SHORT REPORT

Haptoglobin polymorphism and diabetic nephropathyin Brazilian diabetic patients

VANIA P.A. WOBETO, PRISCILA M.D. GARCIA,

TANIA R. ZACCARIOTTO & MARIA DE FATIMA SONATI

Clinical Pathology Department, School of Medical Sciences, State University of Campinas-UNICAMP,

Campinas, State of Sao Paulo, Brazil

(Received 20 January 2009; revised 23 March 2009; accepted 9 April 2009)

AbstractDiabetic nephropathy (DN) has an important impact on morbidity/mortality in diabetic patients.Genetic factors are probably involved in the development of this microvascular complication.Haptoglobin (Hp) is a genetically polymorphic glycoprotein that forms stable complexes with plasma-free hemoglobin (Hb) providing protection against heme-induced oxidative stress and kidney damage.The aim of the present study was to investigate the existence of association between the Hp genotypesand the presence of DN in Brazilian diabetic patients. The Hp genotypes of 265 patients, 95 type 1diabetes mellitus (DM1) sufferers with at least 10 years of disease and 170 type 2 diabetes mellitus(DM2) sufferers with at least 5 years of disease were determined by allele-specific PCR; both groupsincluded patients with and without DN. Hp allele and genotype frequencies were compared amongthe patient groups and between the patient groups and a control group of 142 healthy individuals. Noassociation between Hp genotypes and DN could be demonstrated. Additionally, urinary albuminexcretion values and the presence or absence of systemic arterial hypertension (SAH) were comparedamong the patient groups. Again, no significant correlations were found. The Hp polymorphism couldnot be associated with DN in the population studied here.

Keywords: Haptoglobin, diabetes mellitus, diabetic nephropathy, genetic polymorphisms, Brazilianpopulation

Introduction

Diabetes mellitus (DM) is considered the most frequent metabolic disease affecting

the regulation of insulin and glucose and has a prevalence of almost 6% worldwide

(Adeghate et al. 2006). In Brazil the frequency of DM is nearly the same as that in most

Correspondence: Maria de Fatima Sonati, Department of Clinical Pathology, School of Medical Sciences, State University

of Campinas, UNICAMP, Campinas, State of Sao Paulo, Brazil, PO Box 6111, Zip Code 13083-970. E-mail:

sonati@fcm.unicamp.br

ISSN 0301-4460 print/ISSN 1464-5033 online # 2009 Informa UK Ltd.

DOI: 10.1080/03014460902960263

Annals of Human Biology, July�August 2009; 36(4): 437�441

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developed countries and is estimated to be around 8% in the population aged between 30

and 69 years (Malerbi and Franco 1992). Diabetic nephropathy (DN) is an important cause

of morbidity and mortality in diabetic patients. It affects about one-third of these patients

and is the main cause of end-stage renal disease and early death from cardiovascular

complications (Ritz 2006). Hyperglycemia alone is not sufficient for nephropathy to occur,

and there is strong evidence suggesting the involvement of genetic susceptibility factors in

its pathogenesis. The genes that predispose to the development and progression of DN are

subject of intensive ongoing investigation, but the results of these studies to date are

conflicting (Rich 2006).

Haptoglobin (Hp) is a positive acute-phase protein with immunomodulatory and

antioxidant properties. Its best-known biological function is its Hb-binding capacity,

which prevents iron loss and kidney oxidative damage during hemolysis (Langlois and

Delanghe 1996; Sadrzadeh and Bozorgmehr 1996). The human Hp locus is polymorphic

and is located on the long arm of chromosome 16 (16q22); it has two common alleles, HP1

and HP2, which result in three main genotypes/phenotypes (Hp1-1, Hp2-1 and Hp2-2). It

is well established that different Hp proteins have distinct biochemical and biophysical

properties, as well as functional efficiencies (Langlois and Delanghe 1996; Melamed-Frank

et al. 2001).

Some studies have suggested that the Hp2-2 phenotype is associated with susceptibility to

nephropathy in diabetic patients with type 1 (DM1) and type 2 (DM2) diabetes (Nakhoul

et al. 2001; Bessa et al. 2007; Conway et al. 2007). However, there appears to be no

consensus on this observation (Moczulski et al. 2001; Koda et al. 2002; Awadallah et al.

2008). Our aim in the present study was to investigate the existence of association between

Hp polymorphism and DN in Brazilian patients previously investigated for diabetic

retinopathy (DR) (Wobeto et al. 2007).

Subjects and methods

The present study was approved by the local ethics committee. Peripheral blood samples

were collected from diabetic patients attending at UNICAMP University Hospital in

Campinas, state of Sao Paulo, southeastern Brazil. In total, 265 patients were investigated,

95 with DM1 for at least 10 years and 170 with DM2 for at least 5 years (ages varying from

15 to 75 years old; 36% males; 93% of western Eurasian origin; duration of DM 16.1496.5

years). The diagnosis of DM was based on clinical features, laboratory data and the

guidelines of the American Diabetes Association (American Diabetes Association 2004).

Pregnant patients and those aged 76 years or older were excluded from the study.

Glycosylated hemoglobin (HbA1C) was measured using the Bio-Rad Variant II high-

performance liquid chromatography system (Bio-Rad, Hercules, CA, USA) (normal range

4.5�6.2%; patients’ average�8.7692.03%). The level of urinary albumin excretion (UAE)

of the patients during 24 h period was measured by immunoturbidimetry. Normoalbumi-

nuria was defined as a UAE ofB20 mg/min, microalbuminuria as a UAE of]20 to

B200 mg/min and macroalbuminuria as a UAE of]200 mg/min (Ritz 2006). Patients with

micro- or macroalbuminuria without evidence of DR were also excluded from the study

since albuminuria here did not result from diabetic renal disease (Nakhoul et al. 2001).

Diabetic retinopathy had been previously investigated in these patients by indirect

fundoscopy and biomicroscopy and documented by retinography (Wobeto et al. 2007).

Systemic arterial hypertension (SAH) was considered to be present if the systolic blood

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pressure (SBP) was�130 mm Hg and/or diastolic blood pressure (DPB) was�90 mm Hg.

To compare Hp allele and genotype frequencies, a control group of 142 healthy individuals

(aged 18�62 years old; 36% males; 78% of western Eurasian origin), who were previously

studied (Zaccariotto et al. 2006), was included. Genomic DNA samples were obtained

from peripheral blood leukocytes (GFX Genomic Blood Purification kit, GE Healthcare,

Slough, UK). Haptoglobin genotyping was performed by allele-specific polymerase chain

reaction according to Yano et al. (1998).

Statistical analyses were performed with Statistical Analysis System 8.02 for Windows

(SAS Institute Inc., Cary, NC, USA). The chi-square (x2) test and Fisher’s exact test were

used to determine whether there was an association between the categorical variables. The

results of Hardy�Weinberg equilibrium (HWE) were also obtained with the x2-test. Mann�Whitney and ANOVA tests were used to compare continuous variables. p-valuesB0.05

were considered statistically significant. Statistical adjustment was used to remove the

influence of SAH on diabetic patients with different Hp genotypes.

Results

The Hp genotype and allele frequencies found in patients and controls are demonstrated in

Table I. Genotype frequencies are in agreement with the expected values according to

HWE. No significant differences were observed in the genotype frequency distribution

between the patient and the control groups, nor between DM1 and DM2 patients.

The comparisons of the Hp genotype frequencies between patients with and without DN,

among patients with normo-, micro- or macroalbuminuria and between those with and

without SAH are shown in Table II. Again, no statistical differences could be found.

Discussion

The development and progression of DM can lead to end-stage renal disease in diabetic

patients. High blood glucose levels promote the formation of advanced glycation end

products, which are very unstable and reactive (Ritz 2006). Haptoglobin acts as an

antioxidant protecting against the action of free radicals. However, the antioxidative

potential of Hp is type dependent, and this appears to be due to differences in the molecular

shape and size of Hp proteins (Hp1-1, Hp2-1 and Hp2-2) and their ability to migrate across

the endothelial cell barrier. The protein product of the HP1 allele is monovalent and can

only associate with another Hp molecule to form dimers, whereas the HP2 allele product is

bivalent and can associate with two different Hp monomers to form cyclic polymers.

Table I. Haptoglobin genotype and allele frequencies in healthy controls and diabetic patients.

Hp1-1 (%) Hp2-1 (%) Hp2-2 (%) p HP1 HWE p

Controls (n�142) 36 (25.35) 60 (42.26) 46 (32.39) 0.5232 0.4648 0.0700

Patients (n�265) 55 (20.75) 124 (46.78) 86 (32.45) 0.4420 0.4053

DM1 (n�95) 19 (20.00) 42 (44.21) 34 (35.79) 0.6840 0.4210 0.3630

DM2 (n�170) 36 (21.18) 82 (48.23) 52 (30.59) 0.4530 0.7273

Hp polymorphism and diabetic nephropathy 439

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Consequently, the Hp1-1 protein, which is the smallest type of Hp, has the greatest ability

to pass through the vessel wall (Melamed-Frank et al. 2001).

A number of studies have indicated the existence of an association between Hp genotype/

phenotype and susceptibility to, and/or outcome in, important diseases (Sadrzadeh

and Bozorgmehr 1996). It has been suggested that the Hp2-2 protein confers the lowest

protection against oxidative stress and tissue damage, resulting in higher susceptibility to

the development of vascular complications in DM patients with this phenotype. However,

there is a lack of consensus regarding these results (Moczulski et al. 2001; Nakhoul et al.

2001; Koda et al. 2002; Bessa et al. 2007; Conway et al. 2007; Wobeto et al. 2007;

Awadallah et al. 2008).

In relation to DN, Nakhoul et al. (2001) found an increased incidence of this

microvascular complication in Israeli DM1 and DM2 patients with Hp2-2 genotype.

Conway et al. (2007) investigated Irish patients with DM1 and also concluded that the HP2

allele can increase susceptibility to the development of DN. On the other hand, Moczulski et

al. (2001) and Koda et al. (2002) concluded that the Hp1-1 phenotype did not confer a

protective effect against DN in patients with DM1 and DM2, respectively. Recently,

Awadallah et al., studying Jordanian patients, found no association between Hp phenotypes

and markers of DN such as microalbuminuria and increased serum cystatin C levels

(Awadallah et al. 2008).

In the present work, we could not find any significant association between Hp genotypes

and DN even when the patients were divided into DM1 and DM2 patients. The ethnic

composition of the investigated populations and the respective sample sizes may be

responsible for the heterogeneity observed in different studies. We conclude that, in the

population studied here, the Hp genotype cannot be used as a genetic marker for

predisposition to DN.

Acknowledgements

We would like to thank Mrs Cleide A.M. Silva of the Research Committee of the School of

Medical Sciences-UNICAMP for conducting the statistical analysis. Financial support was

provided by FAPESP (fellowship 05/02 907-7) and CNPq/Brazil.

Table II. Hp genotype distribution in diabetic patients with and without DN, with normo-, micro- or macro-

albuminuria, and with and without SAH.

Hp1-1 (%) Hp2-1 (%) Hp2-2 (%) p

DM with DN (n�107) 20 (18.69) 55 (51.40) 32 (29.91) 0.4627

DM without DN (n�158) 35 (22.15) 69 (43.67) 54 (34.18)

DM1 with DN (n�44) 8 (18.18) 20 (45.45) 16 (36.36) 0.9177

DM1 without DN (n�51) 11 (21.57) 22 (43.14) 18 (35.29)

DM2 with DN (n�63) 12 (19.06) 35 (55.56) 16 (25.40) 0.3315

DM2 without DN (n�107) 24 (22.43) 47 (43.93) 36 (33.64)

Normoalbuminuria (n�158) 35 (22.15) 39 (43.67) 54 (34.18)

Macroalbuminuria (n�48) 8 (16.67) 24 (50.00) 16 (33.33) 0.7238

Microalbuminuria (n�59) 12 (20.34) 31 (52.54) 16 (27.12)

DM with SAH (n�181) 40 (22.10) 85 (46.96) 56 (30.94) 0.6363

DM without SAH (n�84) 15 (17.86) 39 (46.43) 30 (35.71)

440 V.P.A. Wobeto et al.

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Declaration of interest: The authors report no conflicts of interest. The authors alone are

responsible for the content and writing of the paper.

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