Association of an Osteopontin gene promoter polymorphism with susceptibility to diabetic nephropathy in Asian Indians

Clinica Chimica Acta 413 (2012) 1600–1604

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Association of an Osteopontin gene promoter polymorphism with susceptibility to diabetic nephropathy in Asian Indians Balneek Singh Cheema a, Sreenivasa Iyengar b, Tarunveer Singh Ahluwalia c, Harbir Singh Kohli b, Rajni Sharma a, Viral N. Shah d, Anil Bhansali d, V. Sakhuja b, Madhu Khullar a,⁎ a

Department of Experimental Medicine and Biotechnology, Post Graduate Institute of Medicine Education & Research, Chandigarh, 160012, India Department of Nephrology, Post Graduate Institute of Medicine Education & Research, Chandigarh, 160012, India Department of Clinical Sciences—Diabetes and Endocrinology, Lund University Diabetes Centre, Clinical Research Centre, (CRC), University Hospital Skane (UMAS), 20502, Malmo, Sweden d Department of Endocrinology, Post Graduate Institute of Medicine Education & Research, Chandigarh, 160012, India b c

a r t i c l e

i n f o

Article history: Received 12 April 2012 Received in revised form 23 April 2012 Accepted 27 April 2012 Available online 3 May 2012 Keywords: Osteopontin Genetic polymorphism Type 2 diabetes Diabetic nephropathy

a b s t r a c t Genetic predisposition has been proposed to be a major determinant in the development of renal complications of diabetes. Osteopontin (OPN) has been suggested to be associated with renal diseases characterized by tubulointerstitial fibrosis and proteinuria. However, information on association of genetic polymorphisms in OPN with diabetic nephropathy is lacking. Thus, the present study was designed with the aim to examine the association of an OPN gene promoter polymorphism with diabetic nephropathy in Asian Indians. OPN C-443T (rs11730582) polymorphism was determined in 1115 type 2 diabetic patients belonging to two independently ascertained cohorts using Real time PCR based Taqman assay. We observed a nearly threefold elevated risk of diabetic nephropathy among carriers of T allele and TT genotype of OPN C‐443T polymorphism. Further, this allele was found to be significantly associated with proteinuria and lower eGFR, a hallmark of diabetic nephropathy, in both our cohorts. This is the first study which suggests that OPN C-443T polymorphism may be a significant risk factor for diabetic nephropathy in type 2 diabetic patients. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Diabetic nephropathy (DN) is the leading cause of end stage renal disease (ESRD) and renal replacement therapy in developing as well as in developed countries [1,2]. Various factors including metabolic and hemodynamic alterations and growth factors have been implicated in the development of diabetic nephropathy with hyperglycemia being the major risk factor. However, epidemiologic and family studies have demonstrated that only 30–35% diabetic subjects develop progressive diabetic nephropathy irrespective of the glycemic control [3] indicating the involvement of genetic factors in its etiology. This is further evidenced by studies showing familial aggregation of diabetic nephropathy [4,5]. Thus, it has been suggested that there may be genetic susceptibility to diabetic nephropathy which may be independent of genetic factors causing type 2 diabetes [6]. Recently, Osteopontin (OPN), a large phosphoglycoprotein adhesion molecule, has emerged as a potentially key pathophysiologic contributor in diabetic nephropathy. Nicholas et al. reported that the profibrotic adhesion molecule, Osteopontin (OPN), was upregulated in kidneys of humans and mice with diabetes and was involved in glomerular damage in a mouse model of diabetic nephropathy [7]. Osteopontin has also been ⁎ Corresponding author. Tel.: + 91 09316131057, + 91 0172 2755229, + 91 0172 2755232; fax: + 91 0172 2744401. E-mail address: [email protected] (M. Khullar). 0009-8981/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2012.04.028

found to be associated with renal diseases characterized by macrophage infiltration, tubulointerstitial fibrosis and proteinuria, as well as diabetic nephropathy [8–10]. There is increasing evidence which suggests that OPN may mediate the fibrotic changes associated with glomerulosclerosis and interstitial fibrosis which are hallmarks of diabetic nephropathy [7]. Thus, OPN appears to be a potential candidate gene in the pathophysiology of DN. Few studies have shown an association of genetic polymorphisms in OPN with renal disease in systemic lupus erythematosus [11], sarcoidosis [12] and vesicoureteric reflux [13]. However, information on association of genetic polymorphisms in OPN with diabetic nephropathy is lacking. Thus, the present study was designed with the aim to examine the association of an OPN gene promoter polymorphism; C-443T with susceptibility to diabetic nephropathy in Asian Indians, a population exhibiting the highest incidence of type 2 diabetes. 2. Methods 2.1. Study population Two independently ascertained type 2 diabetic cohorts of North Indian origin, visiting the Endocrinology and Nephrology clinics of the Postgraduate Institute of Medical Education and Research, Chandigarh between June 2006 to September 2007 (cohort1) and January 2010 to October 2011 (cohort 2) were recruited in this study. Their ethnicity

B.S. Cheema et al. / Clinica Chimica Acta 413 (2012) 1600–1604

was confirmed on the basis of language spoken and ancestral history. The study was approved by Institute ethics committee and written consent was obtained from participating subjects. Inclusion criteria for diabetic nephropathy group were as follows: North Indian origin, age at onset of diabetes >35 years, subjects having type II diabetes for more than or equal to 10 years and diagnosed with diabetic nephropathy. Diabetic patients were divided into two groups according to the following diagnostic criteria: (1) cases of diabetic nephropathy, that is, patients with diabetes (duration of onset of 10 years or more) and nephropathy, indicated by urinary albumin excretion rates (AERs) > 200 μg/min or urinary albumin-to-creatinine ratios (Alb/Cr) >300 μg/Cr; (2) control patients with diabetes (DM) (duration of onset of 10 years or more), but showing normal urinary albumin excretion, that is, AER b20 μg/min or Alb/Cr b30 mg/g Cr. Patients with type 1 diabetes, any known non-diabetic renal disease and nephropathy other than diabetic nephropathy were excluded from the study. It was ensured that the diabetic nephropathy subjects had no microscopic hematuria. In addition to this, the diabetic subjects (without proteinuria) on antihypertensive drug treatment were excluded from the study group to avoid misclassification of phenotype. All the subjects in both groups were age and ethnicity matched. Based upon this definition, a total of 1495 patients with Type 2 Diabetic Mellitus with duration of disease ≥ 10 years were screened for the presence of diabetic nephropathy, of which, 1115 subjects were finally included for the present study. Cohort 1 consisted of 240 patients with diabetic nephropathy and 255 type 2 diabetic patients without nephropathy; cohort 2 had 405 patients with diabetic nephropathy and 215 patients had type 2 diabetes without nephropathy. 2.2. Genotyping Genomic DNA was isolated from peripheral blood lymphocytes using proteinase K digestion and phenol chloroform method. The OPN promoter polymorphism C-443T, (rs11730582) was determined using Real time PCR based Taqman assay (Applied Biosystems, Foster City, CA) following manufacturer's instructions. Positive and negative controls were used in each genotyping run, and 5% of randomly selected samples were re-genotyped by other lab personnel with 100% concordance. The genotypes were also confirmed by randomly sequencing some of the samples. 2.3. Statistical analysis The statistical tests were performed, using SPSS Inc., Chicago, IL version 11.0. We tested the genotype and allele frequencies for deviation from Hardy–Weinberg equilibrium (HWE) proportions by using a Hardy–Weinberg equilibrium calculator. Discrete and continuous variables were compared between cases and controls using Pearson's χ2 test and unpaired t-test or Mann–Whitney U test as appropriate. Comparison of variables between different genotypes was performed using ANOVA for normally distributed data and Kruskal–Wallis test for skewed data. Power of the sample size (subjects) was calculated using the PAWE software (http://linkage.rockefeller.edu/pawe) Multivariate logistic regression was used to compute odds ratio for developing nephropathy by adjusting for potential confounders which include age, gender, HbA1c, duration of diabetes, duration of hypertension, smoking, systolic blood pressure and triglyceride levels. p b 0.05 was considered as statistical significant. 3. Results 3.1. Baseline characteristics of study participants Baseline characteristics of the study participants of cohorts 1 and 2 showed that in both the cohorts, patients with diabetic nephropathy had significantly higher systolic blood pressure, BMI, HbA1c, serum

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creatinine and lower eGFR and duration of hypertension as compared to diabetic patients without nephropathy. All our DNP patients were proteinurics (AER >200 μg/min), indicating a strong association of risk genotypes and alleles with proteinuria. About 77.6% of type 2 diabetics with nephropathy had retinopathy as compared to 34.1% of diabetics without nephropathy. Using multivariate logistic regression, we observed high systolic blood pressure, BMI and serum creatinine to be significantly associated with increased risk of diabetic nephropathy in both the cohorts. However, male sex, high triglyceride level and smoking were found to be significantly associated with increased risk of diabetic nephropathy in patients of cohort 2 only [Table 1]. The number of patients taking sufonylurea, metformin, pioglitazone and angiotensin converting enzyme inhibitors was higher in type 2 diabetes without nephropathy group as compared with type 2 diabetes with nephropathy group in both the cohorts. Numbers of patients on statins were equal in both groups. Use of insulin was common among patients with diabetic nephropathy in both the cohorts as expected. 3.2. Genotype and allele frequency of OPN C-443T polymorphism (rs11730582) Genotype and allele frequencies of the OPN C-443T polymorphism in two independent cohorts (1, 2) are given in Table 2. The genotyping frequencies were not in Hardy–Weinberg equilibrium for both the cohorts. The frequency of ‘T’ allele and TT genotype was significantly higher in diabetic nephropathy patients as compared to type 2 diabetic patients without nephropathy and was associated with proteinuria. We observed approx. a threefold elevated risk of nephropathy among carriers of C443T polymorphism in both the cohorts. The ‘T’ allele and TT genotype showed significant association with increased risk of diabetic nephropathy in both the cohorts even after adjustment for potential confounders such as age, gender, HbA1c, duration of diabetes, duration of hypertension, smoking, systolic blood pressure and triglyceride levels. 3.3. Clinical characteristics of the study subjects according to OPN (rs11730582) genotypes Baseline characteristics did not differ significantly between patients with CC, CT and TT genotypes and in both the cohorts; however, patients with TT genotype had significantly higher overt proteinuria and lower eGFR in both the cohorts [Table 3]. Further, in dominant model (CC vs CT+TT) genotypes, patients with CT+TT genotypes also showed enhanced proteinuria and lower eGFR suggesting an association of T allele with nephropathy (Table 3). Patients with TT genotype also had lower BMI, waist circumference and higher LDL-C however, results were not statistically significant. Also, we observed an association between genotype and proteinuria in the group of patients without diabetic nephropathy. (CC:CT:TT:: 65.1 ± 50.2:72.3± 42.6:88.3 ± 44.2, p b 0.05): cohort 1 (CC:CT:TT:: 68.1± 46.2:74.1 ± 42.0:96.1 ± 43.0, p b 0.05) cohort 2. 4. Discussion Osteopontin (OPN) is emerging as a potentially key pathophysiologic contributor in diabetic nephropathy [7]. In the present study, we examined the association of this OPN gene promoter polymorphism (C-443T) with susceptibility to diabetic nephropathy in Asian Indians. We observed a consistently higher prevalence of T allele and TT genotype of OPN C‐443T polymorphism in two independently ascertained cohorts of patients with diabetic nephropathy, which was associated with a nearly threefold increased risk of diabetic nephropathy. The significant deviation from Hardy–Weinberg equilibrium of genotype distribution in the present population in OPN gene polymorphism may be due to the moderate population size or the allele is rare which can cause a random change in allele frequencies. High frequency of mutation occurring at the specific loci can also cause deviation from equilibrium of genotype distribution in the present population. This is the first study to report an

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B.S. Cheema et al. / Clinica Chimica Acta 413 (2012) 1600–1604

Table 1 Baseline characteristics of study subjects. Cohort 1

Cohort 2

Base line characteristics (Mean ± SD)

DM (255)

DN (240)

p value

Adjusted p value

DM (215)

DN (405)

p value

Adjusted p value

Age (years) Males/females (%)

58.10 ± 8.1 105/150 (41/59) 15.6 ± 5.24

60.1 ± 6.1 94/146 (39/61) 16.3 ± 3.3

0.312 1.0

– –

0.8 0.002

–

54.6 ± 8.2 236/169 (58/42) 15.9 ± 7.1

b 0.05 b 0.05

>0.05

61.9 ± 8.6 99/ 116 (46/54) 15.1 ± 6.3

0.1

–

7.78 ± 6.9

5.5 ± 6.1

b0.05

0.1

7.81 ± 5.7

5.2 ± 5.1

b 0.05

0.4

135.0 ± 17.7 84.2 ± 7.5 23.9 ± 2.8 98.3 ± 19.8 1.15 ± 0.11 7.6 ± 1.1 91.6 ± 11.8 260.3 ± 63.7 207.6 ± 38.3 61.1 ± 16.1 12.1 ± 1.2 98.7 ± 34.9 –

145.4 ± 22.4 88.9 ± 7.6 27.8 ± 2.9 59.8 ± 34.7 4.22 ± 2.08 8.5 ± 2.5 89.0 ± 13.6 255.3 ± 64.3 204.9 ± 39.2 63.5 ± 15.1 10.0 ± 1.6– 100.4 ± 41.8 –

b0.05 b0.05 b0.05 b0.05 b0.05 b0.05 0.2 0.89 0.86 0.74 b0.05 0.2 –

0.02 0.2 0.02 0.1 0.0001 0.2 – – – – 0.1 – –

127.18 ± 15.6 79.9 ± 6.4 21.7 ± 4.4 98.2 ± 19.6 0.8 ± 0.3 7.8 ± 1.4 92.1 ± 12.4 171.4 ± 40.7 144.9 ± 71.2 60.8 ± 14.8 11.6 ± 1.8 99.0 ± 34.7 47.1 ± 10.1

137.34 ± 20.2 80.6 ± 7.7 24.4 ± 4.2 57.9 ± 36.5 4.2 ± 1.4 8.4 ± 2.3 88.1 ± 13.2 184.1 ± 60.7 170.7 ± 96.1 52.6 ± 13.8 10.1 ± 1.7 102.6 ± 43.9 48.9 ± 20.6

b 0.05 0.2 b 0.05 b 0.05 b 0.05 b 0.05 0.1 0.14 b 0.05 0.6 b 0.05 0.22 0.38

0.01 – 0.01 0.09 0.0001 0.1 – – 0.002

– – –

– – –

– – –

6 (2.8%) 13 (6.0%) 196 (91.2%)

19 (4.8%) 56 (13.9%) 330 (81.3%)

b 0.05

0.04

Time since diagnosis of TIID (years) Duration of HT (years) S.B.P. (mm Hg) D.B.P. (mm Hg) BMI (kg/m2) eGFR (ml/min) S.creatinine (mg%) HbA1c (%) WC (cm) TC TG (mg%) HDL (mg%) Hb (gm/dl) LDL-C (mg/dl) HDL-C (mg/dl) Smoking, n (%) Current Ex-smoker Non-smoker

0.2 – –

[M; male, F; female, BMI; body mass index, WC; waist circumference, HTN; hypertension, Hb = hemoglobin, SBP = systolic blood pressure, DBP = diastolic blood pressure, S.creat = serum creatinine, eGFR = estimated glomerular filtration rate by MDRD formula, TC; total cholesterol, TG; triglyceride, LDL-C; low density lipoprotein cholesterol, HDL-C; high density lipoprotein cholesterol, TIID; Type II Diabetes.]. (Pb 0.05 is significant).

observed in our study may be due to the increased OPN levels in these subjects. However, we could not measure OPN expression in these subjects as we did not have access to renal tissue from these subjects as renal biopsy from these subjects was not ethically approved. However, we observed that T allele carriers also showed overt proteinuria and estimated decreased eGFR which are both markers of renal dysfunction, further suggesting an association of T allele with nephropathy. Our results are in agreement with earlier studies in which increased OPN levels were found to be associated with proteinuria as well as diabetic nephropathy [7]. Also, our study shows a modest association between SNP in the OPN promoter region (rs11730582) and enhanced proteinuria and estimated decreased eGFR, supporting the view that promoter SNP in OPN might influence albuminuria and that this may translate into a progressive deterioration of kidney function. Notably, we excluded patients with ESRD from our study. Also, we observed an association between genotype and proteinuria in the group of patients without diabetic nephropathy and this association was independent of eGFR. Although, these patients only have mild proteinuria, but an association with OPN genotype provides evidence that it is proteinuria that is primarily affected by OPN.

association of OPN promoter gene polymorphism with diabetic nephropathy in type 2 diabetic patients. This association between the OPN C-443T promoter polymorphism and the susceptibility to diabetic nephropathy persisted even after adjustments for potential confounders, such as gender, age, and duration of diabetes. We also found significantly enhanced proteinuria and lower eGFR among patients carrying T allele in our cohorts. Thus, our results suggest OPN C-443T polymorphism to be a significant risk factor for diabetic nephropathy in type 2 diabetic patients in Asian Indians. Functional studies suggest that this polymorphism affects OPN transcription and expression. Francesca et al. [14] reported that this polymorphism affected the binding of an unidentified nuclear factor to OPN promoter region and thus alters its transcriptional activity. Melanoma cell lines homozygous for this −443C allele were also reported to have higher binding affinity for c-Myb transcription factor which resulted in increased OPN expression in these cell lines [15]. The increased expression of OPN has been suggested to stimulate TGF-β and matrix deposition in mesangial cells, which could significantly contribute to the pathophysiology of diabetic nephropathy [7]. Thus, the increased risk of diabetic nephropathy among −443T allele Table 2 Allele and genotype frequency of OPN gene polymorphism in subjects with DM and DN. Cohort 1

Cohort 2

DM (n= 255)

DN (n = 240)

OR (95% CI) P

Adjusted OR (95% CI)a

DM (n= 215)

DN (n= 405)

OR (95% CI) P

Adjusted OR (95% CI)a

Allele

C = 454 (0.89)

C = 360 (0.75)

–

– –

C = 378 (0.88)

C = 599 (0.74)

–

–

Frequency

T = 56 (0.11)

T = 120 (0.25)

2.68 (1.87–3.84) p b 0.0001

2.49 (1.78–3.50), p b 0.0001

–

T = 52 (0.12) CC = 172 (0.80)

T = 211 (0.26) CC = 247 (0.61)

–

–

2.06 (1.42–3.57), p = 0.0007

1.6 (1.1–3.1), p = 0.2

CT = 32 (0.15) TT = 11 (0.05)

CT = 105 (0.26) TT = 53 (0.13)

3.57 (1.77–7.22), p = 0.0004

3.1 (1.4–7.1), p = 0.02

Genotype

CC = 206 (0.81)

CC = 151 (0.63)

–

–

Frequency

CT = 41 (0.16) TT = 8 (0.03)

CT = 58 (0.24) TT = 31 (0.13)

1.96 (1.22–3.13) p = 0.007 3.78 (2.05–6.08) p = 0.0001

1.4 (1.1–2.9) p = 0.2 2.9 (1.3–6.7) p = 0.02

a

Adjusted with potential confounders e.g. age, sex, smoking, duration of diabetes, systolic blood pressure, duration of hypertension, and triglyceride levels.

B.S. Cheema et al. / Clinica Chimica Acta 413 (2012) 1600–1604

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Table 3 The baseline characteristics of study subjects according to OPN genotype. Cohort 1

Cohort 2

Variables

CC (151)

CT (58)

TT (31)

P

CT + TT(89)

p

CC (247)

CT (105)

TT (53)

p

CT + TT (158) p

Age (years) Sex (M/F) Duration of DM (years) Duration of HTN (years) SBP (mm Hg) DBP (mm Hg) Proteinuria (mg/24 h) S.creatinine (mg/dl) eGFR (ml/min) BMI (kg/m2) WC (cm) HbA1c (%) TC (mg/dl) TG (mg/dl) LDL-C (mg/dl) HDL-C (mg/dl)

60.2 ± 10.3 68/83 16.0 ± 6.0

59.8 ± 9.8 27/31 16.4 ± 6.1

60.1 ± 11.0 12/19 16.3 ± 6.2

0.7 0.8 0.8

60.9 ± 10.6 39/50 16.3 ± 6.1

0.7 0.7 0.7

54.1 ± 10.2 111/136 12.1 ± 5.9

54.1 ± 9.1 48/57 12.7 ± 6.2

55.1 ± 11.1 20/33 12.1 ± 5.7

0.8 0.8 0.7

54.6 ± 10.1 68/90 12.4 ± 6.0

0.9 0.7 0.7

6.1 ± 5.8

6.0 ± 5.7

5.4 ± 4.9

0.2

5.7 ± 5.3

0.2

6.6 ± 6.0

6.6 ± 6.5

4.7 ± 5.1

0.09

5.7 ± 5.8

0.09

145.6 ± 15.1 89.3 ± 7.2 752.1 ± 107.1 4.0 ± 0.4

145.9 ± 16.0 88.6 ± 7.2 1061.3 ± 143.1 4.2 ± 0.2

146.1 ± 33.8 88.0 ± 8.2 1365.4 ± 112.1 4.1 ± 0.4

0.6 145.9 ± 24.9 0.7 88.8 ± 7.7 b 0.05 1213.4 ± 127.1 0.2 4.2 ± 0.3

0.5 137.4 ± 15.8 0.6 80.6 ± 6.9 b0.05 774.1 ± 107.9 0.2 4.1 ± 0.4

138.9 ± 16.8 81.3 ± 6.8 1011.3 ± 143.4 4.1 ± 0.2

139.7 ± 34.5 81.2 ± 8.6 1287.4 ± 112.4 4.2 ± 0.4

0.9 139.3 ± 25.7 0.6 81.3 ± 7.7 b 0.05 1149.4 ± 127.4 0.1 4.1 ± 0.3

0.8 0.7 b 0.05

66.1 ± 33.8 28.9 ± 4.2

52.9 ± 34.1 26.6 ± 4.1

46.8 ± 32.0 27.9 ± 3.0

b 0.05 49.4 ± 33.1 0.1 26.3 ± 3.6

b0.05 65.3 ± 34.6 0.2 24.9 ± 4.1

53.6 ± 33.6 24.9 ± 4.0

42.3 ± 31.2 22.6 ± 3.2

b 0.05 47.5 ± 32.4 0.09 23.8 ± 3.8

b 0.05 0.4

92.8 ± 12.0 8.9 ± 2.2 251.6 ± 50.9 208.1 ± 72.2 97.8 ± 36.1 68.1 ± 16.0

90.2 ± 13.2 8.1 ± 1.4 250.9 ± 51.0 209.3 ± 72.3 98.6 ± 38.1 66.1 ± 10.1

85.1 ± 12.1 8.2 ± 2.4 254.3 ± 50.3 200.3 ± 71.0 104.0 ± 43.9 64.5 ± 11.4

0.07 0.9 0.9 0.8 0.6 0.7

0.2 0.8 0.8 0.8 0.9 0.8

88.9 ± 13.1 8.0 ± 1.4 171.6 ± 49.4 177.1 ± 74.1 98.2 ± 37.9 45.9 ± 10.9

84.3 ± 11.9 8.2 ± 2.3 173.1 ± 48.4 172.2 ± 67.5 104.5 ± 44.3 43.5 ± 11.4

0.06 0.8 0.8 0.7 0.7 0.7

0.2 0.9 0.9 0.9 0.9 0.9

87.7 ± 12.7 8.2 ± 1.9 252.6 ± 50.7 209.8 ± 72.3 101.3 ± 41.0 65.3 ± 10.8

93.2 ± 12.4 8.5 ± 2.2 172.7 ± 51.2 184.9 ± 71.7 97.2 ± 35.6 48.0 ± 16.2

86.6 ± 12.5 8.1 ± 1.9 172.4 ± 48.9 174.7 ± 70.8 101.4 ± 41.1 45.9 ± 11.2

0.09

[M = male, F = female, BMI = body mass index, WC = waist circumference, DM = diabetes mellitus, HTN = hypertension, SBP = systolic blood pressure, DBP = diastolic blood pressure, eGFR = estimated glomerular filtration rate, TC = total cholesterol, TG = triglyceride, LDL-C = low density lipoprotein cholesterol, HDL-C = high density lipoprotein cholesterol- all the values are in Mean ± SD]. (P b0.05 is significant).

Intriguingly, TT genotype carriers had lower BMI and waist circumference as compared to those with CC genotype. The plausible explanation of this may be that patients with overt proteinuria and decreased eGFR might be on protein restricted diet and diuretics and this may result into lower BMI. This is further substantiated by the chronic kidney disease registry of India, which shows that with progressive decline in eGFR, there is a progressive decrease in BMI [16]. Moreover, Asian Indians have higher body fat compared to Caucasians of similar BMI [17]. However, body fat estimation would have provided a clearer insight into this paradox observation, which is a limitation of the study. Osteopontin (OPN) has been shown to be expressed in various cells including proximal renal tubular cells and has been suggested to play a role in macrophage infiltration and tubulointerstitial fibrosis [8–10]. A few recent studies have shown that OPN C-443T polymorphism is associated with diseases such as lupus erythematosus [11], sarcoidosis [12], vesicoureteric reflux [13], oral squamous cell carcinoma [18] and chronic hepatitis C [19]. OPN-443TT genotype was shown to influence the development and severity of these diseases. There are several strengths and limitations in our study; we had ethnically homogeneous diabetic subjects who were enrolled from a single center, thus avoiding phenotyping errors and bias. The relatively medium size of our case‐control study is a limitation that could introduce type 1 errors; however, very few investigators (mostly the ones involving a multicenter study) have access to a large sample size. Sample size was predetermined for these variants to have a minimum power of 75% [20,21]. Our study was still underpowered for low frequency SNP. Moreover, positive associations observed between OPN promoter C-443T polymorphism and diabetic nephropathy do not seem to be due to chance, as this association was replicated in two independent cohorts in our study and it persisted even after the influence of confounding factors was corrected. The inclusion criterion of absence of microalbuminuria on two or more occasions for the control group ensured a good selection of control diabetic group for the study. The strength of our study is the stringent criteria employed to define nephropathy, replication in two independent well ascertained cohorts from a single centre and homogeneous ethnic population while the limitation is the cross-sectional design of the study. The present study fulfills most of the prerequisites for a good genetic association study as suggested by Bird et al. [22].

Currently, several clinical trials are investigating the role of OPN in diabetic nephropathy; the positive association of OPN C-443T polymorphism with increased risk of diabetic nephropathy in our study further suggests that OPN may be a critical contributor to diabetic nephropathy. Even though it may be a long way before the clinically relevant therapies targeting osteopontin or its promoters to prevent progression of nephropathy come into play but it certainly shows some hope in making the outcome better for patients who progress to ESKD secondary to diabetes related complications. 5. Conclusion In conclusion, the T allele of OPN gene polymorphism; C-443T is an independent risk factor for proteinuria and is associated with higher risk of nephropathy, thus suggesting that OPN might be a good candidate as a biomarker of diabetic nephropathy in Asian north Indian patients with type II diabetes. Disclosure statement No competing financial interests exist. Conflict of interest None. Acknowledgments The study was carried out at Genomics Laboratory, PGIMER, Chandigarh, India. We are grateful to the volunteers of this study, the laboratory staff, and all who gave their time to take part in this study. B.S. Cheema received individual research fellowships from the Department of Biotechnology (DBT), New Delhi, India. References [1] Ritz E, Rychlik I, Locatelli F, Halimi S. End-stage renal failure in type 2 diabetes: a medical catastrophe of worldwide dimensions. Am J Kidney Dis 1999;34:795–808.

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