INFLUENCE OF THE ENDOTHELIAL NITRIC OXIDE SYNTHASE POLYMORPHISM ON THE PROGRESSION OF AUTOSOMAL DOMINANT POLYCYSTIC KIDNEY DISEASE AND IgA NEPHROPATHY

Abstract

Background: The reason of variability of clinical course and progression to end-stage renal failure (ESRF) of two widespread chronic nephropathies–-autosomal dominant polycystic kidney disease (ADPKD) and IgA nephropathy (IGAN) is not clear. The endothelial dysfunction is considered in the number of factors possibly influencing the prognosis of these nephropathies. Our study tried to verify the hypothesis that endothelial nitric oxide synthase (ecNOS) gene polymorphisms in intron 4 could have some relevance to the progression of ADPKD and/or IgA nephropathy. Methods: We examined 128 Czech patients with ADPKD (62 males, 66 females) and 93 patients with IGAN (51 males, 42 females). As a control group we used 100 genetically unrelated healthy subjects (50 men, 50 women, mean age 51.2 ± 8.2). The genomic DNA was amplified by polymerase chain reaction (PCR) and the products were separated on 1.5% agarose gel and visualized by ultraviolet transillumination. We compared homozygous subjects for ecNOSb allele with homozygous and heterozygous subjects for ecNOSa allele. Results: The frequencies of ecNOSa/b + a/a and ecNOSb/b genotypes were 19% (19/100) and 81% (81/100) in the control group. The frequencies of ecNOSa/b + a/a and ecNOSb/b genotypes in ADPKD patients were: 26.6% (8/30) and 73.4% (22/30) in ADPKD patients with normal renal function, 30% (9/30) and 70% (21/30) in ADPKD with ESRF, 35.2% (18/51) and 64.8% (33/51) in young ADPKD patients, 60% (12/20) and 40% (8/20) in ADPKD patients with ESRF later than in 62 years. In IGAN the frequencies of ecNOSa/b + a/a and ecNOSb/b genotypes were 24% (12/50) and 76% (38/50) in IgA with normal renal function and 20.9 % (9/43) and 79.1% (38/43) in IgA with ESRF. Conclusion: Both in ADPKD and IGAN groups there was no significant difference in the frequencies of ecNOS genotypes between patients with normal renal function and age matched patients with ESRF and between patients with normal renal function and control group. The frequency of ecNOS a allele was significantly higher in a number limited group ADPKD patients with ESRF later than in 62 years (Chi-square test p < 0.05). This higher frequency of a allele among ADPKD patients with later onset of ESRF could suggest the trend of positive influence of a allele in ADPKD patients.

• Nitric oxide synthase
• Polymorphism
• Renal failure
• Polycystic kidney disease
• IgA nephropathy
• Arterial hypertension

INTRODUCTION

Nitric oxide (NO) has a major role as a messenger molecule in most human organ systems. In the kidney, as well as other solid organs, NO physiologic concentrations functions as a tonic vasodilator, working essentially instantaneously. NO, a molecular gas, is formed by the action of one of three isoform of nitric oxide synthase (NOS). The isoforms are named upon the cell types in which they were first isolated: neuronal NOS (nNOS), inducible or macrophage NOS (iNOS) and endothelial NOS (ecNOS). All three enzymes are cytochrome P450-like proteins. They facilitate the addition of the guanidino nitrogen of the amino acid arginine to molecular oxygen, producing NO and water. NO exerts its effects locally and transiently due to an extremely short half-life. It readily penetrates the biological membranes of neighboring cells, modulating a number of signaling cascades. The most recognized cellular target of NO is heme-containing soluble guanylate cyclase. The stimulation of this compound enhances the synthesis of cyclic GMP (cGMP) from guanosine triphosphate (GTP), increasing the cytosolic levels of cGMP.

All three NOS isoforms can be expressed in the kidney: (1) nNOS is principally expressed in macula densa and inner medullary collecting duct, (2) iNOS has been localized in several tubule segments, glomerulus and the interlobular and arcuate arteries, (3) ecNOS is expressed in the endothelium of glomerular capillaries, afferent and efferent arterioles and intrarenal arteries. Insight into the role of NO in the kidney is primarily derived from experiments examining the effect of inhibiting NOS. The most important role of NO in the kidney is (1) regulation of renal hemodynamics, (2) modulation of fluid and electrolyte transport. In the rat, acute systemic NOS inhibition causes marked increase in afferent and efferent arteriolar resistance and a fall in glomerular capillary ultrafiltration.[[1]] Chronic systemic blockade of NO bioactivity in rats resulted in significant glomerular capillary hypertension.[[2]] In these models, both angiotensin II and endothelin1 activation contributed the marked increase of efferent arteriolar resistance.[[3]] Enhanced ecNOS activity in glomeruli in diabetic rats contributed via renal vasodilation and glomerular hyperfiltration to more progressive course of the disease.[[4]] Intrarenal NO synthesis is increased during periods of increased salt intake, thereby facilitating renal sodium excretion. NO inhibits sodium entry in the cortical collecting duct, Na–H exchange in the proximal tubule, and Na–K–ATPase activity in different nephronic segments.[[5]] NO also down regulates the responsiveness of the collecting tubule to antidiuretic hormone, facilitating the excretion of water.[[6]] NO synthesized by nNOS in the macula densa blunts the tubuloglomerular feedback response in which increasing sodium chloride delivery to the macula densa lowers the glomerular filtration rate to maintain distal flow at a relatively constant level.[[7]] NO derived from ecNOS activity through the tonic vasodilation as well as through the ability to inhibit platelet activation and adhesion may help to minimize injury in glomerulonephritis. However, NO from iNOS in excess as a response to inflammatory stimuli may exacerbate the damage of cells.[[8]] Cellular injury is most likely due to the formation of peroxynitrite from NO and superoxide radical.

Autosomal dominant polycystic kidney disease (ADPKD) is the most frequent inherited renal disorder. Its clinical course is highly variable. 85% of ADPKD cases is caused by the mutation of the PKD1 gene on chromosome 16, in about 14% cases the PKD2 gene on chromosome 4 is mutated. Nevertheless, the clinical variability cannot be fully explained by these two different genes. The influence of genetic modifiers must be clarified. It was demonstrated that endothelium dependent relaxation of resistance vessels was impaired in Han:SPRD polycystic kidney disease rats.[[9]] Recently the impaired endothelium-dependent relaxation was demonstrated in subcutaneous vessels of patients with ADPKD.[[10]] The impairment of endothelial function was present in ADPKD, even though the subjects were still in the early normotensive phase of the disease. In contrast to inborn ADPKD, IgA nephropathy (IGAN) is the most frequent type of primary glomerulopathies. Its pathogenesis is closely related to the abnormality of IgA regulation and similarly to ADPKD, the search for factors modulating the prognosis of IGAN is in process. Arterial hypertension affects the majority of patients with IGAN and significantly influences the course of the disease; therefore a special interest is paid to the conditions linked to the onset and presence of arterial hypertension in IGAN. In this context it could be presumed, that inappropriate amounts of NO synthesized by endothelial cells possibly contribute to the impairment of the blood pressure regulation in IGAN.

The ecNOS is encoded by a gene located on chromosome 7q35-36 comprising 26 exons that span 21 kb. Two alleles in ecNOS intron 4 were identified: (1) the larger has five tandem 27-bp repeats (allele b), (2) the smaller has only four repeats (allele a). To verify the hypothesis that lower NO plasma level, found in patients with a allele of ecNOS polymorphism, influences the progression of ADPKD and/or IGAN, we compared the frequencies of gene polymorphisms between different groups of ADPKD and/or IGAN patients.

METHODS

A total of 128 Czech patients with ADPKD (62 males, 66 females) and 93 patients with IGAN (51 males, 42 females) entered into this study. After the approval of Czech Ethical Committee in Czech Republic, nephrologists of 10 dialysis centres in our country were asked for their co-operation. Blood from 50 dialyzed patients with ADPKD (24 males and 26 females) and from 43 patients with IGAN (23 males and 20 females) in EDTA tubes was sent for DNA isolation and determination of the ecNOS genotype. Centres were questioned about the age of ESRF. ADPKD patients with ESRF were divided in two groups: 30 with mean age of ESRF 49.9 ± 5 years, and 17 patients with ESRF later than in 62 years. Moreover, we analyzed 81 individuals (43 males and 38 females) from 35 unrelated families with ADPKD and normal renal function (serum creatinine under 110 µmol/L). They were diagnosed by ultrasound (US). US criteria for diagnosis included the presence of at least two cysts in one kidney or one cyst in each kidney in an at-risk person aged younger than 30 years, the presence of at least two cysts in each kidney in an at-risk person aged between 30 and 59 years, and at least four cysts in each kidney for those persons at risk aged 60 years and older. The ADPKD patients with normal renal function were subdivided in two groups: (1) 30 patients with normal renal function (mean age 49.5 ± 9years), age and gender matched to patients with ESRF, and (2) 51 young patients with normal renal function (mean age 25.6 ± 6 years).

We studied the prevalence of AH for each NOS polymorphism in patients with normal renal function. AH was defined as BP higher than 140/90 by repeated measurement or/and normal BP reached by use of antihypertensive drugs.

IGAN: Apart from the patients with IGAN and ESRF we analyzed age and sex matched 50 individuals (28 males and 22 females) with IGAN and normal renal function (serum creatinine under 110 µmol/L). The diagnosis was established by renal biopsy with typical finding of IgA deposits observed on immunofluorescence microscopy. Hundred genetically unrelated healthy Czech subjects (50 men, 50 women, mean age 51.2 ± 8.2 years) were studied as control group.

Genomic DNA was isolated from peripheral-blood lymphocytes by the salting out procedure, as previously described.[[10]] The NOS a/b polymorphism was amplified with flanking primers 5′-AGGCCCTATGGTAGTGCCTTT-3′ and 5-TCTCTTAGTGCTGTGGTCAC-3′. Each reaction mixture was heated to 94°C for 4 min for denaturation and underwent 35 cycles at 94°C for 1 min, annealing at 56°C for 1 min and extension at 72°C for 2 min and final extension at 74°C for 7 min. The polymerase chain reaction (PCR) products were analyzed by 1.5% Promega agarose gel electrophoresis. The fragments were visualized by ethidium bromide staining and ultraviolet transillumination.

STATISTICS

A chi-square test was used to compare frequencies of different genotypes among chosen groups. All results are statistically significant at p < 0.05.

RESULTS

PCR analysis of genomic DNA generated fragments of 393 bp or 420 bp corresponding to the ecNOSa and ecNOSb alleles, respectively. We compared homozygous and heterozygous subjects for ecNOSa allele with homozygous subjects for ecNOSb allele.

The frequencies of ecNOSa/b + a/a and ecNOSb/b genotypes were 19% (19/100) and 81% (81/100) in the control group. ADPKD: The frequencies of ecNOSa/b + a/a and ecNOSb/b genotypes in ADPKD patients were: 26.6% (8/30) and 73.4% (22/30) in ADPKD patients with normal renal function, 30% (9/30) and 70% (21/30) in ADPKD with ESRF, 35.2% (18/51) and 64.8% (33/51) in young ADPKD patients, 60% (12/20) and 40% (8/20) in ADPKD patients with ESRF later than in 62 years. There was no significant difference in the frequencies of ecNOS genotypes between ADPKD patients with normal renal function and age matched patients with ESRF and between ADPKD patients with normal renal function and control group. The frequencies of genotypes did not differ in young ADPKD patients from control group. The frequency of ecNOSa allele was significantly higher in a number limited group ADPKD patients with ESRF later than in 62 years in comparison with ADPKD patients with ESRF in mean age 49.9 ± 5 years.

The frequencies of ecNOSa/b+a/a and ecNOSb/b genotypes in ADPKD patients with normal renal function in respect to HT were: 62.5% (5/8) and 63.6% (14/22), and 44.4% (8/18) and 42.4% (14/33). The different polymorphisms did not differ in prevalence of HT.

IGAN: The frequencies of ecNOSa/b + a/a and ecNOSb/b genotypes in ADPKD patients were: 23.2% (10/43) and 76.8% (33/43) in ADPKD patients with normal renal function and 26% (13/50) and 74% (37/50) in IGAN patients with ESRF. There was no significant difference in the frequencies of ecNOS genotypes between IGAN patients with normal renal function and age matched IGAN patients with ESRF and between IGAN patients with normal renal function and control group (Table 1).

Table 1. The Frequencies of ecNOS Genotypes in ADPKD/IGAN Patients

	ecNOSa/a + a/b Genotypes	ecNOSb/b Genotypes
ADPKD patients with normal renal function	26.6%	73.4%
ADPKD patients with ESRF	30%	70%
Young ADPKD patients with ESRF	35.2%	64.8%
ADPKD patients with ESRF later than in 65 years	60%	40%
IGAN patients with normal renal function	23.2%	76.8%
IGAN patients with ESRF	26%	74%

DISCUSSION

In this study the frequency of a allele was not different in ADPKD/IGAN patients with normal renal function and age matched ADPKD/IGAN patients with ESRF. The frequency of a allele was significantly higher in a limited number of ADPKD patients with ESRF later than in 62 years in comparison with ADPKD patients with ERSF in mean age of 49.9 years. The results suggest that a allele does not play an important role in clinical course of ADPKD and/or IGAN. On the other hand, higher frequency of a allele among patients with later onset of ESRF in ADPKD group could suggest a protective role of a allele. However, we have to realize only limited number of patients in this examined group. We did not confirm the linkage of ADPKD to the PKD1 or PKD2 gene. The higher prevalence of the PKD2 patients with later onset of ESRF could also influence the result.[[11]]

Wang et al.[[12]] recently found higher incidence of a allele among patients with ESRF in comparison to healthy subjects except those caused by diabetic nephropathy. Overproduction of NO may play a role in diabetic hyperfiltration followed by glomerular hypertrophy. Morita et al. suggested that a allele of the ecNOS gene polymorphism may be involved in the progression of IgA nephropathy.[[13]] He found out higher incidence of patients with AH and more frequent advanced histological grading among patients with a allele. However other authors failed to confirm an association between ecNOS gene polymorphism and prognosis in IGAN (and other primary glomerulonephritides).[[14]]

ecNOS gene polymorphism influences the plasma level of NO. This polymorphism is probably a marker of another functional variant of ecNOS. The mean plasma level of NO in subjects who were homozygous for the a allele was found nearly 20% lower than in the subjects with b allele.[[14]] Because of very low frequency of a/a genotype, we compared the subjects with a/a and a/b genotype to b/b genotype, but the difference of NO plasma level is than less significant. The endothelium-dependent relaxation of the small subcutaneous vessels was impaired in ADPKD patients with normal blood pressure and renal function.[[10]] This impairment could contribute to the development of HT and vascular disease later in their life. In the kidneys of rats with polycystic kidney disease decreases the expression of NOS isoenzymes as a number of cysts increases.[[15]] An inducer of NOS, Taxol, inhibits cyst growth in mice.[[16]] These studies suggest the protective role of higher NO plasma level in ADPKD. On the contrary the negative effect of glomerular hyperfiltration as in polycystic rats was not excluded.

Recent study found a decreased plasma NO level in patients with essential AH.[[17]] Based on the results of our study no genotype was found to be a predisposing factor of AH in ADPKD patients. The a allele could be also related to coronary heart diseases.[[18]], [[19]]

In conclusion, the ecNOS polymorphism does not play an important role in a clinical course of ADPKD and IGAN. The higher frequency of a allele among ADPKD patients with later onset of ESRF could suggest the trend of positive influence of a allele in ADPKD patients, but the influence of a allele should be further studied.

Acknowledgment

The authors had the support of grant project GAUK 203469.