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. 2017 Jul;28(7):2144-2157.
doi: 10.1681/ASN.2016050519. Epub 2017 Mar 7.

Targeting mTOR Signaling Can Prevent the Progression of FSGS

Stefan Zschiedrich  1 , Tillmann Bork  1 , Wei Liang  1   2 , Nicola Wanner  1 , Kristina Eulenbruch  1 , Stefan Munder  1 , Björn Hartleben  1 , Oliver Kretz  1   3 , Simon Gerber  4 , Matias Simons  4 , Amandine Viau  1 , Martine Burtin  5 , Changli Wei  6 , Jochen Reiser  6 , Nadja Herbach  7 , Maria-Pia Rastaldi  8 , Clemens D Cohen  9 , Pierre-Louis Tharaux  10 , Fabiola Terzi  5 , Gerd Walz  1 , Markus Gödel  1 , Tobias B Huber  11   3   12   13
Affiliations

Targeting mTOR Signaling Can Prevent the Progression of FSGS

Stefan Zschiedrich et al. J Am Soc Nephrol. 2017 Jul.

Abstract

Mammalian target of rapamycin (mTOR) signaling is involved in a variety of kidney diseases. Clinical trials administering mTOR inhibitors to patients with FSGS, a prototypic podocyte disease, led to conflicting results, ranging from remission to deterioration of kidney function. Here, we combined complex genetic titration of mTOR complex 1 (mTORC1) levels in murine glomerular disease models, pharmacologic studies, and human studies to precisely delineate the role of mTOR in FSGS. mTORC1 target genes were significantly induced in microdissected glomeruli from both patients with FSGS and a murine FSGS model. Furthermore, a mouse model with constitutive mTORC1 activation closely recapitulated human FSGS. Notably, the complete knockout of mTORC1 by induced deletion of both Raptor alleles accelerated the progression of murine FSGS models. However, lowering mTORC1 signaling by deleting just one Raptor allele ameliorated the progression of glomerulosclerosis. Similarly, low-dose treatment with the mTORC1 inhibitor rapamycin efficiently diminished disease progression. Mechanistically, complete pharmacologic inhibition of mTOR in immortalized podocytes shifted the cellular energy metabolism toward reduced rates of oxidative phosphorylation and anaerobic glycolysis, which correlated with increased production of reactive oxygen species. Together, these data suggest that podocyte injury and loss is commonly followed by adaptive mTOR activation. Prolonged mTOR activation, however, results in a metabolic podocyte reprogramming leading to increased cellular stress and dedifferentiation, thus offering a treatment rationale for incomplete mTOR inhibition.

Keywords: Pathophysiology of Renal Disease and Progression; focal segmental glomerulosclerosis; mTOR; mitochondrial function; rapamycin; raptor.

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Figures

Figure 1.
Figure 1.
FSGS is associated with mTORC1 activation in humans and mice. (A) Gene expression of major mTORC1 target genes was significantly upregulated in microdissected glomeruli from patients with primary FSGS in comparison with MCD or healthy living donor controls (LD) (FSGS n=10, MCD n=5, LD n=18) (B) Immunohistochemistry staining of human kidney biopsy samples demonstrated enhanced mTORC1-dependent phosphorylation of S6 in glomeruli of patients with FSGS compared with patients with MCD (scale bars, 50 µm; representative images at high magnification; n=3 patients each). (C) mTORC1-dependent phosphorylation of S6 was enhanced in podocytes (arrows) of CD1 wild-type mice in response to injury after ADR injection (scale bars, 5 µm). (D) FSGS-like glomerular lesions in CD1 wild-type mice in ADR nephropathy (arrows). (E) Proteinuria increased over time as a marker for progressive glomerular damage after ADR injection (n=5). (F) Quantification of glomerulosclerosis. (G and H) There is a loss of podocytes in ADR nephropathy demonstrated as reduced WT1-positive nuclei per glomerulus in absolute numbers as well as after normalization for glomerular area. (I) Glomerular tuft volume is unchanged in ADR nephropathy. *, significant; n.s., not significant.
Figure 2.
Figure 2.
Complete genetic abrogation of mTORC1 activity facilitates FSGS-like disease. (A) Doxycycline-inducible Raptor knock-out model (Raptorflox/flox; NPHS2.rtTA; tetO.Cre) in mice of mixed (C57BL/6J/ICR) genetic background for inducible deletion of mTORC1. (B) Western blot analysis of isolated glomeruli confirmed Raptor deletion as well as reduced mTORC1-dependent phosphorylation of S6. (C) Deletion of Raptor in adult mice (inducible RaptorΔpodocyte) resulted in significantly increased proteinuria and (D and E) accelerated glomerulosclerosis in a model of ADR-induced FSGS (white arrows, areas with focal sclerosis; bars, 50 µm; glomerulosclerosis index after el Nahas et al. at 8 weeks of experiment; n=3). (F and G) Enhanced loss of podocytes in inducible RaptorΔpodocyte is demonstrated by reduced WT1-positive nuclei per glomerulus in absolute numbers as well as after normalization for glomerular area, whereas (H) glomerular tuft volume is not different in both groups. (I) Subtotal nephrectomy (Nx) after inducible deletion of Raptor in podocytes of adult mice (RaptorΔpodocyte) versus Raptor+/+ mice resulted in severe proteinuria (week 5 of experiment, n=7, respectively) and (J) progressive decline of renal function (serum urea measured after 8 weeks of follow up; n=5 and n=8, respectively). (K) Histologic and transmission electron microscopy images of Nx mice confirm severe glomerular lesions and foot process effacement in Raptor-deficient mice. (L–N) Nx in WT animals induces glomerular hypertrophy and podocyte loss, demonstrated as reduced WT1-positive nuclei per glomerulus in absolute numbers as well as after normalization for glomerular area. In contrast, glomerular hypertrophy is blocked in RaptorΔpodocyte, whereas WT1-positive nuclei differ only in absolute values but not after normalization for glomerular area. *, significant; n.s., not significant; WT, wild type.
Figure 3.
Figure 3.
Partial genetic reduction of mTORC1 activity ameliorates FSGS-like disease. (A) Heterozygous deletion of Raptor in podocytes (RaptorHet podocyte) in mice of ICR genetic background (B) reduces RAPTOR protein level in glomerular lysates, and (C) reduces activity of the mTORC1 pathway in ADR FSGS model, expressed as percentage of glomerular area with phosphorylated S6 in immunofluorescence (mean of 20 glomeruli per mouse; n=3 per column). (D) Proteinuria was reduced after heterozygous deletion of Raptor in ADR-adaptive FSGS model (n=7 and n=8, respectively). (E and G) RaptorHet podocyte prevented glomerulosclerosis (glomerulosclerosis index after el Nahas et al. at 8 weeks of experiment; n=4 per column; and PAS staining, white arrows). (F and H) Quantitative stereologic analyses showed reduced podocyte volume in in RaptorHet podocyte but not in wild-type animals (n=3 and n=3, respectively; bars=SD); silver methenamine stainings (white arrows; bar, 20 µm). *, significant; n.s., not significant.
Figure 4.
Figure 4.
Metabolic podocyte reprogramming by mTOR signaling. (A) Mitochondrial function of immortalized cultured podocytes treated with different doses of the mTOR inhibitor Torin 1 for 14 days. Oxygen consumption rate (OCR) was measured at basal level and after the sequential addition of oligomycin (1 μM), FCCP (0.5 μM), and rotenone (Rot; 1.0 μM) + antimycin A (Ant; 1.0 μM; n=3, technical replicates). (B) Combined results underline the role of mTOR for mitochondrial respiratory function (n=3, biologic replicates; NMR, nonmitochondrial respiration). (C) Although total cellular ATP production remains unchanged after low-dose mTOR inhibition with Torin 1, it is suppressed in response to high-dose treatment (n=3, biologic replicates). (D) Protein levels of key components of the respiratory chain in immortalized cultured podocytes treated with different doses of Torin 1 for 14 days. (E) Assessment of oxidative stress by CellROX live cell fluorescence staining and FACS analysis of primary podocytes, displaying significantly induced ROS production (FL4-H fluorescence intensity) after high-dose mTOR inhibition compared with control and low-dose inhibition. (F) Immortalized cultured podocytes treated with ADR 3 µg/ml for indicated time show no changes in pS6. Treatment with mTOR inhibitor Torin 1 100 nM for 6 hours serves as a control. (G) Mitochondrial function of immortalized cultured podocytes treated with ADR 3 µg/ml for 24 hours or vehicle. OCR was measured at basal level and after the sequential addition of oligomycin (1 μM), FCCP (0.5 μM), and rotenone (Rot; 1.0 μM) + antimycin A (Ant; 1.0 μM; n=3, technical replicates). (H) Combined results demonstrate the effect of ADR for mitochondrial respiratory function (n=3, biologic replicates). (I) Mitochondrial function of immortalized cultured podocytes treated with ADR 3 µg/ml or ADR 3 µg/ml + Torin 1 10 nM for 24 hours. OCR was measured at basal level and after the sequential addition of oligomycin (1 μM), FCCP (0.5 μM), and rotenone (Rot; 1.0 μM) + antimycin A (Ant; 1.0 μM; n=3, technical replicates). (J) Combined results demonstrate the beneficial effect of Torin 1 on ADR-induced mitochondrial dysfunction (n=3, biologic replicates). (K) Mitochondrial function of immortalized cultured podocytes treated with ADR 3 µg/ml or ADR + Torin 1 100 nM for 24 hours. OCR was measured at basal level and after the sequential addition of oligomycin (1 μM), FCCP (0.5 μM), and rotenone (Rot; 1.0 μM) + antimycin A (Ant; 1.0 μM; n=3, technical replicates). (L) Combined results demonstrate the additive effect of high-dose Torin 1 treatment on ADR-induced mitochondrial dysfunction (n=3, biologic replicates).
Figure 5.
Figure 5.
Rapamycin treatment prevents FSGS-like lesions in ADR nephropathy. (A) pS6-stained glomerular area (mean of 20 glomeruli per mouse, n=3 per group). (B) Rapamycin ameliorated proteinuria in ADR nephropathy of mice (n=10 per group), and (C and G) preserved glomerular architecture (glomerulosclerosis index after el Nahas et al. at 8 weeks of experiment; n=3; PAS staining, white arrows). (D and E) Reduced loss of podocytes in CD1 wild-type mice due to rapamycin treatment after ADR injection was demonstrated by a reduced number of WT1-positive nuclei per glomerulus in absolute numbers as well as after normalization for glomerular area. (F) Glomerular tuft volume is not influenced by rapamycin treatment. (G) Low dose Rapamycin treatment prevented glomerulosclerosis. WT, wild type.
Figure 6.
Figure 6.
Role of mTORC1 signaling in progressive glomerulosclerosis. (A) Schematic illustration of mTORC1 activity in FSGS. Initial podocyte injury is mediated by various known (secondary) or as yet unknown (primary) insults. Subsequently, mTORC1 is part of an adaptive response to podocyte loss. However, persistent mTOR activation drives podocyte dedifferentiation and further podocyte loss in self-sustained progression of FSGS. (B) Concept for individual and timed dosage effects of mTOR inhibition.
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Comment in

  • Glomerular disease: mTOR in FSGS.
    Allison SJ. Allison SJ. Nat Rev Nephrol. 2017 May;13(5):260. doi: 10.1038/nrneph.2017.42. Epub 2017 Mar 27. Nat Rev Nephrol. 2017. PMID: 28344329 No abstract available.

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