Haptoglobin Genotype and Renal Function Decline in Type 1 Diabetes

The Epidemiology of Diabetes Complications Study was based on a historical cohort of incident cases of childhood-onset (<17 years) type 1 diabetes, diagnosed or seen within 1 year of diagnosis (1950–1980) at Children's Hospital of Pittsburgh (16). The cohort has been shown to be representative of the Allegheny County, Pennsylvania, type 1 diabetes population (17). Subsequent to a first clinical assessment (1986–1988, when average participant age and diabetes duration were 28 and 19 years, respectively), biennial examinations were conducted for 10 years, with a further examination at 18 years. The University of Pittsburgh Institutional Review Board approved the study protocol.

Prior to each clinic visit, participants were sent questionnaires concerning demographic, health care, self-care, and medical history information. Blood pressure was measured with a random zero sphygmomanometer after a 5-min rest (18). Hypertension was defined as ≥140/90 mmHg or use of antihypertensive medication. Stable HbA₁ was measured by ion exchange chromatography (Isolab, Akron, OH) and subsequently by automated highperformance liquid chromatography (Diamat; BioRad, Hercules, CA). The two assays were highly correlated (r = 0.95). HDL cholesterol was determined by a precipitation technique with a modification (19) of the Lipid Research Clinics method (20). Cholesterol and triglycerides were enzymatically measured (21,22). Non-HDL cholesterol was calculated as total minus HDL cholesterol. White blood cell count was obtained using a counter S-plus IV and fibrinogen using a biuret colorimetric procedure and a clotting method.

Urinary albumin was measured by immunonephelometry (23), and creatinine was assayed by an Ectachem 400 Analyzer (Eastman Kodak, Rochester, NY). Microalbuminuria was defined as albumin excretion rate (AER) of 20–200 μg/min (30–300 mg per 24 h) and macroalbuminuria as AER >200 μg/min (>300 mg per 24 h) in at least 2 of 3 validated timed urine collections. In 10% of the samples, urine collections were deemed inadequate based on creatinine excretion and albumin-to-creatinine ratio was used (microalbuminuria, 0.03–0.3 mg/mg; macroalbuminuria, >0.3 mg/mg) (24). ESRD onset was defined as starting dialysis or undergoing renal transplantation. Early renal function decline was defined as the incidence of a decline of ≥30 ml/min per 1.73 m² from baseline estimated glomerular filtration rate (eGFR) based on the Cockcroft-Gault equation (25) among participants with normal or mildly reduced renal function at study entry (stages I and II).

High molecular weight genomic DNA was isolated using the PureGene kit (Gentra Systems, Minneapolis, MN), and Hp was genotyped by an amplification method (26). Genotypes were assigned visually by comparison with controls of known genotype and in a random sample showed excellent agreement (97%) with an Eliza method (27).

Nonnormally distributed variables were logarithmically transformed. Univariate associations were determined using the Student t test and χ² or Fisher exact test, as appropriate. Cox proportional hazards models with backward elimination were constructed to assess the multivariable association between Hp genotype and the incidence of each outcome of interest adjusting for traditional risk factors (including eGFR and AER levels at study entry) and univariately significant variables. Survival time was defined as the time in years from study entry to either an incident event or censorship during the 18-year follow-up. Statistical analyses were conducted using SAS (version 9.1; SAS Institute, Cary, NC).

Of 658 study participants, DNA for Hp genotyping was available for 486 (73.9%). Compared with those without DNA available, individuals with DNA data had a shorter diabetes duration and higher HbA₁, blood pressure, non-HDL cholesterol, serum creatinine, eGFR, and AER. The distribution of the Hp genotype was 12.1% Hp 1/1, 44.4% Hp 2/1, and 43.4% Hp 2/2. Generally, no differences were observed in participant characteristics by Hp genotype at study entry with the exception of younger age and higher non-HDL cholesterol in those with the Hp 2/2 compared with the 2/1 genotype and lower insulin dose per weight in those with the Hp 2/1 compared with the 1/1 genotype.

During 18 years of follow-up, 40.5% (n = 111) developed incident microalbuminuria, 16.7% (n = 62) macroalbuminuria, and 12.2% (n = 58) ESRD. Moreover, 188 (42.0%) exhibited an early decline in renal function (≥30 ml/min per 1.73 m² from baseline eGFR). Descriptive participant characteristics by incidence of microalbuminuria and macroalbuminuria are shown in Table 1 and by renal function decline ≥30 ml/min per 1.73 m² and ESRD incidence in Table 2. Generally, incident case subjects with both microalbuminuria and macroalbuminuria were more likely to have higher levels of HbA₁, non-HDL cholesterol, AER, and inflammatory markers compared with those who remained disease free. Incident case subjects with macroalbuminuria were also older at the time of diabetes onset compared with noncase subjects and had a greater waist-to-hip ratio.

Compared with noncase subjects, incident case subjects with early renal function decline and ESRD were older, with higher systolic blood pressure, non-HDL cholesterol, AER, and inflammatory marker levels and lower HDL cholesterol. Incident case subjects with early renal function decline had an older age of diabetes onset and higher BMI, HbA₁, and eGFR and lower serum creatinine. Conversely, greater diabetes duration and higher levels of diastolic blood pressure and serum creatinine but lower insulin dose per kilogram body weight and eGFR were observed in incident case subjects with ESRD compared with noncase subjects. No univariate association, however, was observed between Hp and the incidence of other renal outcomes at the 0.05 significance level. Hp genotype was also not associated with all-cause mortality (P = 0.80) based on 82 (16.9%) deceased individuals.

Multivariable Cox proportional hazards models (Table 3) showed no association between the Hp genotype and microalbuminuria or macroalbuminuria incidence. Conversely, adjusting for univariately significant risk factors, an increased risk of an early renal function decline was observed for individuals carrying the Hp 2/2 compared with the Hp 1/1 genotype (hazard ratio 1.79 [95% CI = 1.06–3.00]). Similarly, the Hp 2/2 conferred over a twofold increased risk of ESRD compared with the Hp 1/1 genotype (2.45 [1.05–5.73]). The risk associated with the Hp 2/1 reached statistical significance for neither early renal function decline nor ESRD incidence.

To examine the possibility of survival bias, we stratified the cohort by diabetes diagnosis year (prior to or after 1965, wherein mortality was 40 vs. 13%, respectively). With the exception of macroalbuminuria, a trend toward higher incidence rates among the Hp 2/2 compared with the Hp 1/1 genotype was generally observed in those diagnosed after 1965 (less subject to survival bias); however, none of the stratified results were statistically significant (Table 4). Similarly, when conducting cumulative incidence analyses (including prevalent cases in outcomes), results demonstrated nonsignificantly higher rates in those carrying the Hp 2/2 compared with the Hp 1/1 genotype with the exception of macroalbuminuria (Table 4).

In this cohort of subjects with type 1 diabetes, we failed to show an association between the Hp genotype and either microalbuminuria or macroalbuminuria incidence. However, although not univariately significant, approximately a twofold increased risk emerged for outcomes assessing renal function decline and ESRD incidence after multivariable adjustments.

Previous studies assessing the association between the Hp phenotype and the presence or incidence of renal disease have produced discrepant findings. In a small, cross-sectional study of normotensive subjects with type 1 or 2 diabetes, none of those with the Hp 1/1 phenotype exhibited signs of nephropathy (0/18) compared with 27% (10/37) of those with Hp 2/1 and 34% (19/55) of those with Hp 2/2 (P < 0.02) (6,28). Similar results were reported from an Irish type 1 diabetes case-control study (29). Conversely, a Japanese study of individuals with a long duration (>10 years) of type 2 diabetes did not observe an increased risk associated with the common Hp phenotype (P = 0.43) (30). Similarly, we were also not able to detect an association for either microalbuminuria or macroalbuminuria incidence in our cohort of individuals with a long duration of type 1 diabetes, perhaps suggesting that at a more advanced stage of diabetes, early Hp-susceptible cases of microalbuminuria and macroalbuminuria may have been excluded. Indeed, the cumulative incidence of microalbuminuria (including both prevalent cases at study entry and incident cases) appeared higher in our study among participants carrying the Hp 2 allele, although results did not reach statistical significance. However, analogous findings were not observed for the cumulative incidence of macroalbuminuria, suggesting that the Hp 2/2 genotype is not a strong determining factor for progression to macroalbuminuria.

Despite the null associations for the incidence of proteinuria, a strong relationship was noted between the Hp 2/2 genotype and the incidence of both an early decline in renal function and ESRD. Unfortunately, we are not aware of any published reports on the association between the Hp genotype and renal function decline among individuals with diabetes and thus cannot, at present, confirm these findings. However, the possibility of a factor affecting the incidence of renal dysfunction but not that of renal disease per se raises the hypothesis that these are two different disease entities, and thus factors contributing to their development may be distinct. Indeed, almost a decade ago, we suggested that in certain cases tubulopathy may precede glomerulopathy in type 1 diabetes and that even microalbuminuria may be secondary to impaired tubular reabsorption (31). More recently, research studies have shown that reductions in eGFR do occur without preceding microalbuminuria in those with diabetes (32,–34). Importantly, a pathophysiological mechanism has been proposed that could account for the increased rate of renal function decline among individuals with diabetes and the Hp 2/2 genotype (7), based on the recognition that renal proximal tubule cells serve as a (secondary to CD163) default mechanism for clearance of the Hp-Hb complex. Because CD163-mediated clearance of the Hp-Hb complex is impaired in subjects with diabetes and the Hp 2/2 genotype, renal proximal tubule cells are used to a greater extent, resulting in a dramatic increase in iron deposition, oxidative stress, and hypertrophy. In fact, Hp 2/2 diabetic mice have been shown to display significantly increased glomerular and proximal tubular hypertrophy and greater deposition of collagen type IV, smooth muscle actin, and increased renal iron (35). Intriguingly, vitamin E administration was shown to slow diabetic renal disease progression among the Hp 2/2 but not the Hp 1/1 mice.

In conclusion, we observed an association between the Hp genotype and renal function decline in individuals with long-standing type 1 diabetes. A caveat of this study is the lack of independent replication of findings in another cohort. Nevertheless, these results raise the possibility that pharmacological administration of vitamin E, shown to reduce cardiovascular disease outcomes in those with type 2 diabetes with the Hp 2/2 genotype (13–14), may also lead to reduced renal disease risk.

This research was supported by the National Institutes of Health Grant DK34818.

No potential conflicts of interest relevant to this article were reported.

We thank all study participants for their invaluable contributions as well as the Epidemiology of Diabetes Complications Study staff.