Autosomal Dominant Polycystic Kidney Disease Biology Essay

Abstract:

Autosomal dominant polycystic kidney disease (ADPKD) is an inherited disorder worldwide, and it is mainly associated with renal cyst formation. Several studies have also shown that these mutations regulate the physiology of epithelial tissues and determine renal cyst formation and growth in PKD. Nitric oxide (NO) is thought to be an important factor in the deterioration of renal function. Here we investigated this hypothesis by studying the putative role of the 27 base pair tandem repeat polymorphism in intron 4 of the endothelial nitric oxide synthase (NOS3) gene in the South Indian population of 53 ADPKD patients with end-stage renal disease (ESRD) and 94 unrelated controls. The genotyping was performed by polymerase chain reaction and electrophoresis. The data were statistically analysed using the χ2-test. The NOS3 4a allele frequencies are 21.3% and 13.2% respectively for control and ADPKD groups. The risk associated with individual mutant genotypes of the NOS3 VNTR with regards to risk of ADPKD depicted borderline association with predisposition in the dominant model (4b/b vs. 4b/a+4a/a: OR =0.47, 95% CI =0.219 to 1.014, p=0.052). NOS3 27-bp VNTR genotypes showed significant association with ESRD progression in ADPKD. In conclusion, the NOS3 27bp VNTR is associated with ADPKD and ESRD progression caused by ADPKD in south Indian population.

Introduction

Autosomal dominant polycystic kidney disease (ADPKD) is an inherited disorder worldwide, and it is mainly associated with renal cyst formation. ADPKD is caused by mutations of Polycystic Kidney Disease 1 (PKD1) or PKD2 (1). Several studies have also shown that these mutations regulate the physiology of epithelial tissues and determine renal cyst formation and growth in PKD (1, 2). Several line of evidence suggests that the endothelial dysfunction, secondary to an impaired release of NO, exists in ADPKD (3). Nitric oxide (NO) is the molecular counterpart of the endothelium-derived relaxing factor (4). It’s a key signaling molecule that controls blood pressure and nerve impulses (5, 6). Nitric oxide is synthesized by the endothelial NO synthase (eNOS) through the conversion of L-arginine to L-citrulline, using molecular oxygen. The gene coding for eNOS (NOS3) is located on 7q36 (7).  The relationship between eNOS and several micro and macro vascular complications led to the hypothesis that it could be a modifier gene in ADPKD (8-10). Several single nucleotide polymorphisms (SNPs) have been described in the NOS3 gene and some of them have been associated with altered eNOS function, leading to impair NO synthesis. A missense variant in exon 7 (Glu298Asp) and 27-base pair (bp) variable number of tandem repeat (VNTR) in the intron-4 of NOS3 gene are known to altered eNOS expression (11, 12). Previously, the relationship between NOS3 genetic variation and some diseases has been reported. It has also been shown that endothelial nitric oxide synthase (NOS3) 27 bp VNTR might show role a modifier effect on the ADPKD progression (11). There is no data available in Indian populations with reference to the NOS3 gene polymorphisms and their association with ADPKD or its progression. Thus, the aim of the current study is to determine the frequency of NOS3 27-bp VNTR in ADPKD patients and to correlate the SNP frequency with clinical status of the ADPKD patients.

Materials and methods

For the current study comprises of a total of 147 individuals (53 ADPKD and 94 controls) were selected from the department of Nephrology, Sri Ramachandra University, Chennai, India. For each of the study subjects, hematologic, serum biochemical and electrolyte indices were measured. Glomerular filtration rate (eGFR) was assessed based on Modification of Diet in Renal Disease (MDRD) formula (13). The total number of cysts in the ADPKD patients was detected by using ultrasound imaging. Only those ADPKD patients fulfilling the standard diagnostic criteria were included in the study (14). ADPKD patients were divided into three groups such as slow progressor (>63 years), moderate progressor (45-63 years) and rapid progressor (<45 years). Individuals without diabetes, hypertension and kidney related diseases were considered as controls. The study was approved by the Institutional Ethics Committee of the Sri Ramachandra University, Chennai, India. A written informed consent was obtained from the study participants. Three ml of blood sample was collected from all the participants, Phenol chloroform extraction and ethanol precipitation technique was used to extract DNA from whole blood (15). Genotyping for NOS3 27-bp VNTR polymorphism was performed by PCR-electrophoresis methods (16). The amplicons were examined by agarose gel electrophoresis. The amplicons of 393 bps corresponding to 4 a/a homozygotes, 420bps to 4b/b homozygotes and 393 and 420 bp are hetrozygotes. Allele frequencies were determined by direct counting of alleles at each locus. The genotype distribution was evaluated for Hardy–Weinberg’s equilibrium (HWE). The association between NOS3 27-bp VNTR polymorphism and ADPKD was analysed using χ2-test. Odds ratios and 95% CI were calculated. All statistical analyses were performed with SPSS statistical software version 17.0 (SPSS Inc, Chicago, Illinois) for Windows.

Results:

The baseline characteristics of ADPKD patients and controls were documented in Table 1. The mean age in ADPKD cases were 49.20±10.1, while in control group 52.1±12.2. The NOS3 27-bp VNTR genotypes are following the Hardy-Weinberg equilibrium in control group as well as in ADPKD group (Table 2). The NOS3 VNTR mutation was found in 36 of 94 control subjects and in the ADPKD group 12 of 53 subjects. The NOS3 4a allele frequencies are 21.3% and 13.2% respectively for control and ADPKD groups. The risk associated with individual mutant genotypes of the NOS3 VNTR with regards to risk of ADPKD depicted borderline association with predisposition in the dominant model (4b/b vs. 4b/a+4a/a: OR =0.47, 95% CI =0.219 to 1.014, p=0.052). The NOS3 VNTR did not show significant association with ADPKD at allele level (4b vs. 4a: OR = 0.563, 95% CI =0.290–0.109, p = 0.086) (Table 2). Distribution of these genotypes among different ESRD groups also revealed significant association between NOS3 27-bp VNTR genotypes and ESRD progression in ADPKD (Table 3).

Discussion:

In view of the strong association of ADPKD with hypertension, the eNOS enzyme responsible for nitric oxide production, variation in its expression and activity can be linked to hypertension. Hence NOS3 gene has long been thought to be a candidate gene for ADPKD. In this study, after investigating 94 controls and 53 ADPKD subjects we demonstrate that there is a positive association between NOS3 VNTR polymorphism and ADPKD as well as ESRD progression in ADPKD. The results were consistent to those previously reported studies. The first study linking this SNP and hypertension was published by Miyamoto and co workers (17). Though these findings in an were not substantiated in Australian (18) or a Scandinavian (19) populations further evidence of an association of the NOS3 allele, and with essential hypertension resistant to conventional treatment, found in a Czech population (20). Following the conflicting results generated from these association studies Intron 4 VNTR and haplotype analysis of several other SNPs of NOS3 showed significant association with daytime systolic blood pressure (21).

In the present study the age at renal failure is also significantly associated with NOS3 genotypes in ADPKD. An independent study using the intron 4 VNTR polymorphism in unrelated ADPKD patients from Belgium and the north of France, has also reported similar findings (11, 22). Several studies have demonstrated that a decrease of NO synthesis and release may be important in the progression of renal disease (23), and a significant endothelial dysfunction has been documented in ADPKD patients (Wang et al. 2000). Direct analysis of NOS3 gene polymorphisms in ADPKD patients also revealed inconclusive results from many populations. Although no direct association between NOS3 VNTR and ADPKD, patients who carried a allele showed faster ESRD progression in the group of ADPKD (24). The −786T>C SNP, in the promoter of the NOS3 gene, is a functional polymorphism, C allele being associated with higher gene expression (25).

Another functional polymorphism in the NOS3 is E298D, the D allele is known to lower the enzyme activity by post-translational modifications (11). NOS3 gene polymorphisms in ADPKD disease progression has been noticed in few studies (26). Furthermore, the association was not detected in another series (27, 28). These inconsistent results might have originated from different inclusion criteria, and population heterogeneity and sample ascertainment (27). In addition, the allelic frequency variations in NOS3 gene are different among the world populations, making the association studies as inconclusive or population specific.  In conclusion, the NOS3 27bp VNTR is associated with ADPKD and ESRD progression caused by ADPKD in south Indian population.