Attenuation of Extracellular Matrix Accumulation in Diabetic Nephropathy by the Advanced Glycation End Product Cross-Link Breaker ALT-711 via a Protein Kinase C-α−Dependent Pathway
Article Figures & Tables
Figures
- FIG. 1.
Representative immunoblots stained for PKC-α (80 kDa; A), phosphorylated PKC-α (82 kDa; B), PKC-βI (80 kDa; C), PKC-βII (80 kDa; D), PKC-ε (90 kDa; E), and β-tubulin (F) in kidneys at 32 weeks.
- FIG. 2.
Representative photomicrographs of renal cortical immunostaining at 32 weeks for PKC-α in control (A), diabetic (B; arrow demonstrates cytosolic distribution), DAG (C), and DALT (D) groups, and for phosphorylated PKC-α in control (E), diabetic (F; arrow demonstrates membranous translocation), DAG (G), and DALT (H) groups. Magnification ×200.
- FIG. 3.
Representative photomicrographs of renal cortical immunostaining for PKC-βI at 32 weeks in fixed sections from control (A), diabetic (B), DAG (C), and DALT (D) groups, and in frozen sections from control (E) and diabetic (F) groups. Magnification ×200.
- FIG. 4.
A: Representative immunoblot stained for renal cortical VEGF (45 kDa) (bottom row) and β-tubulin (top row) at 32 weeks. The graph (AG) represents the densitometric analysis (means ± SE) of individual Western blots of VEGF, expressed as relative optical density (ROD). *P < 0.001 vs. control; †P < 0.001 vs. diabetic; ‡P < 0.001 for DALT vs. DAG. B: Morphometric computer-aided analysis of renal glomerular VEGF immunostaining in rats at 32 week, expressed as the percent area of the glomerulus. Data are means ± SE. *P < 0.005 vs. control; †P < 0.01 vs. diabetic; ‡P < 0.001 for DALT vs. DAG. C: Morphometric computer-aided analysis renal cortical fibronectin at 32 weeks. *P < 0.001 vs. control group; †P < 0.005 vs. diabetic group; ‡P < 0.001 vs. diabetic; §P < 0.05 for DALT vs. DAG. D: Laminin immunostaining expressed as percent area. Data are means ± SE. *P < 0.001 vs. control group; †P < 0.001 vs. diabetic group; ‡P < 0.01 for DALT vs. DAG; §P < 0.05 vs. C.
- FIG. 5.
A: Confocal photomicrographs of PKC-α translocation and expression showing the effects of treatment with ALT-711 and aminoguanidine (AG) on VSMCs incubated in low (5 mmol/l) and high (25 mmol/l) glucose. B: Semiquantitative analysis of immunofluorescence (>30 cells of two independent experiments) 15 h after exposure to low-glucose (LG) and high-glucose (HG) concentrations with and without treatment. Data are means ± SE. *P < 0.0001 vs. LG; †P < 0.0001 for HG vs. HG +ALT; ‡P < 0.0001 for HG + ALT vs. HG + AG.
Tables
- TABLE 1
Functional and structural data from all groups
Control group CAG (0–32 weeks) CALT (16–32 weeks) Diabetic group DAG (0–32 weeks) DALT (16–32 weeks) Plasma glucose (mmol/l) 6.3 ± 0.2 5.9 ± 0.1 6.3 ± 0.1 28.4 ± 1.5* 30.0 ± 1.4* 26.9 ± 1.6* HbA1c (%) 4.5 ± 0.2 4.0 ± 0.2 5.9 ± 0.2 15.2 ± 0.8* 14.7 ± 0.9* 15.9 ± 0.7* AER (mg/24 h) Week 16 1.6 (1.5−1.7) 0.6 (0.5−1.5) 0.9 (0.6−1.6) 7.9 (4.8−21.6)* 1.6 (1.0−3.1)†‡ 8.5 (5.7−14.5)* Week 32 3.9 (2.2−10.6) 1.5 (0.6−3.2)§ 8.0 (3.6−9.4) 59.7 (31.6−88.0)* 3.4 (1.7−7.9)† 21.8 (11.1−32.6)†‖ Mean systolic blood pressure (mmHg) 115 ± 5 116 ± 7 114 ± 6 161 ± 4* 142 ± 6*¶ 145 ± 6*¶ Glomerular filtration rate (ml · min−1 · g kidney wt−1 2.2 ± 0.2 1.8 ± 0.2 2.4 ± 0.1 2.6 ± 0.1# 2.8 ± 0.2# 2.3 ± 0.2 Kidney−to−body weight ratio (×10−3) 2.9 ± 0.1 2.7 ± 0.2 2.9 ± 0.1 5.7 ± 0.2* 5.2 ± 0.4*†† 5.0 ± 0.2*¶ Renal AGEs (% area) 7.7 ± 0.4 7.3 ± 1.6 8.1 ± 0.9 14.9 ± 0.6* 8.3 ± 0.8† 7.2 ± 0.7† Data are means ± SE or median (interquartile range). Data for renal AGEs represent morphometric computer-aided analysis of renal cortical AGE (CML) immunostaining.
- *
* P < 0.001 vs control group;
- †
† P < 0.001 vs diabetic group;
- ‡
‡ P < 0.001 for DAG vs. DALT;
- §
§ P < 0.001 vs. CALT;
- ¶
¶ P < 0.01 vs. diabetic group;
- ‖
‖ P < 0.05 vs. DAG;
- #
# P < 0.05 vs. control group;
- ††
†† P < 0.05 vs. diabetic group.
- TABLE 2
Densitometric analysis of Western blots for PKC isoforms
Control group CAG (0−32 weeks) CALT (16−32 weeks) Diabetic group DAG (0−32 weeks) DALT (16−32 weeks) PKC-α 1.0 ± 0.1 1.2 ± 0.1 1.2 ± 0.1 1.8 ± 0.2* 1.5 ± 0.02† 1.4 ± 0.02† Phosphorylated PKC-α 1.0 ± 0.1 2.1 ± 0.1* 0.7 ± 0.1 1.8 ± 0.3* 2.8 ± 0.5* 1.0 ± 0.3† PKC-βI 1.0 ± 0.1 0.8 ± 0.1 0.8 ± 0.1 1.7 ± 0.1* 1.1 ± 0.02‡ 1.0 ± 0.1‡ PKC-βII 1.0 ± 0.1 0.7 ± 0.1§ 0.7 ± 0.1§ 1.3 ± 0.002§ 1.0 ± 0.1† 0.9 ± 0.1† PKC-ε 1.0 ± 0.1 1.2 ± 0.04 1.1 ± 0.1 1.7 ± 0.06* 1.1 ± 0.04‡ 1.3 ± 0.1‡ Data are means ± SE and are expressed as relative optical density.
- *
* P < 0.01,
- §
§ P < 0.05 vs. control group;
- †
† P < 0.05,
- ‡
‡ P < 0.01 vs. diabetic group.
In this Issue
