The Human Peroxisome Proliferator-Activated Receptor γ2 (PPARγ2) Pro12Ala Polymorphism Is Associated With Decreased Risk of Diabetic Nephropathy in Patients With Type 2 Diabetes

A case-control study was conducted in 316 patients with type 2 diabetes (according to World Health Organization criteria) (8) who were selected from a sample of >1,200 patients participating in a multicenter research protocol in the Rio Grande do Sul state (Brazil). The aim was to identify the genetic and nongenetic risk factors associated with diabetes complications. All type 2 diabetic patients with proteinuria (AER >200 μg/min) and serum creatinine >2.0 mg/dl and all patients with normoalbuminuria (AER <20 μg/min) and known diabetes duration of at least 10 years were studied. The 104 proteinuric patients with serum creatinine >2.0 mg/dl (78 on dialysis) were defined as case subjects, and the 212 normoalbuminuric patients with a known diabetes duration of at least 10 years were defined as control subjects.

This study was approved by the ethics committee of the Hospital de Clínicas de Porto Alegre and the corresponding committees at the institutions from which DNA samples were obtained. Written informed consent was obtained from all participants.

Patients underwent a standardized clinical and laboratory evaluation that, to summarize, consisted of a questionnaire, physical examination, and blood collection. Sitting blood pressure was measured twice to the nearest 2 mmHg after a 5-min rest using a standard mercury sphygmomanometer (phases I and V of Korotkoff). The mean value of three measurements was used to calculate systolic and diastolic blood pressure. Hypertension was defined as blood pressure levels ≥140/90 mmHg (9) or the use of antihypertensive medication. Retinopathy was assessed by ophthalmoscopic examination through dilated pupils in 293 patients and classified as absent, nonproliferative, or proliferative. AER was measured in 24-h timed sterile urine samples by immunoturbidimetry (Sera-Pak immuno microalbuminuria; Bayer, Tarrytown, NY). AER values ≥200 μg/min were confirmed in a second urine sample. Glucose was measured by the glucose oxidase method, HbA_1c by an ion exchange high-performance liquid chromatography (Merck-Hitachi L-9100 GhB analyzer, reference range 2.7–4.3; Merck, Darmstadt, Germany) and serum creatinine by Jaffé’s reaction.

Genomic DNA was extracted from peripheral blood. The PPARγ2 Pro12Ala polymorphism was detected as described elsewhere (10). The PCR products were digested overnight at 37°C with BstU-1 enzyme, electrophoresed on a 2.5% agarose gel, and stained with ethidium bromide. The expected products after digestion were 270 bp for normal homozygous (Pro/Pro), 227 bp and 43 bp for Pro12Ala homozygous (Ala/Ala), and 270 bp, 227 bp, and 43 bp for heterozygous (Pro/Ala). The frequency of the Ala allele was 10.0% in white and 7.5% in nonwhite volunteer blood donors (P = 0.357).

The clinical and laboratory characteristics of the case and control subjects were compared by unpaired Student’s t test or χ² test, as appropriate. Genotype distribution in the case and control subjects was compared by χ² test. Multiple logistic regression analysis was used to determine the independent factors associated with diabetic nephropathy. Data are expressed as mean ± SD or as number of patients with a given characteristic. Triglycerides, not normally distributed, were logarithmically transformed before analysis and are presented as median and range. The test for Hardy-Weinberg equilibrium was performed as previously described (11). P values <0.05 were considered significant. SPSS version 10.0 (SPSS, Chicago, IL) was used for the analyses.

Patients with diabetic nephropathy (case subjects) were more often men (P < 0.0001) and, as expected, more often hypertensive (P < 0.0001) than long-standing normoalbuminuric patients (control subjects) (Table 1). There was no difference in the proportion of patients using antihypertensive drugs between case (72.9%) and control (64.3%; P = 0.241) subjects. However, case subjects used ACE inhibitors or angiotensin receptor blockers more often (47.2%) than control subjects (29.2%; P = 0.013). Diabetic retinopathy was also more frequent in case (88% with any retinopathy, 72% with proliferative retinopathy) than in control (42% with any retinopathy, 7% with proliferative retinopathy; P < 0.001) subjects. Diabetes treatment in case and control subjects consisted of diet (16.9 vs. 3.2%), oral agents (16.9 vs. 55.2%), or insulin (66.2 vs. 41.6%; P < 0.001), respectively. None of the patients included in this study were using thiazolidonedione compounds.

The genotypic distribution of the PPARγ2 Pro12Ala gene polymorphism was in Hardy-Weinberg equilibrium. The frequency of the Ala allele in these type 2 diabetic patients was 9%. The frequency of Ala carriers (Ala/Ala and Pro/Ala) was higher among normoalbuminuric (20.3%) than diabetic nephropathy (10.6%; P = 0.031) patients. The Ala allele was also more frequent in control subjects (10.8%) than in diabetic nephropathy case subjects (5.8%; P = 0.038). There was no difference in the Ala allele frequency between patients with chronic renal insufficiency (11.5%) and patients on dialysis (10.3%). The odds ratio (OR) of having diabetic nephropathy for Ala allele carriers was 0.465 compared with noncarriers (95% CI 0.229–0.945; P = 0.034). Multiple logistic regression analysis showed that Ala carriers (OR 0.425 [95% CI 0.196–0.974]; P = 0.043) and male sex (3.94 [2.30–6.76]; P < 0.001) were independently associated with diabetic nephropathy. These results did not change when normoalbuminuric patients using ACE inhibitors or angiotensin receptor blockers or nonwhite patients were excluded from the analyses.

There were no differences between Ala carriers and noncarriers regarding age, diabetes duration, sex distribution, BMI, waist-to-hip ratio, fasting plasma glucose, HbA_1c, total cholesterol, triglycerides, serum creatinine, and blood pressure levels (Table 2). Also, there was no difference regarding antihypertensive use (63.3 vs. 68.8%; P = 0.558) or ACE inhibitor or angiotensin receptor blocker use (29.4 vs. 37.7%; P = 0.361) between Ala carriers and noncarriers, respectively. There was no difference in the antihyperglycemic regimen used by Ala carriers (10.3% diet, 51.3% oral agents, and 38.5% insulin) or noncarriers (7.3% diet, 40.6% oral agents, and 52.1% insulin; P = 0.295).

The decreased risk of diabetic nephropathy among PPARγ2 Ala12 allele carriers suggests that this genetic variation in PPARγ2 has a protective role in relation to the risk of diabetic nephropathy in patients with type 2 diabetes. This confirms and extends the observations that Caucasian type 2 diabetic PPARγ2 Pro12Ala carriers have lower AER levels (7) and, presumably, lower diabetic nephropathy risk. Although no difference in the frequency of Ala allele carriers was observed between normoalbuminuric and ESRD patients (7), this latter study was underpowered to detect this association because only a small number of ESRD patients (n = 44) were evaluated. Further, some of the normoalbuminuric patients may have had a short diabetes duration (interquartile diabetes duration ranging from 4 to 17 years) (7) and may, in fact, be at risk of progressing to diabetic nephropathy (12). The association between the PPARγ2 Pro12Ala polymorphism and macroalbuminuria, seen only when patients with diabetes duration ≥20 years were analyzed (7), is in agreement with this thesis. A large study of Japanese type 2 diabetic patients did not find association between diabetic nephropathy and the PPARγ2 Pro12Ala polymorphism (13). However, diabetic nephropathy in this study (13) was defined as an AER >10 μg/ml. This could have led to patients with clinically insignificant diabetic renal involvement being included in the diabetic nephropathy group (13).

The mechanisms by which the PPARγ2 Pro12Ala polymorphism could lead to diabetic nephropathy are unknown. The PPARγ2 Pro12Ala polymorphism has been associated with higher insulin sensitivity (5,14) and decreased risk of type 2 diabetes (6,10,13–17). Since insulin resistance may be a risk factor for diabetic nephropathy in type 2 diabetes (18,19), improved insulin sensitivity could be the link between the PPARγ2 Pro12Ala polymorphism and decreased risk of diabetic nephropathy. It would also be reasonable to hypothesize that the diabetic nephropathy protective effect of the PPARγ2 Pro12Ala polymorphism could be dependent on a decreased risk of hypertension among Ala carriers versus noncarriers. However, there was no difference in the frequency of hypertension between Ala carriers and noncarriers in the current study. Another possibility would be that this polymorphism could modulate the harmful effects of hypertension in the kidney by altering, for example, the expression of plasminogen activator inhibitor 1 and transforming growth factor-β.

However, caution needs to be exercised when interpreting the results of the present study because association studies require special attention in their design (20,21). It is particularly important to have an appropriate sample size, allowing the detection of true differences between groups, and to adopt strict selection criteria in order to include in the study only patients with well-defined phenotypes (20). In this regard, the current study evaluated a large sample of carefully characterized type 2 diabetic patients, selecting as control subjects only normoalbuminuric patients with a long diabetes duration and as case subjects only patients with clearly established diabetic nephropathy. A possible limitation of the current report would be the inclusion of some patients in the normoalbuminuric group who were, in fact, microalbuminuric patients that had their AER reduced by the use of ACE inhibitors or angiotensin receptor blockers. Nonetheless, as argued above, the association between PPARγ2 Pro12Ala polymorphism and diabetes complications risk seems to make biological sense. Moreover, this association has been previously reported by Herrmann et al. (7) in a study that found lower AER levels among Ala carriers in a large sample of Caucasian type 2 diabetic patients, indicating a protective effect of the Ala12 allele in relation to diabetic nephropathy.

In conclusion, the presence of the Ala allele of the PPARγ2 Pro12Ala polymorphism is associated with a decreased risk of diabetic nephropathy in patients with type 2 diabetes. This association needs to be confirmed in other patient populations. More basic studies will be required to uncover the molecular mechanisms underlying the association between PPARγ2 Pro12Ala polymorphism and diabetic nephropathy risk.

This work was partially supported by grants from Projeto de Núcleo de Excelência do Ministério de Ciência e Tecnologia and Hospital de Clínicas de Porto Alegre. L.A.C. was a recipient of a scholarship from Fundação Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior.

We thank Paula Eichler and Emanuele Kuhn for technical support and Professor Michael Mauer for his thoughtful comments.