Protective effect of statin therapy on connective tissue growth factor induction by diabetes in vivo and high glucose in vitro.

Transcriptional activity of connective tissue growth factor (CTGF) promoter in transfected HEK293 cells was determined by luciferase assays. Secreted CTGF in cultured human mesangial cells was measured by enzyme-linked immunosorbent assay (ELISA). CTGF in urine and plasma was also measured in 405 subjects with/without type 2 diabetes. Our results showed that high glucose significantly increased transcription of the promoter in the transfected cells by more than 2.5-folds (p < 0.0005). CTGF secretion was induced by high glucose in the cells (p < 0.0005). These increases were inhibited by simvastatin. Urine CTGF was positively associated with plasma CTGF in both type 2 diabetes (p = 0.0005) and controls (p = 0.01). Urine CTGF levels in patients with macroalbuminuria were significantly higher than patients without macroalbuminuria (p < 0.05). In conclusion, our in vitro study suggests that statin may have a renal-protective effect through the inhibition of CTGF expression. Urine CTGF may be a good marker for the prediction of diabetic nephropathy.

Polymorphisms of myo-inositol oxygenase gene are associated with Type 1 diabetes mellitus.

Myo-inositol oxygenase (MIOX) is the first and rate-limiting enzyme in myo-inositol (MI) metabolism pathway. The increase in MIOX enzyme activity is in proportion to serum glucose concentrations and may be responsible for the MI depletion found in the diabetic complications. The aim was to investigate whether single nucleotide polymorphisms (SNPs) in the MIOX gene are associated with Type 1 diabetes mellitus (T1D) and its complications. Four hundred thirty Caucasian patients with T1D were recruited: 172 patients had diabetic nephropathy, 140 had diabetic retinopathy/neuropathy, 118 patients had diabetes for ≥20 years without microvascular complications and 224 were normal controls. Three SNPs, rs761745 (C/T), and rs2232873 (A/G) in the promoter and rs1055271 (C/G) in the 3'-untranslated were genotyped commercially. The frequencies of the CC genotype (0.36 vs. 0.44; P=.034) and C allele (0.60 vs. 0.68; P=.011) of rs761745 were significantly lower in patients with T1D compared with normal controls. Patients with T1D had a decreased frequency of the combination genotypes of CC (rs761745), GG (rs2232873) and GC (rs1055271) compared with the normal controls (0.13 vs. 0.22, P=.0027, Pc=0.014). The haplotypes with C/G/G and C/G/C were less common in patients with T1D compared to normal controls (0.59 vs. 0.70, P=.021) and the haplotypes with T/G/C and T/G/G ware more common in patients with T1D compared to normal controls (0.37 vs. 0.26; P=.021). In summary, our results demonstrated that the polymorphism (rs761745) in the promoter region of MIOX gene may be associated with the development of T1D in our studied population.

High glucose induction of DNA-binding activity of the transcription factor NFkappaB in patients with diabetic nephropathy.

The aim of this study was to investigate whether high glucose induces aldose reductase (AKR1B1) expression through NFkappaB, which may contribute to the pathogenesis of diabetic nephropathy. 34 Caucasoid patients with type 1 diabetes were recruited; 20 nephropaths and 14 long-term uncomplicated subjects. Peripheral blood mononuclear cells (PBMCs) were cultured under normal or high glucose (25 mmol/l of d-glucose) with or without an aldose reductase inhibitor (ARI). High glucose increased NFkappaB binding activities in the PBMCs from nephropaths compared to the uncomplicated subjects (1.77+/-0.22 vs. 1.16+/-0.04, p=0.02). ARI induced a substantially greater decrease of NFkappaB binding activities in the nephropaths compared to the uncomplicated subjects (0.58+/-0.06 vs. 0.79+/-0.06, p=0.032). AKR1B1 protein levels in the nephropaths were increased under high glucose conditions and decreased in the presence of an ARI, whilst the silencing of the NFkappaB p65 gene in vitro reduced the transcriptional activities of AKR1B1 in luciferase assays. These results show that NFkappaB induces AKR1B1expression under high glucose conditions, and the pattern of expression differs between nephropaths and the uncomplicated subjects.

Elevated activity of transcription factor nuclear factor of activated T-cells 5 (NFAT5) and diabetic nephropathy.

The expression of aldose reductase is tightly regulated by the transcription factor tonicity response element binding protein (TonEBP/NFAT5) binding to three osmotic response elements (OREs; OREA, OREB, and OREC) in the gene. The aim was to investigate the contribution of NFAT5 to the pathogenesis of diabetic nephropathy. Peripheral blood mononuclear cells (PBMCs) were isolated from the following subjects: 44 Caucasoid patients with type 1 diabetes, of whom 26 had nephropathy and 18 had no nephropathy after a diabetes duration of 20 years, and 13 normal healthy control subjects. In addition, human mesangial cells (HMCs) were isolated from the normal lobe of 10 kidneys following radical nephrectomy for renal cell carcinoma. Nuclear and cytoplasmic proteins were extracted from PBMCs and HMCs and cultured in either normal or high-glucose (31 mmol/l D-glucose) conditions for 5 days. NFAT5 binding activity was quantitated using electrophoretic mobility shift assays for each of the OREs. Western blotting was used to measure aldose reductase and sorbitol dehydrogenase protein levels. There were significant fold increases in DNA binding activities of NFAT5 to OREB (2.06 +/- 0.03 vs. 1.33 +/- 0.18, P = 0.033) and OREC (1.94 +/- 0.21 vs. 1.39 +/- 0.11, P = 0.024) in PBMCs from patients with diabetic nephropathy compared with diabetic control subjects cultured under high glucose. Aldose reductase and sorbitol dehydrogenase protein levels in the patients with diabetic nephropathy were significantly increased in PBMCs cultured in high-glucose conditions. In HMCs cultured under high glucose, there were significant increases in NFAT5 binding activities to OREA, OREB, and OREC by 1.38 +/- 0.22-, 1.84 +/- 0.44-, and 2.38 +/- 1.15-fold, respectively. Similar results were found in HMCs exposed to high glucose (aldose reductase 1.30 +/- 0.06-fold and sorbitol dehydrogenease 1.54 +/- 0.24-fold increases). Finally, the silencing of the NFAT5 gene in vitro reduced the expression of the aldose reductase gene. In conclusion, these results show that aldose reductase is upregulated by the transcriptional factor NFAT5 under high-glucose conditions in both PBMCs and HMCs.

Association of the p22phox component of NAD(P)H oxidase with susceptibility to diabetic nephropathy in patients with type 1 diabetes.

OBJECTIVE: Increased production of reactive oxygen species (ROS) in diabetes is thought to play a major role in the pathogenesis of diabetic microvascular complications such as nephropathy and retinopathy. The NAD(P)H oxidase complex is an important source of ROS in the vasculature. The p22 subunit is polymorphic with a C242T variant that changes histidine-72 for a tyrosine in the potential heme binding site, together with a A640G in the 3' untranslated region. The aim was to investigate the frequency of these polymorphisms in 268 patients with type 1 diabetes with or without microvascular complications. RESEARCH DESIGN AND METHODS: There was a highly significant increase in the frequency of the T/T242 genotype in patients with nephropathy compared with those with retinopathy alone or no microvascular disease after 20 years' diabetes duration (uncomplicated) or normal healthy control subjects (33.3 vs. 6.5, 5.7, and 0.0%, respectively, P < 0.000001). Furthermore, the T242/G640 haplotype was found in 39.4% of the patients with nephropathy but in only 26.5% of the patients with retinopathy and 15.3 and 10.6% of the uncomplicated and normal control subjects, respectively. RESULTS: When these variants of NAD(P)H oxidase were analyzed together with aldose reductase (5'ALR2) susceptibility genotypes, >46.0% of the patients with nephropathy possessed a T242 allele with the Z-2 5'ALR2 allele compared with only 11.2% of the uncomplicated patients (P < 0.00003). CONCLUSIONS: In conclusion, these results suggest NAD(P)H oxidase together with the polyol pathway may contribute to the pathogenesis of diabetic nephropathy.

The response of antioxidant genes to hyperglycemia is abnormal in patients with type 1 diabetes and diabetic nephropathy.

Increased flux of glucose through the polyol pathway may cause generation of excess reactive oxygen species (ROS), leading to tissue damage. Abnormalities in expression of enzymes that protect against oxidant damage may accentuate the oxidative injury. The expression of catalase (CAT), CuZn superoxide-dismutase (CuZnSOD), glutathione peroxidase (GPX), and Mn superoxide-dismutase (MnSOD) mRNA was quantified in peripheral blood mononuclear cells-obtained from 26 patients with type 1 diabetes and nephropathy, 15 with no microvascular complications after 20 years' duration of diabetes, and 10 normal healthy control subjects-that were exposed in vitro to hyperglycemia (HG) (31 mmol/l D-glucose). Under HG, there was a twofold increase in the expression of CAT, CuZnSOD, and GPX mRNA in the patients without complications and the control subjects versus patients with nephropathy (P < 0.0001), and MnSOD did not change in any of the groups. The aldose reductase inhibitor zopolrestat partially restored the levels of CAT, CuZnSOD, and GPX mRNA in the patients with nephropathy (P < 0.05). There was a highly significant correlation between increased aldose reductase (ALR2) expression, CAT, CuZnSOD, and GPX mRNA levels under HG conditions and polymorphisms of ALR2 in the patients with nephropathy (P < 0.00001). In conclusion, these results suggest that high glucose flux through aldose reductase inhibits the expression of antioxidant enzymes.