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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.

Glucose transporter 1 (GLUT1) activity has been implicated in renal hypertrophy and extracellular matrix formation in mesangial cells. Recent studies have suggested that polymorphisms in the GLUT1 gene are associated with susceptibility to diabetic nephropathy (DN) in patients with diabetes mellitus. In this study, a novel polymorphism (A-2841T) in the 5' flanking region of GLUT1 was examined in 288 patients with Type 1 diabetes mellitus (T1DM) and 101 normal controls. The polymorphisms were amplified and the fragment digested with the enzyme HpyCH4V. There was a highly significant increase in the frequency of the TT-2841 genotype in patients with nephropathy (n=131) compared with those with either no microvascular complications after a 20-year duration of diabetes (uncomplicated; n=72; 54.5% vs. 2.7%, chi=79.4, P

Polymorphisms of the glucose transporter (GLUT1) gene are associated with diabetic nephropathy.BackgroundDiabetic nephropathy (DN) is a major cause of morbidity and mortality in patients with type 1 diabetes mellitus. Recent studies suggest that genetic factors, including polymorphisms in the flanking region of the aldose reductase gene (5′ALR2), play an important role in the pathogenesis of nephropathy. Glucose transporter (GLUT1) activity has been implicated in renal hypertrophy and extracellular matrix formation in mesangial cells. The aim was to investigate the frequency of a polymorphism within the GLUT1 gene in 186 Caucasoid patients with type 1 diabetes and 104 normal controls.MethodsAmplimers flanking the Xba-I polymorphic site in the second intron were employed to amplify DNA from subjects. The amplified DNA was restricted with endonuclease Xba-I, separated by gel electrophoresis, and visualized. In the absence of an Xba-I site, a fragment of 1.1 kilobase was seen, whereas fragments of 0.9 and 0.2 were generated if the Xba-I site was present.ResultsThere was a highly significant increase in the frequency of the 1.1 allele in those patients with nephropathy (N = 70) compared with those with no proteinuria or retinopathy after 20 years of diabetes (uncomplicated N = 44, 61.4 vs. 40.9%, respectively, P < 0.001). The 1.1/1.1 genotype was also significantly increased in the nephropathy group compared with the uncomplicated group of patients (37.1 vs. 13.6%, respectively, P < 0.01). The frequency of the 1.1/1.1 genotype was similar in 30 patients with retinopathy but not nephropathy when compared with the uncomplicated group of patients (13.6 vs. 16.7%). Furthermore, only 8 out of 49 patients with DN had the Z+2 5′ALR2 DN “protective” allele and the 0.9 GLUT1 allele in contrast to 21 out of 39 uncomplicated patients (P < 0.0002).ConclusionThese results suggest that the GLUT1 gene together with the aldose reductase gene are associated with susceptibility to DN in patients with type 1 diabetes.

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.

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.

Nuclear factor kappa B (NFkappaB) is an important transcription factor that is involved in the response to oxidative stress and inflammation. Recent studies suggest that it may be involved in the development of diabetic microvascular complications. A highly polymorphic (CA) dinucleotide repeat microsatellite has been identified in the regulatory region of the NFkappaB gene. The aim of this study was to investigate whether this polymorphic region was associated with susceptibility to type 1 diabetes, or its late complications. Genomic DNA was extracted from the peripheral blood of 217 patients with type 1 diabetes mellitus (T1DM) and 111 normal healthy controls. In our population 18 alleles (A1-A18) were identified. There was a highly significant decrease in the frequency of allele 146 bp (A14) in type 1 diabetes (0.03) compared with the normal controls (0.28) (chi(2) = 79.8, Pc = 0.00001). In contrast, the frequency of the allele 138 bp (A10) was significantly increased in patients with type 1 diabetes (0.17) compared with the normal controls (0.02) (chi(2) = 32.8, P < 0.00000). These results demonstrate that the NFkappaB gene may play a role in the susceptibility to type 1 diabetes: individuals with the A10 allele may be more likely to develop diabetes compared with the A14 allele.

We have investigated the frequency of the angiotensin I-converting enzyme (ACE) insertion/ deletion (I/D) polymorphism in 249 patients with type I diabetes and 162 normal healthy controls. There was no significant difference in the frequency of ACE genotypes between those patients with diabetic nephropathy (n = 72) (nephropaths) compared to those with no proteinuria after 20 years duration of diabetes (n = 86) (normoalbuminurics). There was, however, a significant difference in the frequency of ACE genotypes between the short-duration and long-term normoalbuminuric group (chi = 11.5, p = 0.001). Analysis of the ACE genotypes with respect to age and duration of diabetes showed that homozygosity for the insertion (I/I) genotype was significantly decreased with longer duration of diabetes (r2 = 92.7%, p < 0.009). No association was found with age in the normal controls. In conclusion, these results suggest that the ACE locus may be associated with longevity and survival in patients with type I diabetes rather than diabetic nephropathy or microvascular disease per se.

Aldose reductase (ALR2), the first enzyme of the polyol pathway, may plan an important role in the pathogenesis of diabetic microvascular complications. The gene coding for ALR2 has been localized to chromosome 7q35. Using an ALR2 probe in conjunction with the restriction endonuclease Bam-HI, we have investigated the ALR2 locus of 128 patients with type I diabetes. A significant decrease in the frequency of the 8.2 kilobase (kb) Bam-HI ALR2 genotype and 8.2 kb allele was found in patients with nephropathy (nephropaths) compared to those with retinopathy alone (retinopaths) (p0.05 and 0.25, respectively). We have previously shown that an RFLP of the T-cell antigen receptor constant beta-chain (TCRBC) locus, which is also localized to chromosome 7q35, is strongly associated with susceptibility to microvascular complications. The 128 patients were genotyped using the restriction endonuclease Bgl-II and a TCRBC probe. The 10/9.2-8.2 kb TCRBC-ALR2 genotype was significantly decreased in the nephropaths compared to the retinopaths (13.7% versus 43.6%, chi 2 = 10.1, p0.0025). The 10/9.2 and 9.2/9.2 kb TCRBC-ALR2 haplotypes were increased in the nephropaths compared to the retinopaths (32.5% versus 8.9% chi 2 = 10.9, p0.001). These results suggest that chromosome 7q35 harbors a gene(s) that is involved in the pathogenesis of microvascular complications. Interestingly, the gene coding for endothelial nitric oxide synthase has recently been localized to the same chromosomal region as ALR2.

Aldose reductase expression is induced by hyperglycemia in diabetic nephropathy.BackgroundDespite good metabolic control, many patients with type 1 diabetes still develop nephropathy, implicating a role for genetic factors. Recent studies examining the regulatory region of the aldose reductase (ALR2) gene, the rate-limiting enzyme of the polyol pathway, support its role as a candidate gene for nephropathy. Here we report the quantitation of ALR2, together with sorbitol dehydrogenase mRNA in the peripheral blood mononuclear cells (PBMCs) of type 1 diabetic patients with (N = 29) and without nephropathy (N = 11) following stimulation with high levels of d-glucose.MethodsPBMCs from patients and normal controls were cultured for five days with phytohemagglutinin in either normoglycemia (11mmol/L d-glucose) or supplemented with 10mmol/L d-glucose (moderate hyperglyemia) or 20mmol/L d-glucose (hyperglycemia). The RNA was extracted and analyzed by ribonuclease protection assay.ResultsALR2 mRNA levels were significantly elevated with increasing d-glucose concentration (normal to hyperglycemic) in those patients with nephropathy (P < 0.0001). In marked contrast, in those without nephropathy and in the normal healthy controls, there was no change in mRNA expression. Furthermore, those patients with nephropathy and the Z–2/X susceptibility genotype had the greatest increase in ALR2 mRNA compared with those with low-risk genotypes (P < 0.007).ConclusionThese results show that patients with nephropathy exhibit marked disturbances in the expression of the enzyme components of the polyol pathway. Ultimately this leads to tissue damage and ischemia.

The aim of this study was to investigate whether high glucose induces aldose reductase (AKR1B1) expression through NFκB, 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 NFκB 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 NFκB 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 NFκB p65 gene in vitro reduced the transcriptional activities of AKR1B1 in luciferase assays. These results show that NFκB induces AKR1B1expression under high glucose conditions, and the pattern of expression differs between nephropaths and the uncomplicated subjects.