Association of the p22phox Component of NAD(P)H Oxidase With Susceptibility to Diabetic Nephropathy in Patients With Type 1 Diabetes

DNA samples from 274 British Caucasian patients with type 1 diabetes were randomly taken for analysis. A total of 131 samples from normal healthy British Caucasian subjects was randomly obtained for control frequencies. The normal control subjects consisted of DNA from cord blood samples collected sequentially after obstetric delivery from the Obstetric Department, Derriford Hospital (Plymouth, U.K.). Local ethical committee approval had been obtained. The clinical features of the patients are shown in Table 1. Patient classification is summarized as follows.

These patients (n = 70) have had type 1 diabetes for at least 20 years but remained free of retinopathy (fewer than five dots or blots per fundus), proteinuria (negative on urine Albustix on at least three consecutive occasions over the previous 12 months), and overt neuropathy. Overt neuropathy was defined if there was any clinical evidence of peripheral or autonomic neuropathy.

These patients (n = 117) had type 1 diabetes for >10 years. All had proteinuria (urine Albustix positive on at least three consecutive occasions over 12 months or three successive total urinary protein excretion rates >0.5 g/24 h) in the absence of hematuria or infection on midstream urine samples. Diabetic nephropathy was always associated with retinopathy.

These patients (n = 46) had retinopathy defined as more than five dots or blots per eye; hard or soft exudates, new vessels, or fluorescein angiographic evidence of maculopathy or previous laser treatment for preproliferative or proliferative retinopathy; and maculopathy or vitreous hemorrhage. Both a diabetologist and ophthalmologist performed fundoscopy.

These patients (n = 36) had a duration of diabetes ≤10 years but with no evidence of retinopathy, proteinuria, or overt neuropathy.

High-molecular weight DNA was prepared from 10 ml peripheral blood using Nucleon extraction kits (Tepnel; Wythenshawe, Manchester, U.K.). Two sets of amplimers were designed to specifically amplify the p22 PHOX gene; the first set was used to amplify the region containing the C242T polymorphic site and the second set to amplify the region containing the A640G polymorphism.

The amplification reaction for C242T was performed in 30-μl volumes containing the amplimers 5′-TGC TTG TGG GTA AAC CAA GGC CGG TG-3′ and 5′-AAC ACT GAG GTA AGT GGG GGT GGC TCC TGT-3′, 10 mmol/l dNTPs (Invitrogen, Paisley, Scotland), 10× buffer solution, 10 mmol/l MgCl₂, and 1 unit Taq polymerase (HT Biotech, Cambridge, U.K.). The samples were subjected to an initial denaturation at 96°C for 2 min, followed by 30 cycles of amplification in a three-step reaction consisting of denaturation for 30 s at 94°C, annealing for 1 min at 56°C, and extension for 1 min at 72°C in an I Cycler Thermal Cycler (Biorad, Hemel Hempstead, U.K.). Amplification resulted in a 348-bp fragment that included the RsaI polymorphic site.

The amplification reaction for A640G was performed in 30-μl volumes containing the amplimers 5′-AGC AGT GGA CGC CCA TCG AGC CCA A-3′ and 5′-CGC TGC GTT TAT TGC AGG TGG GTG C-3′. The PCR and conditions were the same as above. Amplification resulted in a 258-bp fragment that included the DraIII polymorphic site.

The C242T polymorphism results in the creation of the RsaI recognition site through a cystenine-to-thymine transition. Ten units of RsaI (New England Biolabs, Hertfordshire, U.K.) were used to digest the PCR products for 3 h at 37°C. The digested fragments were separated by gel electrophoresis on a 1.5% agarose gel (Invitrogen). The bands were visualized under ultraviolet light and scored. The presence of the RsaI site resulted in fragments of 160 and 188 bp in size; if the RsaI site was not present, a fragment of 348 bp was visualized.

The A640G polymorphism results in the DraIII site being abolished through an adenine-to-guanine transition. Ten units of DraIII (New England Biolabs) were used to digest the PCR products for 3 h at 37°C. The digested fragments were separated by gel electrophoresis on a 1.5% agarose gel. The bands were visualized under ultraviolet light and scored. If the DraIII site was present, two fragments of 227 and 31 bp were produced; if the site was not present, a fragment of 258 bp was visualized.

The χ² test and contingency tables were used to compare the frequency of alleles and genotypes in the patient subgroups and normal control subjects. The P values were corrected for the number of comparisons made (Pc) using the Bonferroni inequality method (20). Pc values <0.05 were considered significant. Where appropriate, the odds ratio was calculated. The frequency of the C242T and A640G genotypes were in Hardy-Weinberg equilibrium.

The frequency of the C242T and A640G genotypes and alleles of the NAD(P)H p22 PHOX gene in patients with type 1 diabetes is shown in Tables 2 and 3. For the C242T locus, three genotypes were identified and designated homozygous C/C 242, homozygous T/T 242, and heterozygous C/T 242. Similarly, for the A640G locus the genotypes detected were homozygous A/A 640, homozygous G/G 640, and heterozygous A/G 640. There was a marked increase in the frequency of the T/T 242 homozygous genotype in those patients with nephropathy compared with the long-term uncomplicated subjects and those with retinopathy alone (33.3 vs. 5.7 and 6.5%, respectively, P < 0.0000001). The frequency of this genotype in the short-duration patients was similar to the uncomplicated subjects and was completely absent in the normal healthy control subjects (11.4, 5.7, and 0.0%, respectively). The patients with nephropathy also had an increased frequency of the heterozygous genotype T/C 242, although this genotype was also increased in the patients with retinopathy (47.9 and 65.2%, respectively). The C/C 242 genotype was the most common genotype in the uncomplicated patients and normal healthy control subjects (58.6 and 67.2%, respectively, P < 0.0000001, Pc = 0.0000004). The differences in the frequency of the A640G genotypes was less marked between the subject groups, although the A/A 640 genotype was found in 21.4% of the patients with nephropathy and only 8.6% of the uncomplicated subjects. The deviations in the A242T genotypes created marked differences in the C242T allelic frequencies between the groups. As expected, the T242 allele was significantly increased in the patients with nephropathy when compared with any of the other groups (57.3 vs. 23.6% uncomplicated, 39.1% patients with retinopathy, and 16.4% normal healthy control subjects, P < 0.0000001, Pc = 0.0000004). There were no significant differences in the frequency of the A640G alleles between the patient groups.

Table 4 shows the frequency of the C242T/A640G haplotypes in the patient subgroups. There was a marked increase in the frequency of the T242/G640 haplotype in the patients with nephropathy compared with the uncomplicated patients (39.4 vs. 15.3%, P < 0.00001, Pc = 0.00012). The patients with retinopathy and short-duration patients had intermediate frequencies of this haplotype (26.5 and 22.9%, respectively), whereas it was present in only 10.6% of the normal control subjects. In contrast, the C242/G640 haplotype occurred in 63.7% of the uncomplicated patients and 29.8% of the patients with nephropathy (P < 0.000001, Pc = 0.000012). Again, the patients with retinopathy and short-duration patients had intermediate frequencies of 42.6 and 52.9%, whereas the normal control subjects had the same frequency as the uncomplicated patients (63.7%). The frequency of T242/A640 was slightly increased in the patients with nephropathy compared with the uncomplicated subjects (18.7 vs. 4.85%, P < 0.001, Pc = 0.012), but C242/A640 was similar between all the groups.

When the NADPH oxidase and the 5′ALR2 aldose reductase genotypes were combined, >46.0% of the patients with nephropathy carried a T242 allele with the Z-2 5′ALR2 allele compared with only 11.2% of the uncomplicated patients (P < 0.00003, Pc = 0.0003) (Table 5). In contrast, 52.9% of the uncomplicated subjects carried the C242 allele with the “protective” Z+2 5′ALR2 allele compared with only 12.9% of the patients with nephropathy (P < 0.000001, Pc = 0.00003). This association was less marked in the patients with retinopathy. Interestingly, in the patients with nephropathy, 93.5% (72/75) had either the T242 or Z-2 allele compared with 62.2% (33/53) of the uncomplicated patients.

To our knowledge, this is the first report investigating the p22phox C242T polymorphism of the NAD(P)H oxidase complex in patients with type 1 diabetes and coexisting nephropathy, retinopathy, or no microvascular complications. The results obtained suggest that the T242 allele of p22phox contributes to the susceptibility to diabetic nephropathy, while the C242 allele may have a protective role. In addition, there may be an additive effect with the 5′ ALR2 susceptibility allele Z-2. Interestingly, the association was only found with diabetic nephropathy; those patients with retinopathy and normal renal function showed no difference from those with no complications after 20 years’ duration of diabetes. Previous studies have focused on patients with coronary heart disease. It is well known that coronary heart disease occurs at a much higher frequency in diabetic patients with rather than without diabetic nephropathy. This study was not designed to look at the frequency of coronary heart disease in the patient cohorts, but it is possible that the association is a reflection of the high incidence of macrovascular disease in patients with nephropathy rather than renal disease itself.

It has previously been shown that the T242 allele is associated with reduced oxidase activity in saphenous veins, while most studies in atherosclerosis have postulated an increase in activity. Clearly, the role of NAD(P)H oxidase in disease manifestation is complex and involves other molecular and biochemical systems such as the polyol pathway and pro- and antioxidant systems. For example, excess flux through the polyol pathway due to increased expression and activity of ALR2 may lead to the depletion of the cofactors NADPH and NAD+, as well as the increased production of ROS. Consequently, it might be predicted that less NADPH was available as cofactor for NAD(P)H oxidase, but this might be counterbalanced by the increased abundance of NADH. The latter has been suggested as the preferred substrate for NAD(P)H oxidase, although there have been studies that suggest otherwise (10,11,21–23). The flux through the entire polyol pathway and the build-up of NADH may be important rather than the single enzyme reactions. While this may explain part of the relationship between the polyol pathway and NAD(P)H oxidase, it still assumes that excess ROS will be generated. However, the T242 allele appears to be associated with reduced activity. The balance between pro- and antioxidant defenses is clearly important, although the precise nature of these systems has not been elucidated. There is a growing list of genes that are regulated through redox-sensitive pathways that are not always due to an increase in ROS. NAD(P)H oxidase may have a role in cellular processes, including apoptosis and cell proliferation, that may be involved in the disease manisfestation. There is also the possibility that the association is not related to the T242 allele. Our data show that the entire NAD(P)H oxidase haplotype, including the G640 allele, is important. This raises the possibility that additional polymorphisms may exist within the gene that contribute to the association. Previous studies have not considered the role of the haplotype but instead have focused on the individual alleles. It would be interesting to ascertain the activity of the haplotypes rather than the individual alleles.

In conclusion, this is the first report of an association between the phox22 subunit of NAD(P)H oxidase and susceptibility to diabetic nephropathy, whereas retinopathy and normal renal function show no association. There may be an interaction between the NAD(P)H oxidase and polyol pathway that may increase the risk of developing nephropathy or possibly macrovascular disease in patients with diabetes.

This work was funded in part by a grant from the Northcott Devon Medical Foundation.