Table 1.
Biochemical and physical characteristics of the four groups at the end of the 12 weeks experimental period.
Figure 1.
Changes in glomerular phenotypes in fenofibrate-treated db/db mice.
Glomerular mesangial fractional area, TGF-β1 and collagen IV (Col IV) expression, and a F4/80-positive cell infiltration in the glomerulus in the cortical area of the db/m and db/db mice, with or without fenofibrate treatment. A) Representative sections stained with periodic acid-Schiff reagent and representative immunohistochemical staining for TGF-β1, type IV collagen, and F4/80-positive cells (dark brown) are shown (original magnification 400x). B-E) Quantitative analyses of the results for the mesangial fractional area (%), TGF-β1, type IV collagen, and F4/80-positive cells (fold) are shown. Abbreviations: Col IV, type IV collagen; PAS, periodic acid-Schiff stain, **p<0.01 vs. the db/m Cont (db/m), db/m Feno (db/m+Feno), and db/db Feno (db/db+Feno) mice.
Figure 2.
PPARα, Phospho-AMPK Thr172, total AMPK, PGC-1α, ERR-1α, SREBP-1, ChREBP-1, and intra-renal lipid levels in the renal cortex of the db/m and db/db mice with or without fenofibrate treatment.
Protein lysates (30 µg) from renal cortex were separated by SDS-PAGE and analyzed by Western blotting. A) Representative Western blotting is shown for PPARα, phospho-Thr172 AMPK, total AMPK, PGC-1α, ERR-1α, and β-actin. B–E) Quantitative analyses are shown for PPARα/β-actin (B), phospho-Thr172 AMPK/total AMPK (C), PGC-1α/β-actin (D), and ERR-1α/β-actin (E). *p<0.05 and **p<0.01 vs. the db/m control, db/m Feno, and db/db Feno mice. F–H) Representative Western blot of SREBP-1, ChREBP-1 and β-actin, (F) and the corresponding quantitative analyses (G and H). The level of pACC in the kidney are shown (I). J–L) Quantitative analyses of intra-renal total cholesterol (J), free fatty acid (FFA) (K), and triglyceride (L) concentrations. *p<0.05 and **p<0.01 vs. the db/m control, db/m Feno, and db/db Feno mice; †† p<0.01 vs. the db/m control and db/m Feno mice. Abbreviations: pAMPK, phospho-Thr172 AMPK.
Figure 3.
PI3K activity, PI3K, phospho–Akt Ser472, total Akt, phospho-FoxO3a Ser253 and total FoxO3a, and phospho-FoxO1 Ser256 total FoxO1 in the renal cortex of the db/m and db/db mice with or without fenofibrate treatment.
Protein lysates (40 µg) from renal cortex were separated by SDS-PAGE and analyzed by Western blotting. A) Representative Western blot showing PI3K, phospho-Ser472 Akt, total Akt, and β-actin levels. B) PI3K activity. C–D) Quantitative analyses of the results from the Western blot: PI3K/β-actin (C); phospho-Ser472 Akt/total Akt (D). E) Representative Western blot showing phospho-Ser253 FoxO3a, total FoxO3a, phospho-Ser256 FoxO1, and total FoxO1 and β-actin levels. F–G) Quantitative analyses of the results from the Western blot: phospho-Ser253 FoxO3a/total FoxO3a (F); phospho-Ser256 FoxO1/total FoxO1 (G). *p<0.05 and **p<0.01 vs. db/m control, db/m Feno, and db/db Feno mice. † p<0.05 vs. db/m control, db/m Feno and db/db mice. Abbreviations: pAkt, phospho-Ser472 Akt; pFoxO1, phospho-Ser256; pFoxO3a, phospho-Ser253 FoxO3a.
Figure 4.
Pro-apoptotic BAX, anti-apoptotic BCL-2, and TUNEL assay in the renal cortex of db/m and db/db mice with or without fenofibrate treatment.
Protein lysates (40 µg) from renal cortex were separated by SDS-PAGE and analyzed by Western blotting. A) Representative Western blot analysis of the BAX, BCL-2, and β-actin levels. B) The quantitative analyses of the results of the BCL-2/BAX ratio are shown. C) Representative immunohistochemical staining for TUNEL-positive cells and the quantitative analyses of the results are shown. **p<0.01 vs. db/m control, db/m Feno, and db/db Feno mice.
Figure 5.
Intra-renal SOD1, SOD2, and 24-hr urinary 8-isoprostane concentrations in db/m and db/db mice with or without fenofibrate treatment.
Protein lysates (10 µg) from renal cortex were separated by SDS-PAGE and analyzed by Western blotting. A–C) Representative Western blot analysis of the SOD1 and SOD2 and the quantitative analyses of the results are shown: SOD1 (B) and SOD2 (C). D) The 24-hr urinary 8-isoprostane concentrations of the study mice are shown. *p<0.05 and **p<0.01 vs. the db/m control, db/m Feno, and db/db Feno mice. Abbreviations: Cont, control.
Figure 6.
The effect of fenofibrate on intracellular signaling, apoptosis, and oxidative stress (8-isoprostane concentrations in the cultured media) in the mesangial cells cultured in low-glucose (5 mmol/l D-glucose) or high-glucose (30 mmol/l D-glucose) conditions with or without fenofibrate treatment (50 µg/ml).
PPARα, Phospho-Thr172 AMPK, total AMPK, PGC-1α, ERR-1α, PI3K, phospho-Ser472 Akt, total Akt, phospho-Ser253 FoxO3a, total FoxO3a, SREBP-1, and ChREBP-1 levels were assessed in the cultured mesangial cells. Protein lysates (10 µg) were separated by SDS-PAGE and analyzed by Western blotting. A) Representative Western blot analysis and quantitative analyses of PPARα are shown. C-I) Representative Western blot analysis of phospho-Thr172 AMPK, total AMPK, PGC-1α ERR-1α, PI3K, phospho-Ser472 Akt, total Akt, phospho-Ser253 FoxO3a, and total FoxO3a and β-actin levels in the cultured mesangial cells and the quantitative analyses of the results are shown; phospho-Thr172 AMPK, total AMPK (D); PGC-1α (E); ERR-1α (F); PI3K (G); phospho-Ser472 Akt, total Akt (H); phospho-Ser253 FoxO3a and total FoxO3a (I). J-L) Representative Western blot analysis of SREBP-1 and ChREBP-1 in cultured mesangial cells and the quantitative analyses of the results. M–N) The quantitative analyses of the TUNEL-positive cells (M) in the cultured mesangial cells and 8-isoprostane concentrations (N) from the cell-culture media of the study are shown. *p<0.05 and **p<0.01 compared with low glucose. Abbreviations: HG, high glucose; LG, low glucose; MN, mannitol; pAMPK, phospho-Thr172 AMPK; pAkt, phospho-Ser472 Akt; pFoxO3a, phospho-Ser253 FoxO3a.
Figure 7.
Immunoblot for phospho-AMPK, total AMPK, and PGC-1α in AMPKα1 siRNA, AMPKα2 siRNA, or PGC-1α siRNA knock-down mesangial cells (NMS) in a high-glucose environment with fenofibrate treatment.
The cultured mesangial cells were transfected with 50/l control siRNA or 50 nmol/l Ampkα1, Ampkα2, or PGC-1α siRNA using transfection reagent (G-Fectin) and stimulated with fenofibrate in high-glucose media. Approximately 48 hr after transfection, the levels of phospho-Thr172 AMPK, total AMPK, SIRT1, and PGC-1α signaling in the high-glucose media stimulated with fenofibrate, were analyzed. Protein lysates (10 µg) from the cultured mesangial cells were separated by SDS-PAGE and analyzed by Western blotting. A–C) Representative Western blot analysis of phospho-Thr172 AMPK, total AMPK, PGC-1α, and β-actin levels, (A) and the quantitative analyses of the results are also shown: phospho-Thr172 AMPK and total AMPK (B); PGC-1α (C). *p<0.05 compared with other groups. Abbreviations: HG, high glucose; Feno, fenofibrate; pAMPK, phospho-Thr172 AMPK.
Figure 8.
PPARα, Phospho-AMPK Thr172, total AMPK, PGC-1α, and phospho-FoxO3a Ser253 and total FoxO3a levels in the renal medullar of the db/m and db/db mice with or without fenofibrate treatment.
Protein lysates (30 µg) from renal medullar were separated by SDS-PAGE and analyzed by Western blotting. A) Representative Western blotting is shown for PPARα, phospho-Thr172 AMPK, total AMPK, PGC-1α, phospho-FoxO3a Ser253, total FoxO3a, and β-actin. B-E) Quantitative analyses are shown for PPARα/β-actin (B), phospho-Thr172 AMPK/total AMPK (C), PGC-1α/β-actin (D), and phospho-FoxO3a Ser253 and total FoxO3a (E). *p<0.05 and **p<0.01 vs. the db/m control, db/m Feno, and db/db Feno mice.