Human Molecular Genetics, 2002, Vol. 11, No. 3 229-241
© 2002 Oxford University Press
Modifier effect of ENOS in autosomal dominant polycystic kidney disease
Division of Nephrology and 1Division of Endocrine Surgery, and 2Unit of Pharmacology and Therapeutics, Université catholique de Louvain Medical School, Brussels, Belgium, 3Division of Nephrology, KUL Medical School, Leuven, Belgium, 4Complexo Hospitalario Universitario de Santiago, Santiago de Compostela, Spain, 5Division of Nephrology, Hôpital Necker, Paris, France, 6Department of Urology, University of Tokyo, Japan and 7Department of Biology, University of Mons-Hainaut, Mons, Belgium
Received September 10, 2001; Revised and Accepted November 29, 2001.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
A significant phenotypical variability is observed in autosomal dominant polycystic kidney disease (ADPKD). ADPKD is associated with altered endothelial-dependent vasodilation and decreased vascular production of nitric oxide (NO). Thus, ENOS, the gene coding for the endothelial nitric oxide synthase (eNOS), could have a modifier effect in ADPKD. In order to test this hypothesis, we genotyped 173 unrelated ADPKD patients from Belgium and the north of France for the Glu298Asp, intron 4 VNTR and T-786C polymorphisms of ENOS and looked for their influence on the age at end-stage renal disease (ESRD). In males (n = 93), the Glu298Asp polymorphism was associated with a lower age at ESRD (Glu/Asp + Asp/Asp: 49.0 ± 1.2 years, n = 53; Glu/Glu: 53.5 ± 1.5 years, n = 40; simple regression, P = 0.02; multiple regression, P = 0.006). This effect was confirmed in a subset of males linked to PKD1 and reaching ESRD before age 45, and by a cumulative renal survival analysis in PKD1-linked families. Further studies demonstrated that NO synthase (NOS) activity was decreased in renal artery samples from ADPKD males harbouring the Asp298 allele, in association with post-translational modifications and partial cleavage of eNOS. No significant effect of the other polymorphisms was found in males, and no polymorphism influenced the age at ESRD in females. In conclusion, the frequent Glu298Asp polymorphism of ENOS is associated with a 5 year lower mean age at ESRD in this subset of ADPKD males. This effect could be due to a decreased NOS activity and a partial cleavage of eNOS, leading to a further decrease in the vascular production of NO.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common monogenic hereditary diseases (prevalence: 1:400–1:1000). It is characterized by the development of multiple cysts in both kidneys. These cysts grow slowly over decades, until renal failure occurs. By the age of 60 years, about half the patients with ADPKD have progressed to end-stage renal disease (ESRD). In Europe and North America, ADPKD is responsible for 10% of the patients requiring renal replacement therapy (1).
Genetic and molecular studies have revealed that ADPKD is due to mutations in two major genes, PKD1, responsible for 
85% of cases, and PKD2, which accounts for the vast majority of the remaining cases (1). The proteins encoded by PKD1 and PKD2 (polycystins 1 and 2, respectively) probably interact in the plasma membrane to participate in a signal transduction pathway controlling renal tubular cell maturation (2).
One of the most striking features in ADPKD is the substantial variability in the severity of renal phenotype, primarily assessed by the age at ESRD. This variability is observed among families, family members and even dizygotic twins (3,4). Interfamilial phenotypic variability may be explained by distinct effects of mutations in PKD1 and PKD2 and also by the nature of the mutation itself. Indeed, PKD2 is clinically milder than PKD1 disease, as witnessed by a later age at ESRD and a lower prevalence of hypertension (5). In addition, different mutations in the same gene are associated with differences in the mean age at ESRD in both PKD1 (5) and PKD2 families (6). Intrafamilial phenotypic variability could be explained by at least two mechanisms: the second hit event and the effect of modifier genes. If cyst formation is triggered by a second hit, i.e. a somatic mutation in the allele unaffected by germline mutation (7), micro-environmental or genetic factors determining the rate of second hit could be a valuable explanation. Alternatively, modifier genes could exert either a protective or a deleterious effect on the renal phenotype. The role of modifier genes has been shown in other hereditary diseases, including cystic fibrosis, familial Mediterranean fever or familial hypercholesterolemia (8), and modifier loci have recently been demonstrated in several mouse models of polycystic kidney disease (9,10).
The release of nitric oxide (NO) by endothelial cells plays a critical role in the control of local haemodynamics and systemic blood pressure (11,12). ADPKD is associated with an alteration of the endothelium-dependent vasodilation, that has been attributed to a decreased production of NO by the endothelial NO synthase (eNOS) (13). Furthermore, hypertension occurs before renal impairment in 60% of ADPKD patients, and is a predictor of renal function decline (14). Thus, small changes in NO levels may play a role in the progression of renal disease, and ENOS, the gene encoding eNOS, could be a modifier gene in ADPKD. If this hypothesis proves to be correct, the influence of ENOS in ADPKD would be expected to be more critical in males than in premenopausal women. Indeed, both endothelium-dependent vasodilation (15,16) and total body production of NO (17) are decreased in men, which arguably renders them more sensitive than women to small modifications of NO production.
In order to investigate a potential modifier effect of ENOS in ADPKD, we assessed the influence of the three most studied polymorphisms of ENOS (Glu298Asp, intron 4 VNTR, T-786C) on the age at ESRD in males and females from a large series of unrelated ADPKD patients recruited in Belgium and the north of France. We further analysed the NOS activity and the expression of eNOS at the mRNA and protein levels in renal artery samples from ADPKD patients according to genotype at the Glu298Asp locus.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Characteristics of the ADPKD population studied
A total of 182 unrelated patients at ESRD were recruited in three academic centres in Belgium and France. From 57 potentially informative families, linkage to PKD1 and PKD2 was established in 27 and nine families, respectively. Patients belonging to PKD2 families were excluded from the study population, which therefore consisted of 173 unrelated patients (93 males and 80 females). Mean age at ESRD was not significantly different in the three centres (Saint Luc Academic Hospital, Brussels: 50.5 ± 1.0, n = 102; U.Z. Gasthuisberg, Leuven: 55.0 ± 1.8, n = 35; Necker Hospital, Paris: 51.3 ± 1.6, n = 36, P = 0.1) and was not significantly different in males and females (Table 1).
|
Distribution of ENOS polymorphisms and linkage disequilibrium
The distribution of the three diallelic polymorphisms of ENOS (Fig. 1) is shown in Table 1. For each polymorphism, the observed genotype frequencies did not deviate from the Hardy–Weinberg equilibrium (Glu298Asp:
21df = 0.3, P = 0.7; intron 4 VNTR: 21df = 0.01, P = 1; T-786C: 21df = 0.7, P = 0.3) and were similar to those previously described in Caucasian populations (18). The genotype frequencies were not significantly different according to gender (Table 1) and centre (P = 0.3, 0.1 and 0.9 for the Glu298Asp, intron 4 VNTR and T-786 polymorphisms, respectively). The three polymorphisms were in significant linkage disequilibrium (P < 0.001). The a allele of intron 4 VNTR was strongly associated with the Glu allele of the Glu298Asp polymorphism and the C allele of the T-786C polymorphism. In contrast, the Glu298Asp and T-786C polymorphisms were in only weak to moderate linkage disequilibrium (Table 2).
|
|
Influence of ENOS polymorphisms on the age at ESRD in ADPKD
Male patients with ADPKD harbouring the Asp allele of the Glu298Asp polymorphism had a significant, 4.5 year lower mean age at ESRD than Glu/Glu patients (Table 3). The age at ESRD was increased, though less significantly, in patients harbouring the a allele of intron 4 VNTR, as compared to patients with the bb genotype. The T-786C polymorphism had no effect on the age at ESRD in males (Table 3). The distribution of the Glu298Asp polymorphism was significantly different when ADPKD male patients were distinguished according to age at ESRD, with 50 years of age being the cut-off. Patients harbouring the (Glu/Asp + Asp/Asp) genotypes were significantly overrepresented (67%, 31/46) in the subset of patients reaching ESRD before age 50, compared to patients at ESRD after age 50 (47%, 22/47). In female ADPKD patients, none of the polymorphisms of ENOS had an effect on the age at ESRD (Table 3).
|
The effect of the Glu298Asp polymorphism on the age at ESRD in the male subset was confirmed by multiple regression analysis in models including either intron 4 VNTR (P = 0.025, r2 = 0.08) or T-786C (P = 0.006, r2 = 0.13) polymorphisms. The model including Glu298Asp and T-786C, the two polymorphisms in weak to moderate linkage disequilibrium, explained a higher proportion of the variance (r2) and was thus retained as the best fit model. It yielded a P-value of 0.006 for combined (Glu/Asp + Asp/Asp) versus Glu/Glu genotypes across the three genotypes of T-786C (Table 4). In contrast, multiple regression analysis did not confirm the effect of intron 4 VNTR in models including either of the two other polymorphisms. These findings were confirmed by haplotype analysis for the Glu298Asp and T-786C polymorphisms (P = 0.02, data not shown).
|
Of note, the effect of the Glu298Asp polymorphism on the age at ESRD was mainly due to Glu/Asp patients (48.5 ± 1.3 years, n = 45). The mean age at ESRD of the eight patients harbouring the Asp298 allele at the homozygous state was 52.1 ± 2.9 years. Two of them reached ESRD at age 61 and 67 years: since they belong to non-informative families, it was impossible to rule out PKD2 linkage in these two particular patients. The mean age at ESRD in the remaining six Asp/Asp patients was 48.2 ± 1.7 years, similar to that found in the Glu/Asp subgroup. The mean age at ESRD in the Glu/Glu patients (53.5 ± 1.5 years, n = 40) was similar to that reported previously for PKD1 families in a similar population (6).
The effect of the Glu298Asp polymorphism was also investigated in the subset of male patients belonging to PKD1-linked families. In this subset (n = 17), the Glu/Asp and Asp/Asp genotypes were associated with a 8 year lower age at ESRD (45.8 ± 2.8 versus 54.1 ± 3.1 years, P = 0.067). Because previous studies in similar ethnic groups showed that the prevalence of PKD2 mutations is negligible in ADPKD patients reaching ESRD before the age of 45 years (1,4,6), we performed the same analysis for the subset of male patients linked to PKD1 and those with ESRD before the age of 45 years. In this subset (n = 37), the Glu/Asp and Asp/Asp genotypes were associated with a 6 year lower age at ESRD (42.4 ± 1.3 versus 48.5 ± 2.6 years, P = 0.03). As in the whole population, the effect of the Glu298Asp polymorphism was not observed in the subsets of female patients linked to PKD1. It should be noted that the distribution of the Glu298Asp genotypes was not significantly different in the whole ADPKD population studied and in the subgroups including PKD1-linked patients.
In addition to the analyses primarily performed on unrelated ADPKD patients at ESRD, 38 affected male patients with (n = 19) or without renal failure (n = 19) belonging to the 27 PKD1-linked ADPKD families were included in a Kaplan–Meier cumulative renal survival analysis (Fig. 2). Although performed on a small set of patients, this analysis confirmed that cumulative renal survival was significantly lower in patients harbouring the Glu/Asp + Asp/Asp genotypes [44.8 ± 1.7, 95% CI (41.5; 48.1), n = 19] compared to patients harbouring the Glu/Glu genotype [53.3 ± 2.3, 95% CI (48.8; 57.7), n = 19, P = 0.03] of the Glu298Asp polymorphism.
|
Correlations between Glu298Asp genotype and clinical parameters in ADPKD
Clinical parameters related to renal function and blood pressure were obtained in subsets of ADPKD patients, in an attempt to look for clinical factors underlying the lower age at ESRD observed in patients harbouring the Glu298Asp polymorphism.
The profile of renal function loss was obtained in 22 unrelated ADPKD males (Fig. 3). As in the whole male ADPKD subset, the mean age at ESRD was significantly lower in the (Glu/Asp + Asp/Asp) subgroup (45.1 ± 1.9 years, n = 12) than in the Glu/Glu subgroup (53.1 ± 2.7 years, n = 10; P = 0.02). The 1/creatinine slope was steeper in the former subgroup (–0.0065 versus –0.0049 in the Glu/Glu subgroup).
|
Several parameters assessing indirectly the severity of hypertension were available in a subset of 37 ADPKD patients. As expected, a trend towards increased septum (P = 0.3) and posterior wall thickness (P = 0.1) was observed in males (n = 22) versus females (n = 15). In males, a significantly lower age at ESRD was found again in the (Glu/Asp + Asp/Asp) subgroup (46.7 ± 2.0 years, n = 12 versus 53.7 ± 2.7 years, n = 10; P = 0.04). However, no significant difference regarding indexes of left ventricular hypertrophy, estimated duration of hypertension and number of classes of anti-hypertensive drugs was observed according to genotype (data not shown).
NOS enzymatic activities and expression of eNOS in renal arteries
To substantiate the molecular mechanism underlying the influence of the Glu298Asp polymorphism, we assessed both the NOS enzymatic activity and the expression of ENOS at the mRNA and protein levels in renal arteries from ADPKD patients differing for the Glu298Asp genotype. Because the expression of eNOS is influenced by estrogens (16) and age (19), investigations were conducted on samples matched for gender and age.
A panel of well characterized monoclonal and polyclonal antibodies raised against human eNOS was used to demonstrate that eNOS is the predominant NOS isoform expressed in control and ADPKD renal arteries (Fig. 4A–C). Immunostaining in renal arteries from ADPKD patients with the Glu/Glu genotype confirmed the distribution of eNOS in the endothelium, where it co-localized with factor VIII (Fig. 4D).
|
The L-citrulline assay was used to determine Ca2+-dependent NOS activity, which relies on eNOS and neuronal NOS (nNOS), in renal artery extracts from nine ADPKD patients matched for age (Fig. 5A). This analysis showed that Ca2+-dependent NOS activity was systematically decreased in renal arteries of ADPKD patients harbouring the Glu/Asp genotype (–42%, n = 3) or the Asp/Asp genotype (–63%, n = 4) as compared with two ADPKD patients harbouring the Glu/Glu genotype. Similar results were observed for total NOS activity, reflecting the minimal Ca2+-independent NOS activity in these samples. As expected, Ca2+-dependent NOS activity decreased with age, irrespective of the genotype (data not shown).
|
The renal artery samples that were used for the L-citrulline assay were submitted to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) to investigate the expression of eNOS by immunoblotting, using both monoclonal and polyclonal antibodies against human eNOS. As shown in Figure 5B, the Asp/Asp samples were characterized by a significant decrease in the expression of full-length eNOS at 140 kDa. This was paralleled by a major increase in the amount of an immunoreactive band for eNOS at
70 kDa. The apparition of a similar 70 kDa band in endothelial cell lysates submitted to thawing–freezing cycles suggested that it might correspond to proteolytic cleavage of eNOS. It must be noted that the immunoblot pattern for eNOS in Asp/Asp samples, including the major immunoreactive band for eNOS at 70 kDa, was identical when using the lithium dodecyl sulfate (LDS) sample buffer system (20) designed to limit acidic hydrolysis (Fig. 5B). In contrast with the modified expression of eNOS at the protein level (Fig. 5B), real-time quantitative RT–PCR revealed that ENOS mRNA expression levels were not significantly different in age-matched renal arteries belonging to the different Glu298Asp genotypes (1.08- and 1.12-fold the Glu/Glu artery level in Glu/Asp and Asp/Asp arteries, respectively) (data not shown). The hypothesis of increased proteolytic cleavage of eNOS in renal artery samples harbouring the Asp/Asp genotype was substantiated by immunoprecipitation with polyclonal antibodies directed against the N-terminus or C-terminus domains of human eNOS, followed by immunoblotting with the monoclonal antibody against the C-terminus of eNOS (Fig. 5C). Immunoprecipitation with both antibodies yielded the full-length eNOS (140 kDa) in all samples, whereas a 70 kDa fragment containing the C-terminus of eNOS was identified in the Asp/Asp sample only. Immunoprecipitation with anti-caveolin antibodies yielded full-length eNOS in both samples, whereas no signal was observed when the precipitation was performed with non-immune rabbit IgG or in absence of primary antibody (beads only) (Fig. 5C).
The expression of eNOS was further assessed by immunostaining in renal arteries from two ADPKD males harbouring the Asp/Asp genotype (Fig. 6). In contrast with the staining observed in Glu/Glu arteries (Fig. 4D) and the preserved expression of endothelial factor VIII (Fig. 6A, C and E), endothelial staining for eNOS was reduced in renal arteries (B) and not detected in intra-parietal vasa vasorum (D and F).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
We have investigated the effect of the three most characterized polymorphisms of ENOS on the age at ESRD in a large series of unrelated ADPKD patients. No effect on the age at ESRD was found for the intron 4 and T-786C polymorphisms. In contrast, the Glu298Asp polymorphism was associated with a significantly lower age at ESRD in ADPKD males and was over-represented in the subset of ADPKD males reaching ESRD before age 50 years. The effect of the Glu298Asp polymorphism in males with ADPKD was confirmed in subgroups of unrelated patients linked to PKD1, as well as by a renal survival analysis in PKD1-linked families.
The Glu298Asp polymorphism of ENOS has already been associated with hypertension (21), myocardial infarction (22) and carotid atherosclerosis (23), three conditions characterized by endothelial dysfunction. Our data support an influence of the Glu298Asp polymorphism on the progression of ADPKD, another disease associated with endothelial dysfunction (13). The production of NO through eNOS is regulated by estrogens (24,25), which might explain a gender sensitivity to NO (16) and our observation that the effect of the Glu298Asp polymorphism was restricted to ADPKD males. These data are also consistent with the observation that, in the Han:SPRD rat model of ADPKD (26), NOS inhibition with L-NAME had a deleterious effect on the progression of renal disease only in males (27).
In order to find a molecular basis for the lower age at ESRD observed in patients harbouring the Glu298Asp polymorphism, we investigated activity and expression of eNOS in renal arteries of ADPKD males according to the genotype at the Glu298Asp locus (Fig. 5). Contrasting with a similar expression of ENOS at the mRNA level, the Ca2+-dependent NOS activity was systematically lower in patients with the Glu/Asp or Asp/Asp genotype. Furthermore, the Asp/Asp samples were characterized on immunoblot by a decrease in full-length eNOS and a parallel increase in a smaller immunoreactive band at 70 kDa, which was identified as a proteolytic fragment of eNOS by immunoprecipitation. The modified expression of eNOS in these samples was confirmed by immunostaining (Fig. 6), with a reduced endothelial staining in renal arteries and a lack of signal in the endothelium lining vasa vasorum.
These data confirm the in vitro demonstration of increased degradation of eNOS in patients harbouring the Asp298 polymorphism (28). The mechanism of this degradation remains to be elucidated. The glutamic acid at position 298 of eNOS is conserved among species. Recent structural data suggest that the residue at 298 is located between the ß-strands 8 and 9 of eNOS, and is thus fully solvent-exposed, which could make it more sensitive to proteolysis (29). Alternatively, the Asp298 residue could increase the sensitivity of eNOS to acidic hydrolysis as it leads to the formation of an Asp298–Pro299 motif, known to sensitize proteins to acidic hydrolysis (20). However, the latter hypothesis appears unlikely, as our immunoblot results were similar when using the LDS-electrophoresis sample buffer to maintain a neutral pH throughout the experiment (Fig. 5). Taken together, these results suggest that lower enzymatic activity and/or partial cleavage of eNOS could be responsible for a decreased NO release and increased endothelial dysfunction in patients harbouring the Asp298 allele. In the absence of clinical arguments suggesting increased systemic blood pressure, one might postulate that the accelerated renal function degradation observed in these patients could be mediated by a local effect on renal microcirculation.
The two other polymorphisms of ENOS tested in this study, intron 4 VNTR and T-786C, do not appear to influence the renal phenotype of ADPKD. The finding of an increased age at ESRD in ADPKD males harbouring the a allele of intron 4 VNTR, previously associated with hypertension (30) and coronary artery disease (31), was not confirmed by multiple regression analysis. It probably reflects linkage disequilibrium with the Glu298 allele (Table 2). We found no effect of the T-786C promoter polymorphism, which has been associated with a modest (40%) decrease in promoter activity and decreased plasma NO metabolites in some studies (32) but not in others (33). It must be noted that the mouse equivalent of ENOS is located on chromosome 5, thus outside the modifier loci identified in murine models of polycystic kidney disease (9,10). However, these models are all recessive and thus are not a faithful equivalent of human ADPKD.
Only a few candidate genes likely to influence the progression of human ADPKD have been investigated thus far (34). In vitro data have suggested that CFTR channel mediates fluid secretion in ADPKD cysts (35) and an apparent protective effect of rare CF mutations on renal decline in ADPKD has been reported (36). However, we were unable to confirm that the most common 
F508 mutation of CF affects the progression of ADPKD (37). Similarly, initial results suggesting an effect of the ACE I/D polymorphism in ADPKD (38) have been disputed and now appear unlikely (39,40). Such conflicting results might be, at least in part, explained by the lack of power due to the small number of patients included and/or the inclusion of patients belonging to a small number of large PKD1 families (34). This last strategy could indeed lead to spurious results due to under- or over-representation of a given polymorphism in a few large families (41).
In order to overcome these potential biases, we primarily included a large number of unrelated patients, even from uninformative families in terms of linkage to PKD1 or PKD2. Nevertheless, linkage analysis was performed in all informative families, in order to exclude PKD2 families. The latter accounted for 15% of the families tested, in agreement with the expected prevalence in our population (6). Whereas an over-representation of PKD2 patients in the Glu/Glu subset could still contribute to a higher age at ESRD, this hypothesis appears unlikely, as the mean age of the Glu/Glu group (53.5 years) is similar to that found in PKD1-linked patients (6). Furthermore, the effect of the Glu298Asp polymorphism is maintained when examining subsets of ADPKD males linked to PKD1 and those reaching ESRD before the age of 45 years. Previous studies in a similar population have indeed shown that the prevalence of PKD2 mutations is negligible in the latter (6,34,38). Finally, a Kaplan–Meier analysis performed on all affected males belonging to our PKD1-linked families confirmed that cumulative renal survival was significantly lower in patients harbouring the Asp allele of the Glu298Asp polymorphism.
It must be emphasized that the association of the Glu298Asp polymorphism with a faster renal decline in ADPKD males cannot be extrapolated without further studies in other populations with different genetic backgrounds. This is particularly true if the Glu298Asp polymorphism is not the causal polymorphism, but only a marker in linkage disequilibrium with the modifier locus. In particular, it would be worthwhile to look for the influence of ENOS in ADPKD patients from Japan, in which the Asp298 allele is significantly less frequent than in Caucasians (allelic frequency: 14 versus 31%, P < 0.001) and is not in linkage disequilibrium with the intron 4 VNTR (A.Persu and S.Horie, unpublished data).
In conclusion, in male patients from a large ADPKD subset from Belgium and the north of France, the Glu298Asp polymorphism of ENOS is associated with a 5 year lower mean age at ESRD. The NOS activity is significantly decreased in renal artery samples from ADPKD males harbouring the Asp298 allele, in association with post-translational modifications and a partial cleavage of eNOS. It is tempting to speculate that the resulting decrease in NO production may enhance the endothelial dysfunction associated with ADPKD, leading to alteration of intrarenal and/or systemic haemodynamics. This in turn will result in a faster decline in renal function. If the effect of the Glu298Asp polymorphism is confirmed in independent populations, ENOS would appear as the first modifier gene in ADPKD.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Study population
Patients were recruited from September 1998 to September 2000 in Saint-Luc Academic Hospital, Brussels (Belgium), U.Z. Gasthuisberg, Leuven (Belgium) and Necker Hospital, Paris (France). All Caucasian patients affected with ADPKD on renal replacement therapy (dialysis or renal transplantation) in the three centres during the recruitment period were included. The diagnosis of ADPKD was established on the basis of bilateral enlarged cystic kidneys and a family history suggestive of autosomal dominant inheritance (1). The age at ESRD was defined as the age at starting renal replacement therapy (creatinine clearance 10 ml/min). All patients recruited in this first phase were unrelated. A detailed follow-up for at least 2 years before ESRD was available for all patients included.
Linkage analysis for PKD1 and PKD2 was performed in informative families, whenever possible, using microsatellites SM7, AC2.5 and KG8 for PKD1 (42) and D4S1534, D4S231, D4S414, and D4S1563 for PKD2 (43). LOD scores were calculated by the Mlink program (Columbia University, New York, NY). Data available from the linked markers were combined by means of a Bayesian weighting formula to estimate the likelihood that a family shows linkage to PKD1 or PKD2. The prevalence estimates used for PKD1, PKD2 and a hypothetical PKD3 gene were 0.84, 0.15 and 0.01, respectively (5,6). In order to perform a Kaplan–Meier renal survival analysis, we subsequently recruited all affected males (according to the diagnostic criteria for ADPKD defined above) within the families linked to PKD1.
The use of DNA and renal artery samples was approved by the Ethical Review Board of each centre involved, and informed consent was obtained from all patients included.
DNA extraction and genotyping for ENOS polymorphisms
DNA was extracted from peripheral blood samples (Gentra, Minneapolis, MN). PCR for ENOS polymorphisms was carried out in a 20 µl volume with 100 ng of genomic DNA, 10 pM of each primer, 1.25 mM dNTP (Roche Diagnostics, Mannheim, Germany), 1 U Taq polymerase (Roche) and 2 µl of 10x buffer containing 15 mM MgCl2 (Roche). Genotyping for the intron 4 VNTR was performed using primers and conditions described previously (31,44). Genotyping for Glu298Asp was performed by PCR, followed by BanII (Life Technologies, Carlsbad, CA) digestion, and additional control using MboI digestion, as described (21). Genotyping for T-786C was obtained by PCR followed by allele-specific oligonucleotide hybridization (ASO), as described earlier (45). Three patients found to harbour the three possible genotypes (TT, CT and CC) by sequencing were used as positive controls. The localization of the three polymorphisms in ENOS and representative gels or ASO are shown in Figure 1.
Statistical analysis
All data were analysed using the SPSS statistical software (version 10.0; SPSS, Chicago, IL), and P-values <0.05 were considered as significant. The ages at ESRD were normally distributed in males and females, and compared by two-tailed Student’s t-test. For each polymorphism, allele frequencies were calculated from the genotype. Allele and genotype distribution in male and female subsets were compared by chi-square (2) test. As functional changes induced at the protein/mRNA level are observed whether the polymorphisms were present at the homozygous or the heterozygous state (28,32,46), statistical analysis was performed by combining heterozygotes and homozygotes rare allele carriers.
Hardy–Weinberg equilibrium was tested by a 2 test. Pairwise linkage disequilibrium between the different polymorphisms was tested and the linkage coefficient D' was obtained as described. D' is the fraction of maximum linkage that could occur between two loci, given the allelic frequencies. The sign added in front of D' is positive if the less frequent alleles at both loci are preferentially associated and negative if the less frequent allele at one locus is associated with the most frequent allele at the other locus (47).
Simple linear regression analysis was used to assess the individual effect of each ENOS polymorphism (independent variable) on age at ESRD (dependent variable). Multiple linear regression analysis was performed to investigate the joint effects of ENOS polymorphisms on the age at ESRD; variables that significantly influenced the age at ESRD were selected by a forward stepwise procedure. The increase in the model r2 was used to explain the proportion of the variance added by each polymorphism, and allowed us to select the model which explains the highest proportion of the variance. To deal with the categorical nature of a polymorphism, dummy variables were generated for each polymorphism.
Haplotype analysis was performed by analysis of variance for the Glu298Asp and T-786C polymorphisms (48). In the absence of the parental genotypes, four haplotypes (H1–H4) were constructed as follows: H1 (Glu in T background) = Glu/Glu–TT genotype; H2 (Asp in T background) = Glu/Asp–TT and Asp/Asp–TT genotypes; H3 (Glu in C background) = Glu/Glu–CT and Glu/Glu–CC genotypes; and H4 (Asp in C background) = Glu/Asp–CT, Asp/Asp–CT, Glu/Asp–CC and Asp/Asp–CC genotypes. The H1 haplotype (genotype Glu/Glu–TT) was used as the reference haplotype (48).
Differences in genotype distribution between patients having reached ESRD before or after 50 years of age were tested by 2 test. Cumulative renal survival analysis in males belonging to PKD1-linked families was performed using the Kaplan–Meier method, and the log-rank test was used to compare renal survival according to the genotype at the Glu298Asp locus. The influence of the genotype on echocardiographic parameters (either normally distributed, or becoming normal after log-transformation) was tested using two-tailed Student’s t-test.
Continuous variables were expressed as mean ± SEM.
Clinical parameters
The rate of decline in renal function loss over time was indicated by the 1/plasma creatinine slope for individual patients. Patients were included in the analysis only if at least five determinations of plasma creatinine were available before ESRD, and the 1/plasma creatinine slope was obtained by linear regression. Septum and posterior wall thickness, as well as telesystolic and telediastolic diameters, were measured by M-Mode echocardiography (49), performed at the time of initiation of dialysis in ADPKD patients (Acuson Sequoia, Moutain View, CA). Classes of antihypertensive therapy included diuretics, ß-blockers, calcium antagonists, ACE inhibitors and central acting agents.
Cell and tissue samples and processing
Renal artery samples from nephrectomy specimens were obtained in 12 ADPKD males at the time of transplantation, and in four controls (two males, two females) at the time of surgery for renal cell carcinoma. Part of each sample was fixed for 6 h at 4°C in 4% paraformaldehyde in 0.1 M phosphate buffer, rinsed, and embedded in paraffin (37). Most of the sample was used for protein extraction (for NOS assay and immunoblot), as previously described by Combet et al. (50). Lysates from bovine aortic endothelial cells (BAEC) were obtained from Transduction Laboratories (Lexington, KY). The protein concentrations were determined using the Bradford method (Bio-Rad, Melville, NY) with BSA as standard.
Antibodies
NOS isoforms were detected with mouse monoclonal antibodies against human eNOS (C-terminus) and nNOS, and mouse inducible NOS (iNOS) (Transduction Laboratories) and affinity-purified rabbit polyclonal antibodies against the N-terminus (Santa Cruz Biotechnology, Santa Cruz, CA) or C-terminus (Transduction Laboratories) of human eNOS (50). Both the monoclonal (C-terminus) and polyclonal (N-terminus) antibodies were used for detecting eNOS by immunoblotting. Additional antibodies included rabbit antibodies against human Factor VIII (Dako, Glostrup, Denmark); monoclonal anti-ß-actin (Sigma, St Louis, MO) and rabbit polyclonal anti-human caveolin 1 (Transduction Laboratories). Peroxidase-labeled IgG were from Dako and avidin–biotin peroxidase kits from Vector (Burlingame, CA).
Immunohistochemistry
The reactivity for eNOS and Factor VIII in renal arteries from ADPKD patients was investigated using immunoperoxidase, as described by Persu et al. (37). Following incubation in 0.3% H2O2 (30 min) and 10% normal serum in PBS (20 min) at room temperature, rehydrated paraffin sections were incubated successively with primary antibodies, biotinylated goat anti-rabbit or horse anti-mouse IgG, avidin–biotin peroxidase complex, and after washing, aminoethylcarbazole (Vector). Sections were examined under a Leica DMR coupled to a Leica MPS60 photomicrographic system (Leica, Heerbrugg, Switzerland). The specificity of the immunolabeling was confirmed by incubation without primary or secondary antibody, or with non-immune rabbit or mouse IgG (Vector).
Measurement of NOS activity
NOS activity was assayed in renal artery samples by the conversion of L-[3H]arginine (Amersham, Little Chalfont, UK) to L-[3H]citrulline as previously described by Combet et al. (50). The NOS activity (pmol citrulline produced/mg protein/min) was calculated from the counts obtained with and without 1 mM L-NMMA. Assays were performed with 1 mM CaCl2 and, alternatively, without Ca2+ (0 mM CaCl2, 2 mM EGTA, 2 mM EDTA) to measure total versus Ca2+-independent NOS activities and calculate Ca2+-dependent NOS activity. Both inter-assay and intra-assay variability were <10%. All determinations were performed in duplicate in the samples obtained from nine ADPKD patients.
Immunoblotting
SDS–PAGE and immunoblotting were performed as described previously (20,50), using either the Laemmli loading buffer or the LDS sample buffer. Cell lysates and renal artery samples prepared as described above were separated by SDS–PAGE and transferred to nitrocellulose. The membranes were blocked for 30 min at room temperature, and incubated overnight at 4°C with the primary antibody. After washing, membranes were incubated for 1 h at room temperature with peroxidase-labeled secondary antibodies (1:5000 dilution), and immunoblots were visualized with enhanced chemiluminescence (Amersham). The specificity of the bands corresponding to eNOS was verified by incubation (i) with non-immune rabbit or mouse IgG (Vector) and (ii) with affinity-purified anti-eNOS antibodies pre-adsorbed with a 5-fold excess of the cognate versus an unrelated peptide in 100 µl of PBS for 2 h at room temperature. All immunoblots were performed at least in duplicate.
Immunoprecipitation
Samples from ADPKD renal arteries were homogenized in ice-cold lysis buffer (20 mM Tris–HCl pH 7.4, 2.5 mM EDTA, 0.5% Triton X-100, 1% sodium deoxycholate, 100 mM NaCl, 10 mM NaF, 1 mM Na3VO4, 20 mM molybdate) containing CompleteR protease inhibitors (Roche Diagnostics, Brussels, Belgium), followed by a brief sonication. Insoluble material was removed by centrifugation (12 000 g, 10 min) at 4°C. Equal amounts (1 mg total protein) were incubated either with polyclonal anti-eNOS, anti-caveolin1 antibodies or non-immune rabbit IgG (Vector) for 2 h at 4°C. After centrifugation (12 000 g, 10 min), protein G–Sepharose (Zymed Laboratories, San Francisco, CA) was added to the supernatant (50 µl per tube) and incubated overnight at 4°C. The immune complexes were washed four times and boiled in SDS–PAGE sample buffer for 5 min before immublotting as described above.
Real-time quantitative PCR analyses for ENOS
Total RNA was extracted from five renal arteries from ADPKD males matched for age (Glu/Glu, n = 2; Glu/Asp, n = 2; Asp/Asp, n = 1) using TRIzol reagent (Life Technologies, Rockville, MD). cDNA was synthesized from total RNA using random hexamers and Superscript First-strand reverse transcriptase (Invitrogen, Groningen, The Netherlands). Sequences of matching primer pairs and TaqMan probes were selected using the Primer Express software (PE Applied Biosystems, Foster City, CA); probes were labelled with a reporter dye (FAM) at their 5' ends and a quencher dye (TAMRA) at their 3' ends. The gene-specific primers were as follows: ENOS SENS, 5'-cgcagcgccgtgaag-3'; ENOS ANTISENS, 5'-accacgtcatactcatccatacac-3'; ENOS TaqMan probe, 5'-FAM-cctcgctcatgggcacggtg-TAMRA-3'; GAPDH SENS, 5'-cctgttcgacagtcagccg-3'; GAPDH ANTISENS, 5'-cgaccaaatccgttgactcc-3'; GAPDH TaqMan probe, 5'-FAM-agccacatcgctcagacaccatgg-TAMRA-3'. Real-time quantitative PCR analyses were performed in duplicate as previously described by Moniotte et al. (51) with 250 nM TaqMan probe and 300 nM of both sense and antisense primers in a final volume of 50 µl using the TaqMan Universal PCR Master Mix (Applied Biosystems). PCR conditions were 50°C for 2 min, 95°C for 10 min followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. Fluorescence data were obtained and analysed with the Abi PRISM 5700 Sequence Detection System instrument (PE Applied Biosystems). To exclude amplification from contaminating genomic DNA, samples of RNA that had not been reverse-transcribed were run in parallel PCR reactions: these controls always remained negative. Results were expressed as Ct (number of cycles needed to generate a fluorescent signal above a predefined threshold). Relative quantitation for a given gene, expressed as fold-variation over control, was calculated using the 2-Ct formula after normalization to GAPDH (Ct) and determination of the difference in Ct (Ct) between control and polymorphism-bearing arteries.
|
ACKNOWLEDGEMENTS |
|---|
We are indebted to the ADPKD patients, their families, and the nurses and staff of the nephrology units involved. We would like to thank Professors X.Jeunemaitre, J.-P.Grünfeld, M.Vikkula and M.Jadoul for fruitful discussions, and F.Meunier for statistical advice. We are grateful to Drs J.Bagon, J.-L.Christophe, Ch.Cuvelier, J.-J.Lafontaine, G.Loute, F.Reginster, J.Vanwalleghem and the members of the Leuvense Samenwerking Groep voor Transplantatie for information and DNA samples. Professor J.-P.Squifflet, and Drs J.Malaise and B.Tombal provided renal artery samples. Dr P.Moulin performed staining for Factor VIII. We also thank Dr M.-T.Vo Cong for her contribution in patient recruitment and database completion. We are grateful to Y.Cnops, H.Debaix, L.Wenderickx and A.Herelixka for excellent technical assistance. R.W. is a Research Associate from the FNRS. This work was supported by the Communauté Française de Belgique, the Société de Néphrologie, the Belgian agencies FNRS and FRSM, the Action de Recherches Concertées 00/05-260, the Xunta de Galicia (XUGA 90201B98) and Ministerio de Ciencia y Tecnología (PM98-0028).
|
FOOTNOTES |
|---|
+ To whom correspondence should be addressed. Tel: +32 2 764 18 55; Fax: +32 2 764 54 55; Email: devuyst@nefr.ucl.ac.be
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
1 Pirson,Y., Chauveau,D. and Grünfeld,J.P. (1998) Autosomal dominant polycystic kidney disease. In Cameron,J.S., Davison,A.M., Grünfeld,J.P., Kerr,D.N.S. and Ritz,E. (eds), Oxford Textbook of Clinical Nephrology. Oxford University Press, Oxford, UK, pp. 2393–2415.
2 Harris,P.C. (1999) Autosomal dominant polycystic kidney disease: clues to pathogenesis. Hum. Mol. Genet., 10, 1861–1866.
3 Milutinovic,J., Rust,P.F., Fialkow,P.H., Agodoa,L.Y., Phillips,L.A., Rudd,T.G. and Sutherland,S. (1992) Intrafamilial phenotypic expression of autosomal dominant polycystic kidney disease. Am. J. Kidney Dis., 19, 465–472.[Web of Science][Medline]
4 Torra,R., Darnell,A., Estivill,X., Botey,A. and Revert,L. (1995) Interfamilial and intrafamilial variability of clinical expression in ADPKD. Contrib. Nephrol., 115, 97–101.[Medline]
5 Hateboer,N., Lazarou,L.P., Williams,A.J., Holmans,P. and Ravine,D. (1999) Familial phenotype differences in PKD1. Kidney Int., 56, 34–40.[Web of Science][Medline]
6 Hateboer,N., van Dijk,M.A., Bogdanova,N., Coto E, Saggar-Malik,A.K., San Millan,J., Torra,R., Breuning,M. and Ravine,D. for the European PKD1–PKD2 Study Group (1999) Comparison of phenotypes of polycystic kidney disease types 1 and 2. Lancet, 353, 103–107.[Web of Science][Medline]
7 Qian,F., Watnick,T.J., Onuchic,L.F. and Germino,G.G. (1996) The molecular basis of focal cyst formation in human autosomal dominant polycystic kidney disease type I. Cell, 87, 979–987.[Web of Science][Medline]
8 Nadeau,J.H. (2001) Modifier genes in mice and humans. Nat. Rev. Genet., 2, 165–174.[Web of Science][Medline]
9 Woo,D.D., Nguyen,D.K., Khatibi,N. and Olsen,P. (1997) Genetic identification of two major modifier loci of polycystic kidney disease progression in pcy mice. J. Clin. Invest., 100, 1934–1940.[Web of Science][Medline]
10 Upadhya,P., Churchill,G., Birkenmeier,E.H., Barker,J.A. and Frankel,W.N. (1999) Genetic modifiers of polycystic kidney disease in intersubspecific KAT2J mutants. Genomics, 58, 129–137.[Web of Science][Medline]
11 Vallance,P., Collier,J. and Moncada,S. (1989) Effects of endothelium-derived nitric oxide on peripheral arteriolar tone in man. Lancet, 2, 997–1000.[Web of Science][Medline]
12 Forte,P., Copland,M., Smith,L.M., Milne,E., Sutherland,J. and Benjamin,N. (1997) Basal nitric oxide synthesis in essential hypertension. Lancet, 349, 837–842.[Web of Science][Medline]
13
Wang,D., Iversen,J. and Strandgaard,S. (2000) Endothelium-dependent relaxation of small resistance vessels is impaired in patients with autosomal dominant polycystic kidney disease. J. Am. Soc. Nephrol., 11, 1371–1376.
14
Tevfik,E. and Schrier,R.W. (2001) Hypertension in autosomal dominant polycystic kidney disease: early occurrence and unique aspects. J. Am. Soc. Nephrol., 12, 194–200.
15 Celermajer,D.S., Sorensen,K.E., Bull,C.M., Robinson,J. and Deanfield,J.E (1994) Endothelium dependent dilation in the systemic arteries of asymptomatic subjects relates to coronary risk factors and their interaction. J. Am. Coll. Cardiol., 24, 468–474.
16
Majmudar,N.G., Robson,S.C. and Ford,G.A. (2000) Effects of the menopause, gender and estrogen replacement therapy on vascular nitric oxide activity. J. Clin. Endocrinol. Metab., 85, 1577–1583.
17
Forte,P., Kneale,B.J., Milne,E., Chowienczyk,P.J., Johnston,A., Benjamin,N. and Ritter,J.M. (1998) Evidence for a difference in NO biosynthesis between healthy women and men. Hypertension, 32, 730–734.
18 Wang,X.L. and Wang,J. (2000) Endothelial nitric oxide synthase gene sequence variations and vascular disease. Mol. Genet. Metab., 70, 241–251.[Web of Science][Medline]
19
Barton,M., Cosentino,F., Brandes,R.P., Moreau,P., Shaw,S. and Luscher,T.F. (1997) Anatomic heterogeneity of vascular aging: role of nitric oxide and endothelin. Hypertension, 30, 817–824.
20
Fairchild,T.A., Fulton,D., Fontana,J.T., Gratton,J.P., McCabe,T.J. and Sessa,W.C. (2001) Acidic hydrolysis as a mechanism for the cleavage of the Glu298Asp-variant of human endothelial nitric oxide synthase. J. Biol. Chem., 276, 26674–26679.
21
Miyamoto,Y., Saito,Y., Kajiyama,N., Yoshimura,M., Shimasaki,Y., Nakayama,M., Kamitani,S., Harada,M., Ishikawa,M., Kuwahara,K. et al. (1998) Endothelial nitric oxide synthase gene is positively associated with essential hypertension. Hypertension, 32, 3–8.
22
Hibi,K., Ishigami,T., Tamura,K., Mizushima,S., Nyui,N., Fujita,T., Ochiai,H., Kosuge,M., Watanabe,Y., Yoshii,Y. et al. (1998) Endothelial nitric oxide synthase gene polymorphism and acute myocardial infarction. Hypertension, 32, 521–526.
23
Lembo,G., De Luca,N., Battagli,C., Iovino,G., Aretini,A., Musicco,M., Frati,G., Pompeo,F., Vecchione,C. and Trimarco,B. (2001) A common variant of endothelial nitric oxide synthase (Glu298Asp) is an independent risk factor for carotid atherosclerosis. Stroke, 32, 735–740.
24 Goetz,R.M., Morano,I., Calvino,T., Studer R. and Holtz,J. (1994) Increased expression of eNOS in rat aorta during pregnancy. Biochem. Biophys. Res. Commun., 91, 5212–5216.
25
Chambliss,K.L., Yuhanna,I.S., Mineo,C., Liu,P., German,Z., Sherman,T.S., Mendelsohn,M.E., Anderson,R.G.W. and Shaul,P.W. (2000) Estrogen receptor 
and eNOS are organized into a functional module in caveolae. Circ. Res., 87, E44–E52.
26 Wang,D., Iversen,J. and Strandgaard,S. (1999) Contractility and endothelium-dependent relaxation of resistance vessels in polycystic kidney disease rats. J. Vasc. Res., 36, 502–509.[Web of Science][Medline]
27 Yoshida,I., Bengal,R. and Torres,V.E. (2000) Gender-dependent effect of L-NAME on polycystic kidney disease in Han:SPRD rats. Am. J. Kidney Dis., 35, 930–936.[Web of Science][Medline]
28
Tesauro,M., Thompson,W.C., Rogliani,P., Qi,L., Chaudhary,P.P. and Moss,J. (2000) Intracellular processing of endothelial nitric oxide synthase isoforms associated with differences in severity of cardiopulmonary diseases: cleavage of proteins with aspartate vs. glutamate at position 298. Proc. Natl Acad. Sci. USA, 97, 2832–2835.
29 Fischmann,T.O., Hruza,A., Niu,X.D., Fossetta,J.D., Lunn,C.A., Dolphin,E., Prongay,A.J., Reichert,P., Lundell,D.J., Narula,S.K. and Weber,P.C. (1999) Structural characterization of nitric oxide synthase isoforms reveals striking active-site conservation. Nat. Struct. Biol., 6, 233–242.[Web of Science][Medline]
30 Uwabo,J., Soma,M., Nakayama,T. and Kanmatsuse,K. (1998) Association of a variable number of tandem repeats in the endothelial constitutive nitric oxide synthase gene with essential hypertension in Japanese. Am. J. Hypertension, 11, 125–128.[Web of Science][Medline]
31 Wang,X.L., Sim,A.S., Badenhop,R.F., McCredie,R.M. and Wilcken,D.E. (1996) A smoking-dependent risk of coronary artery disease associated with a polymorphism of the endothelial nitric oxide synthase gene. Nat. Med., 2, 41–45.[Web of Science][Medline]
32
Miyamoto,Y., Saito,Y., Nakayama,M., Shimasaki,Y., Yoshimura,T., Yoshimura,M., Harada,M., Kajiyama,N., Kishimoto,I., Kuwahara,K. et al. (2000) Replication protein A1 reduces transcription of the endothelial nitric oxide synthase gene containing a –786T
C mutation associated with coronary spastic angina. Hum. Mol. Genet., 9, 2629–2637.
33 Sim,A.S., Wang,J., Wilcken,D. and Wang,X.L. (1998) MspI polymorphism in the promoter of the human endothelial constitutive NO synthase gene in Australian Caucasian population. Mol. Genet. Metab., 65, 62.[Web of Science][Medline]
34 Peters,D.J. and Breuning,M.H. (2001) Autosomal dominant polycystic kidney disease: modification of disease progression. Lancet, 358, 1439–1444.[Web of Science][Medline]
35
Hanaoka,K., Devuyst,O., Schwiebert,E.M., Wilson,P.D. and Guggino,W.B. (1996) A role for CFTR in human autosomal dominant polycystic kidney disease. Am. J. Physiol., 270, C389–C399.
36 O’Sullivan,D.A., Torres,V.E., Gabow,P.A., Thibodeau,S.N., King,B.F. and Bergstralh,E.J. (1998) Cystic fibrosis and the phenotypic expression of autosomal dominant polycystic kidney disease. Am. J. Kidney Dis., 32, 976–983.[Web of Science][Medline]
37
Persu,A., Devuyst,O., Lannoy,N., Materne,R., Brosnahan,G., Gabow,P.A., Pirson,Y. and Verellen-Dumoulin,C. (2000) CF gene and cystic fibrosis transmembrane conductance regulator expression in autosomal dominant polycystic kidney disease. J. Am. Soc. Nephrol., 11, 2285–2296.
38 Baboolal,K., Ravine,D., Daniels,J., Williams,N., Holmans,P., Coles,G.A. and Williams,J.D. (1997) Association of the angiotensin I converting enzyme gene deletion polymorphism with early onset of ESRF in PKD1 adult polycystic kidney disease. Kidney Int., 52, 607–613.[Web of Science][Medline]
39 Perez-Oller,L., Torra,R., Badenas,C., Mila,M. and Darnell,A. (1999) Influence of the ACE gene polymorphism in the progression of renal failure in autosomal dominant polycystic kidney disease. Am. J. Kidney Dis., 34, 273–278.[Web of Science][Medline]
40
van Dijk,M.A., Breuning,M.H., Peters,D.J. and Chang,P.C. (2000) The ACE insertion/deletion polymorphism has no influence on progression of renal function loss in autosomal dominant polycystic kidney disease. Nephrol. Dial. Transplant., 15, 836–839.
41 Houlston,R.S. and Tomlinson,I.P.M. (1998) Modifier genes in humans: strategies for identification. Eur. J. Hum. Genet., 6, 80–88.[Web of Science][Medline]
42 Peral,B., Ward,C.J., San Millan,J.L., Thomas,S., Stallings,R.L., Moreno,F. and Harris,P.C. (1994) Evidence of linkage disequilibrium in the Spanish polycystic kidney disease I population. Am. J. Hum. Genet., 54, 899–908.[Web of Science][Medline]
43 Torra,R., Badenas,C., Darnell,A., Nicolau,C., Volpini,V., Revert,L. and Estivill,X. (1996) Linkage, clinical features and prognosis of autosomal dominant polycystic kidney disease types 1and 2. J. Am. Soc. Nephrol., 7, 2142–2151.[Abstract]
44
Wang,Y., Kikuchi,S., Suzuki,H., Nagase,S. and Koyama,A. (1999) Endothelial nitric oxide synthase gene polymorphism in intron 4 affects the progression of renal failure in non-diabetic renal diseases. Nephrol. Dial. Transplant., 14, 2898–2902.
45 Zanchi,A., Moczulski,D.K., Hanna,L.S., Wantman,M., Warram,J.H. and Krolewski,A.S. (2000) Risk of advanced diabetic nephropathy in type 1 diabetes is associated with endothelial nitric oxide synthase gene polymorphism. Kidney Int., 57, 405–413.[Web of Science][Medline]
46 Wang,X.L., Sim,A.S., Wang,M.X., Murrell,G.A., Trudinger,B. and Wang,J. (2000) Genotype-dependent and cigarette specific effects on endothelial nitric oxide synthase gene expression and enzyme activity. FEBS Lett., 471, 45–50.[Web of Science][Medline]
47 Thompson,E.A., Deeb,S., Walker,D. and Motulsky,A.G. (1988) The detection of linkage disequilibrium between closely linked markers: RFLPs at the AI-CIII apolipoprotein genes. Am. J. Hum. Genet., 42, 113–124.[Web of Science][Medline]
48 Razzaghi,H., Aston,C.E, Hamman,R.F. and Kamboh,I.M. (2000) Genetic screening of the lipoprotein lipase gene for mutations associated with high triglyceride/low-HDL cholesterol levels. Hum. Genet., 107, 257–267.[Web of Science][Medline]
49
Sahn,D.J., DeMaria,A., Kisslo,J. and Weyman,A. (1978) Recommendations regarding quantitation in M-mode echocardiography: results of a survey of echocardiographic measurements. Circulation, 58, 1072–1083.
50 Combet,S., Balligand,J.L., Lameire,N., Goffin,E. and Devuyst,O. (2000) A specific method for measurement of nitric oxide synthase enzymatic activity in peritoneal biopsies. Kidney Int., 57, 332–338.[Web of Science][Medline]
51 Moniotte,S., Vaerman,J.-L., Kockx,M.M., Larrouy,D., Langin,D., Noirhomme,P. and Balligand,J.-L. (2001) Real-time RT–PCR for the detection of ß-adrenoceptor messenger RNAs in small human endomyocardial biopsies. J. Mol. Cell. Cardiol., 33, 2121–2133.[Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  B. Tazon-Vega, M. Vilardell, L. Perez-Oller, E. Ars, P. Ruiz, O. Devuyst, X. Lens, P. Fernandez-Llama, J. Ballarin, and R. Torra Study of candidate genes affecting the progression of renal disease in autosomal dominant polycystic kidney disease type 1 Nephrol. Dial. Transplant., June 1, 2007; 22(6): 1567 - 1577. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. K. Ahrabi, S. Terryn, G. Valenti, N. Caron, C. Serradeil-Le Gal, D. Raufaste, S. Nielsen, S. Horie, J.-M. Verbavatz, and O. Devuyst PKD1 Haploinsufficiency Causes a Syndrome of Inappropriate Antidiuresis in Mice J. Am. Soc. Nephrol., June 1, 2007; 18(6): 1740 - 1753. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Rossetti and P. C. Harris Genotype-Phenotype Correlations in Autosomal Dominant and Autosomal Recessive Polycystic Kidney Disease J. Am. Soc. Nephrol., May 1, 2007; 18(5): 1374 - 1380. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. S. Forbes, B. A. Thornhill, M. H. Park, and R. L. Chevalier Lack of Endothelial Nitric-Oxide Synthase Leads to Progressive Focal Renal Injury Am. J. Pathol., January 1, 2007; 170(1): 87 - 99. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. P. Casas, G. L. Cavalleri, L. E. Bautista, L. Smeeth, S. E. Humphries, and A. D. Hingorani Endothelial Nitric Oxide Synthase Gene Polymorphisms and Cardiovascular Disease: A HuGE Review Am. J. Epidemiol., November 15, 2006; 164(10): 921 - 935. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Zhang, R. Lopez-Ridaura, D. J. Hunter, N. Rifai, and F. B. Hu Common variants of the endothelial nitric oxide synthase gene and the risk of coronary heart disease among u.s. Diabetic men. Diabetes, July 1, 2006; 55(7): 2140 - 2147. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L C Jones and A D Hingorani Genetic regulation of endothelial function Heart, October 1, 2005; 91(10): 1275 - 1277. [Full Text] [PDF]
|
|
|
|
|
|
A. Page, H. Reich, J. Zhou, V. Lai, D. C. Cattran, J. W. Scholey, and J. A. Miller Endothelial Nitric Oxide Synthase Gene/Gender Interactions and the Renal Hemodynamic Response to Angiotensin II J. Am. Soc. Nephrol., October 1, 2005; 16(10): 3053 - 3060. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. D. Paterson, R. Magistroni, N. He, K. Wang, A. Johnson, P. R. Fain, E. Dicks, P. Parfrey, P. St. George-Hyslop, and Y. Pei Progressive Loss of Renal Function Is an Age-Dependent Heritable Trait in Type 1 Autosomal Dominant Polycystic Kidney Disease J. Am. Soc. Nephrol., March 1, 2005; 16(3): 755 - 762. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. C. Serrano, J. P. Casas, L. A. Diaz, C. Paez, C. M. Mesa, R. Cifuentes, A. Monterrosa, A. Bautista, E. Hawe, A. D. Hingorani, et al. Endothelial NO Synthase Genotype and Risk of Preeclampsia: A Multicenter Case-Control Study Hypertension, November 1, 2004; 44(5): 702 - 707. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. P. Rossi, G. Maiolino, J. P. Casas, A. D. Hingorani, S. E. Humphries, L. Smeeth, and L. E. Bautista Do Meta-Analyses of Association Studies of Endothelial Nitric Oxide Synthase Variants and Ischemic Heart Disease Provide Conclusive Answers? * Response Circulation, September 14, 2004; 110(11): e305 - e306. [Full Text] [PDF]
|
|
|
|
|
|
G. Gillerot, H. Debaix, and O. Devuyst Genotyping: a new application for the spent dialysate in peritoneal dialysis Nephrol. Dial. Transplant., May 1, 2004; 19(5): 1298 - 1301. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. P. Casas, L. E. Bautista, S. E. Humphries, and A. D. Hingorani Endothelial Nitric Oxide Synthase Genotype and Ischemic Heart Disease: Meta-Analysis of 26 Studies Involving 23028 Subjects Circulation, March 23, 2004; 109(11): 1359 - 1365. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Erbs, A. Linke, G. Schuler, and R. Hambrecht Promoter and Exon 7 Polymorphism of Endothelial Nitric Oxide Synthase: Impact on Endothelial Function and Its Correction by Exercise Training in Patients With Coronary Artery Disease Arterioscler. Thromb. Vasc. Biol., February 1, 2004; 24(2): e12 - 12. [Full Text]
|
|
|
|
|
|
O. Devuyst, A. Persu, and M.-T. Vo-Cong Autosomal dominant polycystic kidney disease: modifier genes and endothelial dysfunction Nephrol. Dial. Transplant., November 1, 2003; 18(11): 2211 - 2215. [Full Text] [PDF]
|
|
|
|
|
|
A. Persu, O. El-Khattabi, T. Messiaen, Y. Pirson, D. Chauveau, and O. Devuyst Influence of ACE (I/D) and G460W polymorphism of {alpha}-adducin in autosomal dominant polycystic kidney disease Nephrol. Dial. Transplant., October 1, 2003; 18(10): 2032 - 2038. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. M. Seccia, G. P. Rossi, E. Noiri, T. Fujita, and K. Tokunaga Endothelial Nitric Oxide Synthase Gene Polymorphisms and Renal Survival * Response: Multifactorial Disease: Glu298asp of Endothelial Nitric Oxide Synthase Hypertension, June 1, 2003; 41 (6): e11 - e12. [Full Text] [PDF]
|
|
|
|
|
|
R. Magistroni, N. He, K. Wang, R. Andrew, A. Johnson, P. Gabow, E. Dicks, P. Parfrey, R. Torra, J. L. San-Millan, et al. Genotype-Renal Function Correlation in Type 2 Autosomal Dominant Polycystic Kidney Disease J. Am. Soc. Nephrol., May 1, 2003; 14(5): 1164 - 1174. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Paolo Rossi, M. Cesari, M. Zanchetta, S. Colonna, G. Maiolino, L. Pedon, M. Cavallin, P. Maiolino, and A. C. Pessina The T-786C endothelial nitric oxide synthase genotype is a novel risk factor for coronary artery disease in Caucasian patients of the GENICA study J. Am. Coll. Cardiol., March 19, 2003; 41(6): 930 - 937. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A.H. Gomma, M.A. Elrayess, C.J. Knight, E. Hawe, K.M. Fox, and S.E. Humphries The endothelial nitric oxide synthase (Glu298Asp and -786T>C) gene polymorphisms are associated with coronary in-stent restenosis Eur. Heart J., December 2, 2002; 23(24): 1955 - 1962. [Abstract] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||