G-699/C polymorphism in the bradykinin-1 receptor gene in patients with renal failure

Heidrun Knigge, Martin Blüthner, Angelika Brüntgens, Heike Sator and Eberhard Ritz

Department of Internal Medicine, Division of Nephrology, Heidelberg, Germany

Abstract

Background. Bradykinin is thought to have protective effects on the progression of renal failure. Of particular interest, it has been reported that one polymorphism in the promoter region of the human kinin B1-receptor gene which is associated with higher activity, is less frequently found in patients with end-stage renal failure. The present study was performed to independently confirm these results.

Design. Crossectional study on 376 healthy controls, 262 non-diabetic dialysis patients and 175 patients with type 1 diabetes >=10 years and microalbuminuria (of whom 21 were dialysis-dependent) and 334 patients with type 2 diabetes >=10 years and nephropathy (of whom 61 were dialysis-dependent).

Methods. Genotyping was performed by polymerase chain reaction, followed by restriction enzyme analysis.

Results. All groups were in Hardy Weinberg equilibrium. The study showed no significant difference in the frequency of the C-allele between controls (0.093) and non-diabetic dialysis patients (0.095). No significant difference in C-allele frequency was observed between controls and patients with type 1 diabetes and microalbuminuria (0.092) or patients with type 2 diabetes and nephropathy (0.099).

Conclusion. In large cohorts of patients with non-diabetic end-stage renal disease and diabetic renal disease with and without end-stage renal failure, no change in the frequency of the C-699 allele of the B-1-receptor gene was found.

Keywords: kinin; kinin receptor; kinin receptor gene polymorphism; renal failure; diabetic nephropathy

Introduction

The role of the systemic and renal kinin systems in the genesis of renal disease is not perfectly understood and marked abnormalities in gene transcription and protein expression of the various components of the system have been noted particularly in diabetic renal disease [1,2]. Against this background a recent communication of Bachvarov et al. is of considerable importance [3]. These authors noted that one member of the G-protein coupled receptor superfamily, the bradykinin receptor subtype 1, exhibited a polymorphism in the promoter region (G-699-C). The frequency of the C-allele was significantly less in a cohort of patients with endstage renal failure [3].

The present study was carried out in a total of 771 renal patients and 376 non-renal controls to examine whether these findings could be confirmed in an independent large cohort of renal patients with non-diabetic and diabetic renal disease.

Patients and methods

Patients
All available dialysis patients were recruited from the dialysis centres in Heidelberg, Heilbronn, Mannheim, Villingen-Schwenningen, and Wiesloch. Of these patients, 21 had type 1 and 61 type 2 diabetes according to National Diabetes Data Group criteria [4]. Of the non-diabetic dialysis patients, 70 had glomerulonephritis, 30 autosomal dominant polycystic kidney disease (ADPKD), 16 analgesic nephropathy, 22 urological problems, and 124 were of unknown or other causes. All patients with type 1 or type 2 diabetes were recruited from the outpatient clinic. Mean body mass index (BMI) was 23.9±3.1 in the control group, 25.3±3.6 in type 1 and 28.7±4.8 in type 2 diabetic patients. All available blood donors from the local blood bank and volunteers were examined as healthy controls. The characteristics of the study population are given in Table 1Go.


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Table 1. Characteristics of the study population

 
The study was approved by the local ethics committee, and all patients and blood donors gave written informed consent.

Methods
High-molecular weight DNA was isolated from 10 ml of ethylenediaminetetraacetic acid (EDTA) blood. Genotyping was performed by polymerase chain reaction (PCR), followed by restriction enzyme analysis of the resulting products [3]. PCR was performed with 200 ng of genomic DNA in a reaction volume of 50 µl. Reaction conditions were per cycle as follows: denaturation at 94°C for 45 s, annealing at 60°C for 45 s, and elongation at 72°C for 45 s. All together, 35 cycles were performed. Reaction products were digested with the restriction enzyme Acil (New England Biolabs) and were analysed on 2% agarose, stained with ethidium bromide, and visualized under ultraviolet illumination. The undigested PCR product (GG genotype) gave a band of 191 bp in size. The completely digested PCR product (CC genotype) generated two bands of 135 bp and 56 bp. Thus, heterozygotes (GC genotype) showed three bands of 191 bp, 135 bp, and 56 bp.

Statistics
Data are given as mean and standard deviation (SD). Genotype distribution, and allele frequencies were compared between the groups using the chi-square test.

Results

The homozygous CC genotype was very rare in the background population and the frequency of the heterozygote GC was considerably less than in the paper of Bachvarov et al. [3]. All groups were in Hardy Weinberg equilibrium. No significant difference was noted between controls and either non-diabetic dialysis patients or type 1 or type 2 diabetic patients with nephropathy (Table 2Go).


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Table 2. Genotypes of the study population

 
The calculated allele frequencies in the control group, i.e. 0.093, were markedly less than in the report of Bachvarov et al. [3]. Again, no significant differences were found between the background control population and non-diabetic dialysis patients or nephropathic patients with type 1 or type 2 diabetes respectively (Table 3Go).


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Table 3. Allele frequencies of the study population

 
Comparisons were also made between the three subgroups and respective control individuals matched for age and gender. No difference in genotype and allele frequency was noted (data not shown).

Discussion

The above study examined larger samples of the background population and of patients, respectively, than the preceding report of Bachvarov et al. [3]. Yet our study failed to confirm a lesser frequency of the 699-C-allele and of GC-heterozygotes amongst patients with renal disease. This was true both for non-diabetic and diabetic renal disease.

Our study had a power of more than 93% to detect a 10% difference in GC genotype distribution between controls and dialysis patients as found in the study of Bachvarov et al. [3], so that a beta error problem appears unlikely. The power to detect such a difference between controls and nephropathic type I and type II diabetic patients was 88% and 95% respectively. We also were very careful in the execution of PCR analysis. While the products were not sequenced we made certain by repeated examinations that the results were well reproducible and consistent.

One potential source of bias can be introduced by the selection of controls. An effort was made to recruit only subjects who had passed detailed health check-ups as blood donors. Bachvarov et al. [3] studied ‘unrelated healthy volunteers’ of non-specified background.

More recently, using the transmission disequilibrium test approach, Zychma et al. [5] noted that the B1R gene G->C substitution at position -699 on the promoter region and the B2R gene C->T transition at position 181 in exon 2 were not stochastically transmitted in 247 family trios, i.e. the less common alleles of both polymorphisms (B1R C and B2R T alleles respectively) were transmitted from heterozygous parents to offspring with end-stage renal disease less frequently than expected. These results cannot be directly compared with ours because of the different design of the study.

This negative finding raises several issues. As to the biological plausibility it is of note that blood pressure is an important promoter of progression. In bradykinin subtype-2 receptor knockout mice, Madeddu et al. [6] found elevated blood pressure, whilst transgenic mice expressing the human B2 receptor cDNA displayed an exaggerated hypotensive response to injection of bradykinin. Whereas the B2-receptor is constitutively expressed, the B1 receptor is expressed primarily upon stimulation by cytokines and growth factors. Inflammatory processes go in kidneys with progressive renal damage, so that involvement of the B1 receptor subtype cannot be completely discounted, but indeed the B2 receptor subtype would be a more attractive candidate for a genetically determined bradykinin receptor related predisposition to progression.

We acknowledge, however, that one cannot exclude the possibility that a gene in linkage disequilibrium with a pathomechanistically uninvolved B2R gene is responsible for the association found by others. In view of the apparent conflict between the results of the present study and of the preceding one [3], a recent discussion of discrepancies between association studies is of interest [7]. It is not unheard of that the results of association studies cannot be replicated. This is not always explained by inappropriate controls or inadequate statistical evaluation. We agree with the opinion [7] that repeated confirmation in independent cohorts is necessary to make association studies or more sophisticated sibship disequilibrium studies entirely convincing.

The failure of the present study to find abnormalities in the B1 receptor genotype distribution calls for further studies in independent populations to definitely confirm or to refute the hypothesis proposed by Bachvarov and coworkers [3].

Acknowledgments

We thank Drs Reichel (Schwenningen/Germany), Zeier (Wiesloch/Germany), Strojek (Zabrze/Poland), Rambausek (Heilbronn/Germany), Schwarzbeck (Mannheim, Germany), Bergis (Mergentheim/Germany), Nawroth (Heidelberg/Germany) for providing information and blood samples of patients. We thank PD Dr G. Zilow (Heidelberg/Germany) for providing blood samples of blood donors.

Notes

Correspondence and offprint requests to: Dr Eberhard Ritz, Department of Internal Medicine, Division of Nephrology, Bergheimerstr. 58, D-69115 Heidelberg, Germany. Back

References

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Received for publication: 31.12.99
Accepted in revised form: 6. 3.00