Article Figures & Data
Figures
- Figure 1.
p18 KO podocytes show unique characteristics in mTOR-related protein expression. (A) Tissue distribution of mTOR-Rag-Ragulator related proteins. Equal amount of proteins from different mouse tissues were applied to SDS-PAGE and different target proteins were examined by Western blotting. GAPDH is used as a loading control. (B) p18fl/fl and p18 KO podocytes were examined for their expression of WT1. Immortalized mouse podocytes (MPC5)41 were used as a positive control and mouse embryonic fibroblasts (MEF) as a negative control. (C) Genomic DNA was extracted from p18fl/fl and p18 KO podocytes. Primers spanning floxed exon 1 were used for PCR. (D) Cell lysates were prepared from p18fl/fl and p18 KO podocytes under basal growing conditions. Equal amounts of protein lysates were used for Western blotting.
- Figure 2.
p18 plays a key role in lysosomal mTOR localization in response to amino acids. (A) Immunostaining of p18 and LAMP2, a lysosomal membrane protein, in p18fl/fl and p18 KO podocytes. (B) Immunostaining of RagC and LAMP2 in p18fl/fl and p18 KO podocytes. (C) Immunostaining to examine mTOR subcellular localization under amino acid deprived or replenished conditions.
- Figure 3.
p18 plays a critical role in acute mTORC1 activation in response to growth factor or amino acids in podocytes. (A) The activity of mTORC1 and mTORC2 in normal growing p18fl/fl and KO podocytes. Cells were harvested under normal growth conditions (after 24 hours in replete growth culture media containing 10% serum and normal concentrations of amino acids). (B) Time course of mTORC1 activation by serum in p18fl/fl and p18 KO podocytes. Cells were serum starved, then stimulated with serum (2%) and harvested at the indicated time points. (C) Time course of mTORC1 activation by insulin stimulation (100 nM). (D) Time course of mTORC1 activation by amino acids (1×).
- Figure 4.
p18 in podocytes is dispensable for glomerular function. (A) Representative periodic acid–Schiff (PAS) staining and TEM images of the glomeruli of age-matched WT and podo-p18 KO mice are shown. (B) Immunohistochemistry analysis for nephrin and synaptopodin localization in the glomeruli of WT and podo-p18 KO mice. (C) 24-hour urine was collected from WT and podo-p18 KO mice at the indicated time points (n>4 for each genotype in each time point) and albumin and creatinine concentrations were measured. The results are shown as ratios of micrograms of albumin by milligrams of creatinine.
- Figure 5.
Ragulator is important for active Rheb-induced mTORC1 activation and podocyte injury in vitro. (A) TSC1 is knocked down in p18fl/fl and p18 KO podocytes. Cells were serum starved or stimulated with insulin (100 nM for 15 minutes). Cells lysates were used for Western blotting to examine mTORC1 activity. (B) Levels of cleaved or noncleaved caspase-3 and Akt phosphorylation were monitored.
- Figure 6.
Ablation of p18 in the podocytes prevents hyper-activation of mTORC1 and podocyte injury in podo-TSC1 KO mice. (A) Immunohistochemistry analysis of mTORC1 activity and Akt activity in the glomeruli from 8-week-old WT, podo-TSC1 KO, and podo-TSC1/p18 DKO mice. (B) Glomeruli were isolated from mice of the above genetic backgrounds. Tissue lysates were analyzed by Western blotting to examine mTORC1 activity and Akt phosphorylation. (C) Renal tissues from the indicated animals were double stained with Bip and synaptopodin, or Desmin and WT1. (D) Immunohistochemistry analysis of nephrin and synaptopodin localization in the glomeruli of the indicated mice. Areas indicated by squares in the Merge column were shown at higher magnification with individual channels. (E) Kidney sections from WT, podo-TSC1 KO, and podo-TSC1/p18 DKO mice were stained with hematoxylin and eosin (H&E), periodic acid–Schiff (PAS), fibronectin (FN), and type IV collagen (COL4). Glomerular tuft area and PAS-positive staining areas within the glomeruli of the indicated mice were quantified. For each genotype, 20 pictures were taken of different fields and used for quantification. *P<0.01 versus other groups; mean±SEM (n= approximately 3–4 mice).
- Figure 7.
Ablation of p18 in the podocytes prevents mTORC1-induced podocyte loss and glomerular dysfunction in podo-TSC1 KO mice. (A) The formation of the podocyte foot process in the indicated 8-week-old male mice was analyzed by TEM. The arrow and arrowhead indicate developed rough ERs and increased ribosomes, respectively, in podo-TSC1 KO podocytes. (B) Ratios (a number of WT1-positive cells/glomerular tuft area) were determined in about 25–35 glomeruli from the indicated animals. The data were expressed as the mean fold change. **P<0.001 versus other groups; mean±SEM (n= approximately 3–5 mice). (C) Urine was collected from the indicated animals at 4 and 8 weeks of age and subjected to SDS-PAGE. Coomassie blue staining was performed to visualize urinary proteins (upper panels). Urinary albumin concentrations in 24-hour urine from the indicated animals were measured. **P<0.001; mean±SEM (n= approximately 5–8 mice).
- Figure 8.
Ablation of p18 in the podocytes prevents mesangial expansion in diabetic mice. (A) Kidney sections from the indicated 20-week-old mice were stained with pS6, synaptopodin, periodic acid–Schiff (PAS), type IV collagen, and fibronectin. (B–D) Quantifications of PAS-, type IV collagen-, and fibronectin-positive area within a glomerulus were shown. Ratios (positive area/ glomerular tuft area) were determined in 40 glomeruli from the indicated mice and expressed as the mean fold change. **P<0.001; mean±SEM (n=4 mice).
- Figure 9.
The effects of p18 ablation in the podocytes on their foot process formation, number, and glomerular function in diabetic mice. (A) Representative WT1 staining and TEM images (foot process and cytoplasm) of the indicated 20-week-old mice. The arrow and arrowhead indicates rough ERs and ribosomes, respectively. (B) 24-hour urine volume of the indicated diabetic mice. Data are expressed as an average of the amount of urine over 3 days. Mean±SEM (n=5 mice). (C) Blood glucose levels in the indicated diabetic mice at 4 weeks after STZ treatment. Mean±SEM, (n=5 mice). (D) Ratios (the number of WT1-positive cells/glomerular tuft area) were determined in 40 glomeruli from the indicated animals. Data is expressed as the mean fold change. *P<0.01 versus WT diabetic mice (WT STZ); mean±SEM (n=5 mice). (E) Urinary albumin and creatinine concentrations were measured in 24-hour urine samples from the indicated animals. Albumin-to-creatinine ratios were shown (n=5 mice). STZ, streptozotocin.
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