker Lysotracker Red. Podocytes exposed to rapamycin also demonstrate increased superoxide activity as shown by Mitosox Red, suggestive of mitochondrial damage. (B) Western blot analysis confirms downregulation of phospho-S6K (pS6K) and inhibition of mTOR activity in podocytes exposed to rapamycin along with upregulation of the autophagosome-associated LC3 isoform. (C) Podocytes exposed to starvation show only a transient increase in ALVs. In contrast, rapamycin-treated cells exhibit prolonged and marked accumulation of ALVs. ALVs are marked by co-localization of the lysosomal dye (Lysotracker Red) and GFP-LC3 (green). (D) Western blot analysis shows recovery of mTOR activity after 6 hours of starvation as evidenced by increasing levels of phospho-S6K. In contrast, rapamycin-treated cells show continued suppression of phospho-S6K.
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