Fig 1.
Generation of podocyte-specific Tsc2 knockout mice, Tsc2Δpodocyte. Homozygous floxed Tsc2 mice were crossed with Nphs2-Cre transgenic mice to generate Tsc2Δpodocyte, Nphs2-Cre+/-; Tsc2flox/flox.
(A) PCR genotyping of genomic DNA from mouse tails. NPHS2-Cre+/-, Tsc2wt/wt mice (Nphs2-Cre) and NPHS2-Cre-/-, Tsc2flox/flox mice (Tsc2flox/flox) were used as controls. F, flox; +, wt. (B) Primary cultured podocytes isolated from Tsc2Δpodocyte and wild-type control mice at 4 weeks of age demonstrated that Tsc2 mRNA was knocked down by over 80% in the podocytes of Tsc2Δpodocyte mice compared with control mice. Results are expressed as means ± s.d. *P < 0.05 compared with age-matched controls. (C) Western blot analysis of primary cultured podocytes isolated from Tsc2Δpodocyte and control mice. TSC2 gives a band at 200 kDa (arrow). Non-specific bands were also detected above the TSC2 band in all samples. β-Tubulin was used as an internal control. (D) Tsc2 mRNA expression in various tissues of Tsc2Δpodocyte and wild-type controls. Expression levels of Tsc2 mRNA in Tsc2Δpodocyte were comparable with those in controls in all the tissues examined, including the renal cortex. White bar, Nphs2-Cre; dotted bar, Tsc2flox/flox; black bar, Tsc2Δpodocyte.
Fig 2.
mTORC1 activation in podocytes causes proteinuria and increased mortality.
(A) Kaplan-Meier survival plots for Tsc2Δpodocyte and control mice. A significant increase in mortality was found in Tsc2Δpodocyte compared with control mice. Nphs2-Cre (black line), n = 9; Tsc2flox/flox (blue line), n = 10; Tsc2Δpodocyte (red line), n = 32. (B) At 3 weeks, Tsc2Δpodocyte became albuminuric. SDS-PAGE gel shows a microliter of urine was loaded for each lane. (C) Seven-week follow-up of Tsc2Δpodocyte for proteinuria (n = 10 per group). Results are expressed as means ± s.d. White bar, Nphs2-Cre; dotted bar, Tsc2flox/flox; black bar, Tsc2Δpodocyte. Results are expressed as means ± s.d. *P < 0.05 compared with age-matched controls.
Fig 3.
mTORC1 activation in podocytes resulted in progressive glomerulosclerosis.
(A) Tsc2Δpodocyte developed progressive glomerulosclerosis between 4 and 8 weeks of age. Renal tissues from Tsc2Δpodocyte and control mice at 4, 6, and 8 weeks of age were stained with periodic acid-Schiff (PAS). Representative glomeruli from Tsc2Δpodocyte and control mice are shown. Various degrees of glomerulosclerosis, partial glomerulosclerosis and protein casts in tubules (asterisks), and glomerulosclerosis with synechia formation (arrowhead) are shown. Scale bar: 50 μm. (B) Transmission electron microscopy (TEM) shows partial flattening and disorganization of podocyte foot processes (arrows). Scale bar: 5 μm. (B, bottom) Glomerular basement membrane thickness in Tsc2Δpodocyte at 4 weeks of age was comparable with that in age-matched control mice (within arrowheads). Scale bar: 1 μm.
Fig 4.
Podocytes in Tsc2Δpodocyte mice were excreted in urine from glomeruli with progression of renal functional decline.
(A) Immunofluorescence staining of podocin and synaptopodin showed decreased signal intensity in Tsc2Δpodocyte mice (arrows). (B, top) The number of podocytes was decreased in the glomeruli of Tsc2Δpodocyte mice. Representative images of WT1 positive (red) and Hoechst 33342-positive (blue) podocytes are shown. (B, bottom) The graph shows the average number of podocytes per glomerulus in each group. Each dot represents the mean number of WT1-positive cells in about 20 glomeruli from the indicated mouse, and horizontal lines represent the mean number of WT1-positive cells in each group. Tsc2Δpodocyte mice had fewer WT1-positive podocytes per glomerulus compared with age-matched controls. *P < 0.05, **P < 0.01 compared with age-matched controls. (C) Western blot analysis of abundance of WT1 in urine from Tsc2Δpodocyte mice. Loaded samples contained equal amounts of creatinine. WT1 gives a band at 52 kDa (arrow).
Fig 5.
mTORC1 hyperactivation led to a decreased autophagic activity in the podocytes.
(A) Primary cultured podocytes were isolated from Tsc2Δpodocyte and wild-type controls, followed by western blot analyses of TSC2, phospho-4EBP1, LC3B type II, p62, phospho-ULK1, and β-tubulin. Arrows indicate specific bands corresponding to each indicated protein. (B) FIP200 and ATG101 mRNA expressions were decreased in the primary cultured podocytes from Tsc2Δpodocyte. (C) Representative fluorescence images of glomeruli of Nphs2-Cre- (top, left), Tsc2flox/flox- (top, right) and Tsc2Δpodocyte-GFP-LC3 transgenic mice (bottom, left) at 4 weeks of age. The white box indicates the location of the magnified figure. Scale bar: 50 μm. (bottom, right) Quantitative analysis of autophagic activity in vivo. Graph bars show the number of GFP-LC3 dots per glomerulus from the indicated mice (n = 3 per group). The number of GFP-LC3 dots per glomerulus was counted in 20 independent visual fields from the indicated mice. Results are expressed as means ± s.d. *P < 0.05 compared with age-matched controls.
Fig 6.
Rapamycin prevented death from renal dysfunction in Tsc2Δpodocyte.
(A) Tsc2Δpodocyte mice treated with rapamycin had improved survival compared to vehicle-treated Tsc2Δpodocyte mice. From 4 to 11 weeks of age, rapamycin was injected intraperitoneally (2 mg/kg body weight) twice a day (bold vertical line). Nphs2-Cre with rapamycin treatment (black line), n = 17; Tsc2flox/flox with rapamycin treatment (blue line), n = 16; Tsc2Δpodocyte with rapamycin treatment (red line), n = 8; vehicle-treated Nphs2-Cre (dashed black line), n = 7; vehicle-treated Tsc2flox/flox (dashed blue line), n = 9; vehicle-treated Tsc2Δpodocyte (dashed red line), n = 26. (B) Eight-week follow-up of rapamycin-treated Tsc2Δpodocyte for proteinuria (n = 6 per group). *P < 0.05 compared with age-matched vehicle-treated Tsc2Δpodocyte. (C) Urine collected from vehicle-treated and rapamycin-treated Tsc2Δpodocyte at 3, 5, and 7 weeks of age was subjected to SDS-PAGE. Arrow indicates the bands corresponding to albumin. (D, left) Renal tissues from Tsc2Δpodocyte and control mice with rapamycin treatment for 7 weeks were stained with PAS. Rapamycin treatment decreased the large amounts of PAS-positive materials present in the mesangial area of Tsc2Δpodocyte. Scale bar: 20 μm. (D, center) TEM analysis of rapamycin-treated Tsc2flox/flox and Tsc2Δpodocyte. Rapamycin treatment restored podocyte hypertrophy and foot process effacement in Tsc2Δpodocyte at 11 weeks of age. Scale bar: 4 μm. (D, right) Representative fluorescence images of glomerulus in Tsc2flox/flox and Tsc2Δpodocyte mice mated with GFP-LC3 transgenic mice 1 week after rapamycin treatment are shown. The white box indicates the location of the magnified figure. Scale bar: 50 μm.
Fig 7.
mTORC1 is activated in podocytes in patients with CKD.
(A) Representative photomicrographs of periodic acid-Schiff (PAS) staining in the kidney of db/db and db/m mice. Db/db mice exhibited glomerulosclerosis at 24 weeks of age. Scale bar: 50 μm. (B) Western blot analysis of p70 S6 kinase (p70S6K) phosphorylation using primary cultured podocytes isolated from db/db and db/m mice at 24 weeks of age. β-tubulin served as the internal control. (C) mTORC1 is activated in glomeruli of obese patients with CKD. Human kidney biopsy specimens from normal controls and obese patients with CKD were immunostained with anti-phospho-S6 ribosomal protein (pS6) antibody and counterstained with hematoxylin. pS6 protein was detected in podocytes (arrowheads), parietal cells lining Bowman’s capsule (arrow), and tubulointerstitial regions (asterisk) of obese patients with CKD. Representative low-magnification (top) and high-magnification (bottom) images are shown.