Ciglitazone, a PPARgamma agonist, ameliorates diabetic nephropathy in part through homocysteine clearance

Abstract

Diabetes and hyperhomocysteinemia (HHcy) are two independent risk factors for glomeruloslerosis and renal insufficiency. Although PPARgamma agonists such as ciglitazone (CZ) are known to modulate diabetic nephropathy, the role of CZ in diabetes-associated HHcy and renopathy is incompletely defined. We tested the hypothesis that induction of PPARgamma by CZ decreases tissue Hcy level; this provides a protective role against diabetic nephropathy. C57BL/6J mice were administered alloxan to create diabetes. Mice were grouped to 0, 1, 10, 12, and 16 wk of treatment; only 12- and 16-wk animals received CZ in drinking water after a 10-wk alloxan treatment. In diabetes, PPARgamma cDNA, mRNA, and protein expression were repressed, whereas an increase in plasma and glomerular Hcy levels was observed. CZ normalized PPARgamma mRNA and protein expression and glomerular level of Hcy, whereas plasma level of Hcy remained unchanged. GFR was dramatically increased at 1-wk diabetic induction, followed by hypofiltration at 10 wk, and was normalized by CZ treatment. This result corroborated with glomerular and preglomerular arteriole histology. A steady-state increase of RVR in diabetic mice became normal with CZ treatment. CZ ameliorated decrease bioavailability of NO in the diabetic animal. Glomerular MMP-2 and MMP-9 activities as well as TIMP-1 expression were increased robustly in diabetic mice and normalized with CZ treatment. Interestingly, TIMP-4 expression was opposite to that of TIMP-1 in diabetic and CZ-treated groups. These results suggested that diabetic nephropathy exacerbated glomerular tissue level of Hcy, and this caused further deterioration of glomerulus. CZ, however, protected diabetic nephropathy in part by activating PPARgamma and clearing glomerular tissue Hcy.

Fig. 1.

A: expression of peroxisome proliferator-activated receptor-γ (PPARγ) gene in diabetic kidney tissues. Mice were treated with alloxan for 10 wk, as stated in materials and methods. RNA was isolated from kidney tissues, and equal amounts of poly (A)⁺ RNA from control and diabetic tissue were hybridized to cDNA array membranes. B: total mRNA was isolated from the kidney samples, and expression of PPARγ mRNA was measured by agarose gel eclectrophoresis; bottom: densitometric analysis of PPARγ mRNA expression. C: PPARγ protein expression was measured by Western blot, as described in the materials and methods; bottom: densitometric analysis of PPARγ expression. Note that PPARγ cDNA, mRNA, and protein show attenuated expression in diabetic kidney tissue, whereas ciglitazone (CZ) normalized PPARγ mRNA and protein expression (n = 6). #P < 0.01 compared with 0 wk.

Fig. 2.

Plasma levels of glucose and homocysteine (Hcy). A: mice were treated with alloxan (65 mg/kg body wt) as shown. Separate groups of alloxan-treated mice received CZ after 10 wk, and blood was collected at 12 and 16 wk. Plasma glucose levels were measured and compared with 0 wk (n = 6 in each group). *P < 0.01 compared with 0 wk. **P < 0.02 compared with 10 wk. B: plasma Hcy was separated with HPLC and measured by a spectrophotometer (n = 6 in each group). *P < 0.05 when compared with 0 wk. Note that CZ ameliorated hyperglycemia; however, CZ did not change the levels of plasma Hcy.

Fig. 3.

Glomerular filtration rate (GFR). A: mice were treated with alloxan (65 mg/kg alloxan body wt), and GFR was measured at different time periods as shown (n = 6 in each group). Mice were acclimatized in metabolic cages for 3 days, and 0.3 mg of inulin-FITC/25 g body wt was given intraperitoneally; urine was collected for 24 h. Separate groups of alloxan-treated mice that received CZ were placed in metabolic cages, and urine was collected at 12 and 16 wk. Plasma and urine inulin-FITC was measured, and GFR was expressed as μl·min⁻¹·g kidney wt⁻¹ (n = 6 in each group). *P < 0.02 compared with 0 wk; **P < 0.05 compared with 1 wk. B: renal vascular resistance (RVR) was measured by renal artery blood flow (ml/min) and pressure (mmHg) and expressed as mmHg·ml⁻¹·min⁻¹. *P < 0.01 compared with 0 wk; **P < 0.02 compared with 10 wk. Note that in this model of diabetes, initially there was hyperfiltration followed by hypofiltration.

Fig. 4.

CZ attenuates glomerular hypertrophy. Six-micrometer sections of kidney tissue samples from different treated groups as shown were stained with Masson-Trichrome stain and visualized under dissecting microscope; 10 × 20 magnification (n = 6 in each group).

Fig. 5.

Glomerular tissue levels of Hcy and nitric oxide (NO). A: kidney was removed from anesthetized mice, and cortical tissues were separated from medullary mass. Cortical tissue homogenates were prepared. Total Hcy was extracted, separated by HPLC, and quantitated by a spectrophotometer. Hcy was expressed as ng/mg of protein. *P < 0.01 compared with 0 wk; **P < 0.01 compared with 10 wk. B: total nitrate/nitrite was measured by Griess method. NO levels were expressed as nM/l of cortical tissue homogenate. Equal amounts of total protein were used. *P < 0.02 compared with 0 wk; **P < 0.05 compared with 0 wk. ***P < 0.05 compared with 10 wk. Note that tissue levels of Hcy were normalized by CZ treatment.

Fig. 6.

A: matrix metalloproteinase (MMP)-2 and -9 analysis. Glomeruli were isolated under a dissecting microscope from different groups of animals as indicated, and glomeruli-extracted protein was analyzed by 1.5% in-gel gelatin zymography (zymography image of MMP-2 and -9). To verify equal amount of loading, homogenates of the gelatin zymography for MMP-2 and -9 groups containing 25 μg of protein from each of the samples were separated by 10% SDS-PAGE gel and transferred to PVDF membrane, and Western blot was performed using anti-β-actin antibody (Western blot image of β-actin). B: densitometric analyses of MMP activity that were shown in A. *P < 0.01 compared with 0 wk (means ± SE; n = 6 in each group); **P < 0.05 compared with 10 wk (means ± SE; n = 6 in each group). Note that both MMP activities were attenuated by CZ at 12 and 16 wk.

Fig. 7.

A: tissue inhibitor of metalloproteinase (TIMP)-1 and -4 analysis. Glomeruli were isolated under a dissecting microscope from different groups of animals as indicated, and glomeruli homogenates were subjected for Western blot analyses using anti-TIMP-1 and anti-TIMP-4 antibody. Membranes were reprobed with anti-β-actin antibody to verify equal amount of loading. A representative β-actin loading control is shown at A, bottom. B: densitometric analyses of TIMP-1 and -4 protein that were shown in A. *P < 0.01 compared with 0 wk (means ± SE; n = 6 in each group); **P < 0.05 compared with 10 wk (means ± SE; n = 6 in each group). Note that TIMP-1 expression was increased at 1 and 10 wk, and this expression was attenuated by CZ at 12 and 16 wk, whereas TIMP-4 expression was reversed.

Fig. 8.

Preglomerular arteriole and tubule histology. Part of kidney from 0 wk (A), 10 wk of alloxan treatment (B), and 10 wk of alloxan plus another 6 wk of CZ treatment (C) was frozen using tissue-freezing medium for cryosection and histological analysis. Six-micrometer sections were stained with Masson-Trichrome. D: preglomerular arterioles were identified under a microscope, and medial/lumen ratio was measured by a digital micrometer and plotted. Bar graph represents mean ± SE of 6 animals/group. *P < 0.01 compared with 0 wk; **P < 0.05 compared with 10 wk. Note that medial/lumen ratio was increased dramatically after 10 wk of alloxan treatment and was ameliorated significantly by CZ treatment at 16 wk.

Fig. 9.

A: typical dose-response curve of phenylepinephrine (PE) in renal artery isolated from 0-wk animal. B: dose-response curve of PE in renal artery isolated from different groups of animal as shown. C: the vessels were precontracted with PE and treated with different doses of acetylcholine (Ach) as shown. *P < 0.01 compared with 0 wk (means ± SE; n = 6 in each group). Note that CZ ameliorated diabetes-induced renal artery contraction and endothelial-mediated renovascular relaxation.