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mTORC1 activation in podocytes is a critical step in the development of diabetic nephropathy in mice - PubMed

mTORC1 activation in podocytes is a critical step in the development of diabetic nephropathy in mice

Ken Inoki et al. J Clin Invest. 2011 Jun.

Abstract

Diabetic nephropathy (DN) is among the most lethal complications that occur in type 1 and type 2 diabetics. Podocyte dysfunction is postulated to be a critical event associated with proteinuria and glomerulosclerosis in glomerular diseases including DN. However, molecular mechanisms of podocyte dysfunction in the development of DN are not well understood. Here we have shown that activity of mTOR complex 1 (mTORC1), a kinase that senses nutrient availability, was enhanced in the podocytes of diabetic animals. Further, podocyte-specific mTORC1 activation induced by ablation of an upstream negative regulator (PcKOTsc1) recapitulated many DN features, including podocyte loss, glomerular basement membrane thickening, mesangial expansion, and proteinuria in nondiabetic young and adult mice. Abnormal mTORC1 activation caused mislocalization of slit diaphragm proteins and induced an epithelial-mesenchymal transition-like phenotypic switch with enhanced ER stress in podocytes. Conversely, reduction of ER stress with a chemical chaperone significantly protected against both the podocyte phenotypic switch and podocyte loss in PcKOTsc1 mice. Finally, genetic reduction of podocyte-specific mTORC1 in diabetic animals suppressed the development of DN. These results indicate that mTORC1 activation in podocytes is a critical event in inducing DN and suggest that reduction of podocyte mTORC1 activity is a potential therapeutic strategy to prevent DN.

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Figures

Figure 1
Figure 1. mTORC1 activation in podocytes, and prevention of podocyte morphological changes by rapamycin treatment in db/db mice.

(A) Foot process effacement in db/db mice is blocked by rapamycin (rapa). Glomeruli from mice at 25 weeks of age with the indicated genotypes were examined by TEM. Foot process width at the level of slit diaphragm was measured (100 foot processes/glomerulus, 4 glomeruli/mouse, 3 male mice/group); *P < 0.001 versus other groups, mean ± SEM. Rapamycin treatment (1 mg/kg, i.p. injection, 3 times/week) was performed from 8 to 25 weeks of age. Pod, podocyte. (B) mTORC1 activity is enhanced in the podocytes of diabetic animals. Double staining with anti–phospho-S6 and anti-WT1 antibodies was performed on frozen sections from mice of the indicated genotypes at 12 weeks of age. The arrows indicate enhanced phospho-S6 signal in the cytoplasm of db/db podocytes. Quantification of glomerular phospho-S6 (green) pixel density divided by glomerular area is shown. Data are expressed as fold induction (4 images per mouse, 3 male mice/group); *P < 0.001, mean ± SEM. Original magnification, ×13,500 (A); ×400 (B).

Figure 2
Figure 2. Characterization of PcKOTsc1 mice.

(A) Enhanced S6 phosphorylation, podocyte hypertrophy, and mesangial expansion in PcKOTsc1 glomeruli. Renal tissues from 4-week-old mice of the indicated genotypes were stained with phospho-S6, WT1, H&E, and PAS as indicated. Representative glomeruli from wild-type, PcKOTsc1 (KO), and rapamycin-treated PcKOTsc1 (KO+rapa) are shown. Rapamycin treatment (1 mg/kg, i.p., 3 times/wk) was performed from 2 to 4 weeks of age. (B) S6 but not Akt phosphorylation was enhanced in PcKOTsc1 glomeruli. Levels of phospho-S6, S6, phospho-Akt (Ser473), and Akt in glomeruli from mice of the indicated genotypes were determined by Western blot. The levels of phosphorylation of each protein were quantified. Data are expressed as fold increase in the ratio of phospho-protein to total protein. *P < 0.001 versus other groups; mean ± SEM, n = 3. (C) Enhanced matrix protein mRNAs in PcKOTsc1 glomeruli. The expression levels of fibronectin (FN) and type IV collagen (COL4) mRNAs in the indicated mouse glomeruli were determined by quantitative RT-PCR. Data were normalized to Hprt1. *P < 0.001 versus other groups; mean ± SEM, n = 7. (D) Enhanced deposition of the matrix proteins in PcKOTsc1 glomeruli. Representative glomerular images of staining for fibronectin and type IV collagen in the indicated animals are shown. The ratio of positive staining area to glomerular area in the indicated animals was determined (10 glomeruli/mouse, 3 mice/group). *P < 0.001 versus other groups, mean ± SEM. Original magnification, ×400 (A and D).

Figure 3
Figure 3. PcKOTsc1 mice show proteinuria and podocyte loss.

(A) Rapamycin treatment prevents proteinuria in PcKOTsc1 mice. Scheme of administration of rapamycin is shown above. Urine (1 μl ) from mice of the indicated genotypes was subjected to SDS-PAGE with Coomassie blue staining (n = 6 each group). veh, vehicle. Asterisk indicates a leak of sample from lane 6. (B) Urinary albumin/creatinine ratio is shown. Urinary albumin and creatinine concentrations in 4-week-old mice were determined by ELISA. *P < 0.001 versus other groups, mean ± SEM, n = 6. Note logarithmic scale for the y axis. Rapamycin treatment was performed from 2 weeks of age as indicated in Figure 2A. (C) TEM analyses reveal abnormal foot process and GBM in PcKOTsc1 and wild-type mice. Representative TEM images of the indicated animals (3 mice/group) from 4 and 8 weeks of age are shown. Rapamycin treatment was as in Figure 2A. Original magnification, ×13,500 (top row), ×7,900 (bottom row). Scale bars: 1 μm. (D) Scanning EM analyses show abnormal podocyte in PcKOTsc1 mice, which was prevented by rapamycin treatment. Rapamycin treatment was as in Figure 2A. Original magnification, ×2,000. Scale bars: 5 μm (top row); 1.25 μm (bottom row). (E) Podocyte loss in PcKOTsc1 mice. The ratio (the number of WT1-positive cells/glomerular tuft area [μm2]) was determined in 15~30 glomeruli from the indicated animals. *P < 0.001 versus wild-type and/or KO with rapamycin treatment, mean ± SEM, n = 3–5 mice.

Figure 4
Figure 4. Rapamycin reverses established phenotypes in PcKOTsc1 mice.

(A and B) Rapamycin treatment ameliorated the established proteinuria in PcKOTsc1 mice. Proteinuria-positive 4-week-old PcKOTsc1 mice (n = 9) were treated with rapamycin for 2 and 6 weeks. The levels of urinary protein from 9 mice were tested at the indicated time points and visualized by Coomassie staining (n = 9) (A), and the ratio of albumin to creatinine concentration is shown (B). *P < 0.001 versus other groups, mean ± SEM, n = 9; note logarithmic scale for y axis. The ratios for 4- and 10-week-old wild-type mice are also shown. (C) H&E staining of renal tissue from 4-week-old untreated and 10-week-old treated PcKOTsc1 mice (rapamycin treatment for 6 weeks). Eosin-positive area in glomerulus was measured in 30 glomeruli from 4-week-old wild-type and PcKOTsc1 mice as well as 10-week-old treated PcKOTsc1 mice. *P < 0.001 versus other groups, no statistical significance between WT 4 weeks and KO with rapamycin 10 weeks, mean ± SEM, n = 3~5. (D) TEM analyses of 10-week-old PcKOTsc1 mice (rapamycin treatment for 6 weeks). Foot process width at the level of slit diaphragm was measured (20~50 foot processes/glomerulus, 3 glomeruli/mouse, 3 mice/group). *P < 0.001 versus other groups, mean ± SEM. (E) Scanning EM analyses of 6-week-old PcKOTsc1 mice (rapamycin treatment for 2 weeks). Representative glomerulus from 2 different rapamycin-treated PcKOTsc1 mice is shown. Original magnification, ×400 (C), ×7,900 (D), ×2,000 (E). Scale bars: 10 μm (top row); 2.5 μm (bottom row).

Figure 5
Figure 5. mTORC1 activation in podocytes causes proteinuria and mesangial expansion in adult mice.

(A) Rapamycin prevents death in PcKOTsc1 mice. Mice were treated with rapamycin as indicated. Survival curve of the indicated animals is shown. Wild-type, n = 50; heterozygous (Het), n = 50; KO, n = 50; KO with rapamycin treatment (off-on), n = 15; KO with rapamycin treatment (off-on-off), n = 10; KO C57BL/6 (B6) background, n = 30. (B) mTORC1 activation in podocytes causes proteinuria and mesangial expansion in adult mice. Withdrawal of rapamycin treatment in adult PcKOTsc1 mice led to rapid onset of proteinuria and glomerulopathy. Scheme of administration of rapamycin is shown at top. Urinary albumin concentrations of the indicated animals were determined (WT 4 wk; KO 15 wk [rapamycin treatment from 2 to 15 weeks]; KO 16 wk, 19 wk, and 21 wk [off-on-off; 1 wk, 4 wk, and 6 wk after withdrawal of rampamycin treatment]). *P < 0.001 versus WT and KO 15 wk, mean ± SD, n = 6. (C) Withdrawal of rapamycin treatment in adult PcKOTsc1 mice led to mesangial expansion. Scheme of administration of rapamycin is shown at top. H&E and PAS staining of the representative glomeruli are shown. PAS-positive area in glomerulus was measured in 30 glomeruli from the indicated animals. *P < 0.001 versus other groups, n = 4. Original magnification, ×400.

Figure 6
Figure 6. Hyperactivation of mTORC1 leads to mislocalization of slit diaphragm proteins.

(A) Nephrin expression at interpodocyte filtration slit area is diminished in PcKOTsc1 mice. Representative electron micrographs of immunogold nephrin staining in 3-week-old mice are shown. Number of immunogold particles for nephrin at the slit diaphragm was measured in 290 slit diaphragms from 2 wild-type and 170 slit diaphragms from 2 PcKOTsc1 mice. *P < 0.001 versus wild-type, mean ± SEM. (B) Nephrin and actin expression in the indicated glomeruli were determined by Western blotting. Levels of nephrin expression in the indicated genotypes were quantified (nephrin/actin). Data are expressed as fold increase. P = 0.67, mean ± SEM, n = 3. (C) Nephrin is mislocalized in PcKOTsc1 podocytes. Confocal microscopic analyses of nephrin and synaptopodin expression in the indicated animals (3 weeks old) are shown. Rapamycin treatment was performed for 1 week. Areas indicated by squares in the Merge column are shown at higher magnification in the right two columns. Original magnification, ×13,500 (A), ×400 (C).

Figure 7
Figure 7. Mislocalization of nephrin by abnormal mTORC1 activation and diabetes.

(A) Membrane localization of nephrin is disrupted by Rheb. Exogenous nephrin expression pattern was analyzed by confocal microscopy. Human nephrin (Myc-Nephrin) was expressed with or without Rheb (HA-Rheb) in COS7 cells. Nephrin and Rheb expression was determined by rabbit polyclonal nephrin and mouse monoclonal HA antibodies, respectively. The arrowheads indicate membrane expression of nephrin. Rapamycin (20 nM) treatment was for 12 hours. (B) Rapamycin treatment restores membrane localization of nephrin in db/db mice. The pattern of nephrin expression of the indicated animals (25 weeks of age) was monitored by confocal microscopy. Rapamycin treatment was performed from 8 to 25 weeks. The arrowheads indicate the accumulation of nephrin in cytoplasm. (C) Membrane localization of nephrin was reduced in human type 2 DN. Nephrin staining showed a granular pattern expression in cytoplasm with reduced membrane expression (NOR, normal subject; DN p1, DN patient 1). Original magnification, ×600 (A), ×400 (B, and C).

Figure 8
Figure 8. Hyperactivation of mTORC1 induces ER stress and EMT-like phenotype in podocytes.

(A) EMT-like phenotypic changes were induced in PcTSC1KO podocytes. Double staining with desmin and WT1 antibodies was performed in renal tissues from the indicated animals and analyzed by confocal microscopy (top row). Arrowheads indicate desmin expression in the podocytes. Rapamycin treatment was performed for a week. ZO-1 expression was analyzed by confocal microscopy (bottom row). Arrowheads indicate the liner expression pattern of ZO-1. (B) Detection of nephrin and podocin mRNA in PcTSC1KO urinary sediments. Twenty-four-hour urine samples were collected from the indicated animals. RT-PCR was performed using purified mRNA from the sediments. Glomerular mRNA was used as a positive control (p). (C) Accumulation of GRP78 in PcKOTsc1 podocytes (3 weeks of age). Double staining using anti-GRP78 and anti-synaptopodin antibodies in the indicated glomeruli is shown. Rapamycin treatment was performed for 1 week. (D) PBA treatment protects podocyte loss in PcKOTsc1 mice. Staining with GRP78 plus synaptopodin, nephrin, H&E, and WT1 is shown in the indicated animals (8 weeks of age). PBA treatment (400 mg/kg/d, i.p.) was performed for 6 weeks. (E) Podocyte number in the indicated glomerular sections from 8-week-old animals was measured. The ratio (number of WT1-positive cells/glomerular tuft area [μm2]) was determined in 15~30 glomeruli from the indicated animals as in Figure 3E. *P < 0.001 versus other groups, mean ± SEM, n = 4~8. (F) Urinary albumin concentrations were measured in the indicated animals. *P < 0.001 versus other groups; no statistical significance was observed between KO and KO with PBA treatment; mean ± SEM, n = 4~8. Original magnification, ×400 (A, C, and D).

Figure 9
Figure 9. Podocyte-specific Raptor-heterozygous mice show resistance to the development of DN.

(A) Podocyte-specific Raptor-heterozygous db/db mice (DM Raptor+/–) develop diabetes to the same extent as control db/db mice. Blood glucose levels in the indicated animals are shown. *P < 0.001 versus DM and DM Raptor+/–; no statistical significance was observed between DM and DM Raptor+/–; mean ± SEM, n = 6~8. (B) Twenty-four-hour urine volume of the indicated animals. Data are expressed as an average of the amount of urine for 3 days. *P < 0.001 versus DM and DM Raptor+/–; no statistical significance was observed between DM and DM Raptor+/–; mean ± SEM, n = 6~8. (C) DM Raptor+/– mice show resistance to the development of mesangial expansion. Representative H&E and PAS staining of the mice at 40 weeks of age is shown. (D) PAS-positive mesangial area was quantified using ImageJ software. PAS staining was performed in the indicated renal tissues, and PAS-positive mesangial area of 10–20 glomeruli cut at the vascular pole were measured. *P < 0.001 versus other groups, mean ± SEM, n = 6~8. (E) DM Raptor+/– mice developed glomerular hypertrophy. Glomerular area was quantified as described in D. *P < 0.001 versus other groups; no statistical significance was observed between DM and DM Raptor+/–; mean ± SEM, n = 6~8. (F) Decreased RAPTOR dosage prevents nephrin mislocalization in DM mice. Confocal microscopic analyses of nephrin and synaptopodin expression in the indicated animals (40 weeks old) are shown. (G) DM Raptor+/– mice show resistance to proteinuria. Urinary albumin and creatinine concentrations were measured in 24-hour urine samples from the indicated animals. Albumin/creatinine ratio is shown. *P < 0.01 versus DM and control, mean ± SEM, n = 6~8. Original magnification, ×400 (C and F).

Comment in

  • The targeted podocyte.

    Fogo AB. Fogo AB. J Clin Invest. 2011 Jun;121(6):2142-5. doi: 10.1172/JCI57935. Epub 2011 May 23. J Clin Invest. 2011. PMID: 21606599 Free PMC article.

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