Genetic analysis of nitric oxide and endothelin in end-stage renal disease
Barry I. Freedman1,,
Hongrun Yu2,
Pamela J. Anderson2,
Bong H. Roh2,
Stephen S. Rich3 and
Donald W. Bowden2
1 Department of Internal Medicine/Nephrology,
2 Department of Biochemistry and
3 Department of Public Health Sciences, The Wake Forest University School of Medicine, Winston-Salem, North Carolina, USA
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Abstract
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Background. Genetic factors have been implicated in the development of the common aetiologies of end-stage renal disease (ESRD), including renal failure attributed to hypertension, diabetes mellitus, systemic lupus erythematosus and human immunodeficiency virus infection. Nitric oxide (NO) and endothelin are powerful vasoactive mediators involved in inflammation and regulation of vascular tone and blood pressure. We evaluated the role of the neuronal constitutive (NOS1) and endothelial constitutive (NOS3) nitric oxide synthase genes and the endothelin-1 (EDN-1) gene in predisposition to chronic renal failure in AfricanAmericans.
Methods. The study population for the linkage and association analyses in ESRD consisted of 361 individuals from 168 multiplex AfricanAmerican families. These individuals comprised 207 unweighted sibling pairs concordant for all-cause ESRD. Microsatellite markers NOS1B (NOS1), D7S636 (NOS3) and CPHD11/2 (EDN-1) were genotyped in the sample. In addition, a mutation, Glu298Asp, in exon 7 of NOS3 and a 27 bp variable number tandem repeat (VNTR) marker in intron 4 of NOS3 were evaluated in the sibling pairs and in an additional 92 unrelated AfricanAmericans with type 2 diabetes mellitus-associated ESRD (singletons). Association analyses utilized the relative predispositional effect method. Model independent linkage analyses were performed using GeneHunter-plus and MapMaker/SIBS (exclusion analysis) software.
Results. Significant evidence for association with ESRD was detected for alleles 7 and 9 of the NOS1 gene (11.9 and 34.2%, respectively, in unrelated probands of ESRD families versus 6.5 and 27.5%, respectively, in race-matched controls, both P<0.01). These associations were maintained when the unrelated first sibling from each family was used in a case-control comparison and was most pronounced in the non-diabetic ESRD cases. The NOS3 and EDN-1 markers failed to provide consistent evidence for association in the sibling pairs and the diabetic ESRD singletons, although we identified two novel endothelial constitutive NOS4 (ecNOS4) VNTR alleles in AfricanAmericans. Significant evidence for linkage was not detected between the NOS genes or the EDN-1 gene in either all-cause ESRD or when the ESRD sibling pairs were stratified by aetiology (type 2 diabetic ESRD or non-diabetic aetiologies).
Conclusion. Based upon the consistent allelic associations, we believe that further evaluation of the NOS1 gene in ESRD susceptibility in AfricanAmericans is warranted.
Keywords: AfricanAmericans; endothelin; end-stage renal disease; genetics; hypertension; nitric oxide synthase
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Introduction
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Endothelin-1 (EDN-1) is a potent constrictor of renal blood vessels. It causes mesangial cell contraction, stimulates extracellular matrix deposition and increases glomerular cell proliferation [1]. Conversely, nitric oxide (NO) dilates renal blood vessels and modulates tubuloglomerular feedback and renin secretion [2]. Abnormalities in the synthesis and/or metabolism of NO and EDN-1 have been implicated in the initiation and progression of various aetiologies of chronic renal disease [35]. Interactions between these two important pathways have been detected that can exacerbate or ameliorate progressive renal dysfunction [6].
There are three known nitric oxide synthase (NOS) genes in humans and they are differentiated by their tissue-specific expression. NOS1, or neuronal constitutive NOS, has a chromosomal location on 12q24 [7]. NOS2, or inducible NOS, is expressed in pathological processes such as inflammation and has a chromosomal location on chromosome 17q11 [8]. NOS3, or endothelial constitutive NOS, has a chromosomal location on 7q3536 [9]. Importantly, each of these three forms of NOS can be expressed in multiple tissues, including kidney [6]. Therefore, all three NOS genes may be relevant in the initiation and progression of chronic renal failure. The NOS3 gene locus appears to be responsible for variations in plasma levels of NOS [10]. Inhibition of NO synthesis by N(G)-nitro-
-arginine methyl ester (
-NAME) produces systemic hypertension, nephrosclerosis and left ventricular hypertrophy in rats [11]. In humans, polymorphisms in the NOS3 gene have been associated with a heightened risk of coronary artery disease in cigarette smokers [12] and with terminal renal failure [13].
EDN-1 is generated by the vascular endothelium and normally contributes to vascular tone and resting blood pressure [1]. The EDN-1 gene is located on chromosome 6 [14]. Chronic renal failure is accompanied by enhanced renal synthesis of EDN-1 and blockade of EDN-1 receptors in streptozotocin-induced diabetic rats reduces systemic blood pressure and urinary protein excretion [15].
We evaluated the roles of the NOS1, NOS3 and EDN-1 genes in renal failure susceptibility in AfricanAmericans using genetic association and linkage analyses. Three polymorphic markers were tested for NOS3 and one marker each was tested for NOS1 and EDN-1. The NOS2 gene was not evaluated, since only one polymorphic marker with a relatively low heterozygosity (0.27) has been described to date [16].
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Materials and methods
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Study population
The study population consisted of 361 AfricanAmerican patients from 168 families. This generated 207 AfricanAmerican sibling pairs concordant for ESRD. Sibling pairs were identified using the ESRD Network 6 (Southeastern Kidney Council, Inc.) sponsored Family History of ESRD' database [17]. Aetiologic determinants included 92 sibling pairs concordant for type 2 diabetes-mellitus-associated ESRD and 115 sibling pairs with non-diabetic aetiologies of ESRD (renal limited or systemic glomerular diseases, hypertension or unknown). Discordant aetiologies of ESRD were observed in the non-diabetic sibling pairs. The cause of ESRD in study patients was assigned by a single investigator (BIF), using previously reported criteria [18]. Exclusion criteria included families containing members with renal failure attributed to adult polycystic kidney disease, Alport's syndrome (hereditary nephritis) or urologic disease (urinary reflux or surgical nephrectomy), affected relatives less than 18 years old, members unable to provide informed consent or with self-reported race other than AfricanAmerican.
DNA was collected from an additional 92 unrelated AfricanAmericans (singletons) with type 2 diabetes mellitus-associated ESRD for association analyses. Eighty-six AfricanAmericans born in North Carolina and employed at the North Carolina Baptist Hospital, which is physically associated with the Wake Forest University School of Medicine, served as controls.
Genotyping
The NOS1 gene was genotyped with a microsatellite marker NOS1B [19]. A highly polymorphic dinucleotide marker was identified in intron 13 of the NOS3 gene by Marsden et al. [8] and has been used in studies involving hypertension [20]. By BLAST-searching GenBank (http://www.ncbi.nlm.nih.gov/), we found that a random polymorphic genomic marker D7S636 [21] amplified the same CA repeat sequence (Fig. 1
). Therefore, we used D7S636 to genotype the NOS3 gene. NOS1 and NOS3 genotyping was performed by polymerase chain reaction (PCR) on an ABI 377 sequencer [22]. The forward primers were labelled with fluorescent dyes and samples were run against sizing standards.

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Fig. 1. A fragment of human genomic DNA equence (GenBank accession no. Z23585) showing that the microsatellite markers D7S636, in bold and italic [21], GT NOS and GT NOSBR, underlined [23] and cNOS-1 and cNOS-2 double underlined [20] all amplify the same CA repeat in intron 13 of the NOS3 gene. Only part of the forward cNOS-1 primer is shown. The 5' part could not be identified in this sequence and may have been a typographic error in the publication.
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We determined that the intron 13 microsatellite marker of NOS3 is D7S636 by BLAST-searching the GenBank using available primer sequences for the NOS3 gene [20,23] (Fig. 1). Nadaud et al. [23] had mapped the NOS3 gene to a 4 cM region between AFM199zd4 (D7S505) and AFM074xg5 (D7S483) on chromosome 7. D7S636 is located between D7S505 and D7S483. This finalizes the exact location of NOS3 at 7q317qter and highlights the redundancy of marker names in the literature.
In addition, an allelic variant, Glu298Asp [24], in exon 7 of NOS3 and a 27 bp variable number tandem repeat (VNTR) marker [13] in intron 4 of NOS3 (allele a of endothelial constitutive NOS4 (ecNOS4)) were evaluated. The PCR products of the NOS3 intron 4 VNTR were run on agarose gels. Different alleles were recognized by their length and were measured with a sizing standard of 1 kb plus ladder (GIBCO BRL). For any new alleles, PCR products were sequenced on the ABI 377 sequencer. The Glu298Asp point alleles were assayed by two restriction enzymes acting upon the mutation site, BanII and MboI [25]. Enzyme incubation lasted overnight. The 248-bp PCR product of the wild type DNA samples was completely digested into a 163 and an 85-bp fragment that could be identified on agarose gels. No digestion occurs when there is a sequence change from a G allele to a T allele. The heterozygous samples carry all three fragments. The heterozygous and homozygous genotypes were confirmed by DNA sequencing, in order to prevent any mistyping due to incomplete digestion of the enzyme. We confirmed both genotypes with MboI. In contrast to BanII, Mbo generated two DNA fragments of 158 and 90 bp, respectively, on the homozygous T carrying DNAs. There was no digestion of the wild type DNA samples. Again, heterozygotes carry all three fragments.
EDN-1 was genotyped using primers CPHD11 and CPHD12 [26]. CPHD11/2 were genotyped manually on denaturing acrylamide gels with one primer end-labelled with [32P]dATP in the PCR reaction [27].
Statistical analyses
Allele association analyses were performed using the relative predispositional effect (RPE) technique [28] in the GAS computer program (Alan Young, Oxford University, UK, 1995). We chose one sibling from each family as a proband and another as a sibling of the proband. Remaining cases in families with more than two siblings were excluded. The two groups were further stratified into type 2 diabetic and non-diabetic ESRD cases.
Phenotype and genotype data available in the 207 sibling pairs from the 168 families were analysed using GeneHunter-plus [29] and MapMaker/SIBS software [30], the latter for exclusion analysis. For GENEHUNTER, the single point method was utilized. In the exclusion analysis, all markers were set as inherited independently, 50 cM, distant from each other except for the D7S636 and ecNOS4 markers (within the NOS3 gene). These last two markers were set at 0.1-cM distance for convenience of the analysis. The disease allele frequency was set at 0.05. The allele frequencies for all markers were calculated from the local controls. In addition to assessing for linkage between each marker and ESRD in the entire family set, we stratified the study population into sibling pairs concordant for type 2 diabetic ESRD and those with non-diabetic aetiologies of ESRD.
In analyses of these data, evidence of discordance of allele sharing significantly greater than 25% (identity by descent (IBD)=0 greater than expected under either the linkage or no linkage hypotheses) was used to detect potential non-paternities. No significant deviations from 25% sharing at IBD=0 were obtained.
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Results
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An allele association analysis was performed for all markers having a frequency of 0.1 or greater in either cases or controls. The alleles for EDN-1, CPHD11 and CPHD12, showed no significant evidence of association with ESRD in any of the groups analysed (all probands, first siblings of probands, or diabetic ESRD singletons). In contrast, several alleles of NOS1B showed significant and consistent association to ESRD (Table 1
). Alleles 7 and 9 of the NOS1B gene were positively associated with ESRD with frequencies of 11.9 and 34.2%, respectively, in all ESRD probands compared with 6.5 and 27.5% in non-renal disease controls (P
0.01 for both). These associations were also observed when comparing the first sibling of each proband with the controls (11.8 and 35.8% in first siblings, respectively; both P
0.01). However, the association of NOS1 allele 7 was confined only to the non-diabetic ESRD probands and their siblings. The allele 7 frequencies were 15.2 and 12.5% in the 99 non-diabetic ESRD probands and their first siblings. These were significantly higher than the 6.5% observed in the controls (P
0.01 for both). The allele 7 frequencies in type 2 diabetic ESRD probands and their siblings, 7.2 and 10.8%, respectively, did not differ significantly from the 6.5% in controls.
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Table 1. Allele frequencies of candidate genes obtained using the relative predispositional effect (RPE) approach
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The endothelial NOS3 markers failed to demonstrate consistent association in the ESRD probands and their first siblings. The T allele of Glu298Asp, previously associated with essential hypertension in a Japanese population [24], was not associated with all-cause, type 2 diabetic-, or non-diabetic aetiologies of ESRD in the probands (there was evidence for association in the first siblings of the ESRD probands with non-diabetic ESRD). Another marker for NOS3, allele 6 of D7S363 was negatively associated with ESRD in the type 2 diabetic probands compared to the controls (5.1 vs 14.5%; P
0.05). However, this association was not consistent in the first siblings of the diabetic ESRD probands (Table 1
). The final NOS3 marker, the a allele of ecNOS4, was associated with all-cause ESRD in both the probands and their siblings compared to controls. The frequency in probands and their siblings was 31 and 30.1%, respectively, compared with 24.4% in controls (P
0.05 for both).
DNA from an additional 92 unrelated type 2 diabetic AfricanAmericans with ESRD (singletons, not from the multiplex families) was evaluated for association with NOS3. Markers included the 27 bp VNTR marker (ecNOS4) and the mutation (Glu298Asp) in exon 7 of ecNOS. No evidence for significant association was observed with either marker in this population (data not shown).
Affected sibling pair analyses did not provide evidence for linkage with any of the markers tested in either the total, diabetic or non-diabetic family sets (Table 2
). Results of the exclusion analysis of the data are shown in Table 3
. The EXCLUDE option is designed to evaluate evidence for linkage to a trait (ESRD) and the region of a chromosome on the basis of specified relative risks [30,31]. These calculations provide the investigator with a sense of whether a specific genetic locus can be excluded from contributing a specific relative risk (
) toward expression of a trait (i.e. ESRD). As shown in Table 3
, the EDN-1 and NOS3 markers could both be excluded at a LOD score of -2 as contributing to ESRD in the total family set using a
=1.2. The NOS1 marker could be excluded at a LOD score of -4.86 in the total family set using
=1.5.
We detected two novel alleles for the NOS3 VNTR in intron 4. They differed from the previously observed a (four repeats) and b (five repeats) alleles. We sequenced the PCR products and found that one allele had six repeats of the 27 bp sequence with the PCR product length of 447 bp. This was designated as the c allele (Table 4
). The second was found in only one individual. It was 339 bp long and had only two of the 27 bp repeats. One repeat differed slightly in nucleotide composition from the published 27 bp repeat sequence of Wang et al. [12].
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Discussion
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One approach to assess whether a gene has an aetiologic role in ESRD is to determine whether individual alleles of a locus are associated with ESRD. This association, if present, may be due to linkage disequilibrium or may be implicated in a disease pathway. Linkage disequilibrium or allelic association can signal linkage between a marker and a disease. In this study, we identified consistent and significant evidence for association between alleles 7 and 9 of the NOS1B gene and ESRD. The positive associations were observed both in the probands from all the ESRD sibling pairs and in their unrelated first siblings. The associations between alleles 7 and 9 were also consistent in the 99 non-diabetic probands as well as in their first siblings. However, the 69 type 2 diabetic probands from these sibling pairs lacked the association with alleles 7 and 9 (Table 1
). The significant increase in frequency of these two alleles suggests that NOS1 may play a role in ESRD susceptibility, particularly in non-diabetic ESRD. The relevance of these findings would have been increased had we been able to demonstrate alterations in plasma, urinary, or tissue NO levels based on the allele detected. Unfortunately, our study population had ESRD requiring either haemodialysis, peritoneal dialysis or kidney transplantation and was composed of individuals with variable levels of residual renal function. Therefore, their serum and urine NO levels would have been difficult to interpret. In addition, the association data were analyased in siblings with multiple aetiologies of ESRD. The clustering of disparate aetiologies of ESRD in single families is observed in AfricanAmericans [18,32,33]. This phenomenon suggests the presence of underlying susceptibility to renal failure in families, independent of the inciting injury. However, it may also lead to the unintentional clustering of other disease risk factors in this heterogeneous group of patients.
We have also evaluated polymorphic markers for the EDN-1, NOS1 and NOS3 genes in linkage studies with ESRD in AfricanAmericans. Significant evidence for linkage was not observed with any of these markers in either all-cause ESRD or in the sibling pairs stratified for type 2 diabetic ESRD or non-diabetic aetiologies of ESRD. Multipoint exclusion analysis complemented the affected sibling pair analyses and confirmed that there was no evidence for linkage of these candidate genes to the common aetiologies of ESRD in AfricanAmericans.
Evidence for association in the absence of evidence for linkage presents the researcher with the problem of evaluating the significance of the association data. Association methods are widely considered to be significantly more sensitive (having greater power) than linkage methods in a comparably sized study [34]. Consequently, it would not be surprising to detect association in the absence of evidence for linkage when, as in the case of this study, a gene contributes a relatively minor component of the risk. For this reason, we suggest that the NOS1-associated risk is but one contributor of genetic susceptibility to ESRD.
A second issue to address is whether the results of the NOS1 polymorphism evaluations represent true association. Association studies have been difficult to interpret due to inconsistent results in the evaluation of similar, but rarely identical study populations. Given that association studies such as this may be detecting relatively modest components of risk, it does not seem surprising that different populations will have varying degrees of risk associated with minor genetic determinants. Consequently, trying to compare studies in which the populations are poorly matched may be unproductive. To our knowledge this is the first evaluation of NOS and EDN in AfricanAmericans with ESRD. Additional studies of the AfricanAmerican population will need to be carried out to conclusively determine the contribution of NOS and EDN genes to ESRD in this population.
Importantly, all three isoforms of NOS have been localized to the kidney [6]. Neuronal constitutive NOS gene activity has been localized to the macula densa region in rat and mouse kidney using isoform-specific antibodies and in situ hybridization [35]. Terada et al. [36] localized NOS1 mRNA predominantly to the inner medullary collecting duct in rats, but also in the glomerulus, inner medullary thin limb, collecting duct and renal vasculature. Therefore, the renal localization of neuronal constitutive NOS could explain its association with renal microvascular disease. We are unaware of other linkage or association analyses with the NOS1 gene in ESRD.
The 7 and 9 alleles of the microsatellite marker NOS1B which show evidence of association with ESRD are, in all likelihood, not mutations which themselves contribute to ESRD susceptibility. More likely these alleles are in linkage disequilibrium with other alleles in the NOS1 gene (exons, introns, 5' promoter region, and 3' untranslated region), which might directly confer susceptibility. It is noteworthy that the genomic structure and regulation of expression of the NOS1 gene are complex (reviewed in [37]). Multiple promoters, alternative splicing, cassette insertions/deletions, and multiple sites for cleavage and polyadenylation in the 3' end of the transcript are involved in regulation of the gene expression. It is attractive to speculate that such unparalleled complexity in regulation of expression is vitally important and that dysregulation of expression in the kidney could contribute to disease processes. In the same vein, however, this remarkable complexity presents a considerable barrier to a detailed molecular analysis of the relationship of NOS1 to renal disease.
The a allele of the ecNOS4 polymorphism in the endothelial constitutive NOS3 gene was also associated with ESRD in the 168 ESRD probands and in their first siblings (Table 1
). The a allele was not associated with ESRD in the 92 AfricanAmerican diabetic singletons. The other markers for the NOS3 gene, D7S363 and Glu298Asp, failed to demonstrate consistent evidence for association to ESRD. Zanchi et al. [38] reported that NOS3 gene polymorphisms influence the risk for development of advanced nephropathy in Caucasian type 1 diabetics. The a allele of NOS3 has also been associated with an increased risk of ESRD [13] and coronary artery disease [12] and a missense Glu298Asp variant has been associated with myocardial infarction [24] in Japanese populations. We find it difficult to determine whether the NOS3 gene plays an important role in the genesis of ESRD in AfricanAmericans, based upon the inconsistent results using the three markers we tested for this gene in multiple groups of patients. The association between microvascular (ESRD) and macrovascular (coronary artery atherosclerosis) disease with the a allele in Japanese and AfricanAmerican populations warrants further analysis of its role in arteriosclerosis. Other analyses have revealed associations between polymorphisms in the NOS3 gene and hypertension in Japanese populations utilizing the CA repeat polymorphisms of the NOS3 gene [20] and the Glu298Asp coding variant [22]. An intron 13 repeat polymorphism has also been associated with familial pregnancy-induced hypertension in Scottish and Icelandic women [39]. Two other studies have failed to detect association between NOS3 and hypertensives residing in the US [40] and France [41]. In our opinion, the lack of consistent association or linkage between ESRD and the three NOS3 markers we analysed makes it less likely that polymorphisms in the NOS3 gene contribute substantially to ESRD susceptibility in AfricanAmericans. The associations between the a allele and ESRD could signify the existence of renal disease-modifying loci near the NOS3 gene.
To our knowledge, the NOS2 gene (inducible NOS) has not been evaluated for its role in the genesis of ESRD. This could relate to the lack of high heterozygosity markers for NOS2 and future studies should evaluate the role of NOS2 in ESRD.
Based upon the association of alleles 7 and 9 of NOS1B with ESRD in AfricanAmericans, we feel the NOS1 gene is an attractive candidate for renal disease susceptibility and warrants additional study in other populations. The association of the a allele of NOS3 with ESRD in AfricanAmericans and the Japanese also raise the possibility of NOS3 contributing to ESRD susceptibility. However, the other NOS3 markers did not reveal an association with ESRD in our AfricanAmerican study population. The EDN-1 gene also did not appear to contribute to ESRD susceptibility in our study population.
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Acknowledgments
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The authors thank the physicians, patients and staff of ESRD Network 6 (The Southeastern Kidney Council, Inc.) treatment facilities for their assistance in collecting clinical information and blood samples. We also thank Mrs Kim Hairston for her secretarial assistance. This work was supported, in part, by grants RO1 HL56266 (BIF) and RO1 DK53591 (DWB).
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Notes
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Correspondence and offprint requests to: Barry I. Freedman, MD, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 271571053, USA. 
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Received for publication: 25. 1.00
Revision received 19. 6.00.