Alessandra Testa

Vitamin D receptor (VDR) gene polymorphism is associated with left ventricular (LV) mass and predicts left ventricular hypertrophy (LVH) progression in end-stage renal disease (ESRD) patients.

Left ventricular hypertrophy (LVH) is a strong cardiovascular risk marker in end‐stage renal disease (ESRD) patients. Vitamin D deficiency and/or disturbed vitamin D signaling has been implicated in LVH in experimental models. Because the BsmI vitamin D receptor VDR gene polymorphism may alter VDR function, we performed a cross‐sectional and longitudinal study in a cohort of 182 dialysis patients to investigate (1) the relationship between BsmI VDR gene polymorphism and left ventricular mass index (LVMI) measured by echocardiography and (2) the predictive power of this polymorphism for progression in LVH over a 18 ± 2 months of follow‐up. As a reference group, we used 175 healthy subjects matched to the study population as for age and sex. The distribution of BsmI genotypes did not significantly deviate from Hardy‐Weinberg equilibrium either in patients or in the control group of healthy subjects. The frequency of the B allele of BsmI polymorphism (40.4%) in dialysis patients was similar to that of healthy control subjects (38.6%), and the number of B alleles was directly related to LVMI (r = 0.20, P = .007). This relationship remained robust (β = 0.19, P = .006) in multivariate analysis adjusting for traditional and nontraditional risk factors and antihypertensive and calcitriol treatment. In the longitudinal study, LVMI rose from 60.1 ± 17.9 to 64.2 ± 19.3 g/m2.7 (P < .001), and again, the number of B alleles was associated with LVMI changes both in crude and in fully adjusted analyses. These cross‐sectional and longitudinal observations coherently support the hypothesis that altered vitamin D signaling is implicated in LVH in ESRD patients.

The ENPP1 Q121 Variant Predicts Major Cardiovascular Events in High-Risk Individuals: Evidence for Interaction With Obesity in Diabetic Patients

OBJECTIVE Insulin resistance (IR) and cardiovascular disease may share a common genetic background. We investigated the role of IR-associated ENPP1 K121Q polymorphism (rs1044498) on cardiovascular disease in high-risk individuals. RESEARCH DESIGN AND METHODS A prospective study (average follow-up, 37 months) was conducted for major cardiovascular events (myocardial infarction [MI], stroke, cardiovascular death) from the Gargano Heart Study (GHS; n = 330 with type 2 diabetes and coronary artery disease), the Tor Vergata Atherosclerosis Study (TVAS; n = 141 who had MI), and the Cardiovascular Risk Extended Evaluation in Dialysis (CREED) database (n = 266 with end-stage renal disease). Age at MI was investigated in cross-sectional studies of 339 type 2 diabetic patients (n = 169 from Italy, n = 170 from the U.S.). RESULTS Incidence of cardiovascular events per 100 person--years was 4.2 in GHS, 10.8 in TVAS, and 11.7 in CREED. Hazard ratios (HRs) for KQ+QQ versus individuals carrying the K121/K121 genotype (KK) individuals were 1.47 (95% CI 0.80–2.70) in GHS, 2.31 (95% CI 1.22–4.34) in TVAS, and 1.36 (95% CI 0.88–2.10) in CREED, and 1.56 (95% CI 1.15–2.12) in the three cohorts combined. In the 395 diabetic patients, the Q121 variant predicted cardiovascular events among obese but not among nonobese individuals (HR 5.94 vs. 0.62, P = 0.003 for interaction). A similar synergism was observed in cross-sectional studies, with age at MI being 3 years younger in Q121 carriers than in KK homozygotes among obese but not among nonobese patients (P = 0.035 for interaction). CONCLUSIONS The ENPP1 K121Q polymorphism is an independent predictor of major cardiovascular events in high-risk individuals. In type 2 diabetes, this effect is exacerbated by obesity. Future larger studies are needed to confirm our finding.

Association of a Polymorphism in a Gene Encoding a Urate Transporter with CKD Progression

BACKGROUND AND OBJECTIVES Hyperuricemia predicts a high risk for CKD progression but there is no large clinical trial in humans indicating that this relationship is causal in nature. The rs734553 single-nucleotide polymorphism (SNP) of the GLUT9 urate transporter gene was strongly associated with uric acid (UA) levels in a large meta-analysis. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS This prospective study adopted the Mendelian randomization approach. The rs734553 SNP was used as an instrumental variable to investigate the relationship between UA and renal outcomes in a cohort of 755 patients with CKD who were enrolled between October 18, 2005, and October 2, 2008. The association between the polymorphism and UA was preliminary confirmed in a series of 211 healthy volunteers enrolled between January 1, 2001, and July 12, 2011, from the same geographic area as the patients with CKD. The study end point was a composite renal-end point (i.e., >30% decrease in the GFR, dialysis, or transplantation). Patients were followed up for a median of 36 months. RESULTS In healthy individuals, serum UA levels were highest in homozygotes for the T allele (risk allele), intermediate in heterozygotes for the same allele, and lowest in those without the risk allele (P<0.001), but no such relationship was found in patients with CKD. In the CKD cohort, homozygotes (TT) and heterozygotes (GT) for the risk allele had a 2.35 times higher risk (hazard ratio, 2.35; 95% confidence interval, 1.25 to 4.42; P=0.008) of CKD progression. The risk for CKD progression by rs734553 remained unmodified in analyses adjusting for proteinuria, GFR, and other classical and CKD-peculiar risk factors. CONCLUSIONS A GLUT9 polymorphism, which is strongly associated with serum UA levels in healthy individuals of the general population with normal renal function, holds a strong predictive power for CKD progression. These findings are compatible with the hypothesis that the link between UA and CKD progression is causal in nature.

Association of IL-6 and a Functional Polymorphism in the IL-6 Gene with Cardiovascular Events in Patients with CKD

BACKGROUND AND OBJECTIVES High serum IL-6 is a major risk factor for cardiovascular disease (CVD) in the general population. This cytokine is substantially increased in patients with CKD, but it is still unknown whether the link between IL-6 and CVD in CKD is causal in nature. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS In a cohort of 755 patients with stages 2-5 CKD, consecutively recruited from 22 nephrology units in southern Italy, this study assessed the relationship of serum IL-6 with history of CVD, as well as with incident cardiovascular (CV) events (mean follow up±SD, 31±10 months) and used the functional polymorphism (-174 G/C) in the promoter of the IL-6 gene to investigate whether the link between IL-6 and CV events is causal. RESULTS In adjusted analyses, serum IL-6 above the median value was associated with history of CVD (P<0.001) and predicted the incidence rate of CV events (hazard ratio, 1.66; 95% confidence interval [95% CI], 1.11 to 2.49; P=0.01). Patients homozygous for the risk allele (C) of the -174 G/C polymorphism had higher levels of IL-6 than did those with other genotypes (P=0.04). Homozygous CC patients more frequently had a history of CVD (odds ratio, 2.15; 95% CI, 1.15 to 4.00; P=0.02) as well as a 87% higher rate of incident CV events (hazard ratio, 1.87; 95% CI, 1.02 to 3.44; P=0.04) compared with other genotypes. CONCLUSIONS In patients with stages 2-5 CKD, high serum IL-6 is associated with history of CVD and predicts incident CV events. The parallel relationship with history of CVD and incident CV events of the -174 G/C polymorphism in the IL-6 gene suggests that IL-6 may be causally involved in the high CV risk in this population.

A polymorphism in the major gene regulating serum uric acid associates with clinic SBP and the white-coat effect in a family-based study.

Objectives: Hyperuricemia associates with hypertension, but it is uncertain whether this relationship is causal in nature. Glucose transporter 9 (GLUT9) gene is a major genetic determinant of plasma uric acid levels in humans. Since polymorphisms are randomly distributed at mating (Mendelian randomization), studies based on GLUT9 polymorphisms may provide unconfounded assessment of the nature of the link between uric acid and hypertension. Methods: We tested the association between uric acid, the rs734555 polymorphism of the GLUT9 gene and arterial pressure in a family-based study including 449 individuals in a genetically homogenous population in Southern Italy. Results: Serum uric acid levels were strongly associated (P < 0.001) with all components of clinic and 24-h ambulatory blood pressures (BPs). However, only clinic SBP and the white-coat effect (the difference in clinic systolic and daytime systolic ambulatory blood pressure monitoring) associations remained significant after adjustment for classical risk factor and the estimated glomerular filtration rate. Serum uric acid was strongly associated with the risk allele (T) of the rs734555 polymorphism (P < 0.001). Furthermore, TT individuals showed higher clinic SBP (129 + SEM 1 mmHg) than GT (125 + 1 mmHg) and GG individuals (122 + 3 mmHg), as well as a higher white-coat effect (P = 0.02), confirming that the association between uric acid and these BP components is unconfounded by environmental risk factors. Conclusion: Results in this family-based study are compatible with the hypothesis that uric acid is a causal risk factor for hypertension. Trials testing uric acid-lowering interventions are needed to definitively establish the causal implication of hyperuricemia in human hypertension.

The GLU298ASP variant of nitric oxide synthase interacts with asymmetric dimethyl arginine in determining cardiovascular mortality in patients with end-stage renal disease.

Objectives Impaired nitric oxide generation and accumulation of the endogenous inhibitor of nitric oxide synthase (NOS), asymmetric dimethylarginine (ADMA), have been identified as strong predictors of cardiovascular outcomes in patients with end-stage renal disease (ESRD). We evaluated the role of endothelial NOS (eNOS) gene polymorphisms and its interaction with plasma ADMA in the high cardiovascular complications rate of these patients. Methods The relationship between the Glu298Asp variant of eNOS and all-cause and cardiovascular mortality was assessed in a cohort study including 261 ESRD patients that were followed up for an average of 42 months. Results During the follow-up period, 138 patients died, 81 of them (i.e. 59% of total deaths) of cardiovascular causes. On univariate Coxs regression analysis, eNOS genotype tended to be related to all-cause death but failed to reach formal statistical significance (P for trend = 0.11). However, eNOS genotype showed a significant association with cardiovascular mortality in statistical models, including traditional risk factors and factors peculiar to ESRD, and became even stronger when plasma ADMA was forced into the Cox model (P = 0.006). Furthermore, the risk of cardiovascular death was maximum in heterozygotes and homozygotes patients carrying the risk allele and in those having high ADMA levels (hazard ratio = 2.71, 95% confidence interval 1.38–5.35, P = 0.004) compared to those having just one of these two risk factors. Conclusions The T allele of the Glu298Asp polymorphism predicts cardiovascular mortality and interacts with plasma ADMA in determining this outcome in dialysis patients.

The deletion polymorphism of the angiotensin-converting enzyme is associated with nephroangiosclerosis

The D allele of the angiotensin-converting enzyme (ACE) gene has been linked with diabetic nephropathy and IgA glomerulonephritis and with faster renal disease progression. The association of this allele with nephroangiosclerosis has been scarcely investigated. We have tested this association in 45 hypertensive patients (all whites) with well defined nephroangiosclerosis (diagnosis established on the basis of renal biopsy in all cases) and moderate to severe renal failure. As studies of genetic association of small size often produce conflicting results, besides a control group of 343 Italian patients with essential hypertension and normal renal function, we elected to use also a very large control group of race-matched subjects taken from a meta-analysis of 27,565 whites. The proportion of patients with the D allele (64%) was higher in patients with nephroangiosclerosis than that in Italian hypertensives (54%) and in whites (54%). DD and DI genotypes were more prevalent in patients than in control groups. The dominant model (DD and DI v II: nephroangiosclerosis v Italian controls: chi2 = 6.19, P = .012; nephroangiosclerosis v whites chi2 = 6.86, P = .009) fitted the data better than the codominant and the recessive model (P < or = .022). The D allele is associated with nephroangiosclerosis with a dominant effect in the sample of patients studied. Although intervention studies are needed to see whether these findings imply a causal association, our data suggest that this allele may at least act as disease marker in nephroangiosclerosis.

Plasma cytokines, glomerular filtration rate and adipose tissue cytokines gene expression in chronic kidney disease (CKD) patients

BACKGROUND AND AIM Systemic inflammation is a hallmark of chronic kidney disease (CKD) and obesity represents a major risk factor for CKD. We investigated the relationship between plasma interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α) and the glomerular filtration rate (GFR) in 75 stage 2-5 CKD patients. METHODS AND RESULTS We studied the steady-state relationship between plasma and subcutaneous adipose tissue (SAT) gene expression of the same cytokines in 19 patients and in 17 well-matched healthy subjects (HS) and compared SAT gene expression of these cytokines and of two additional cytokines (IL-1β and IL-8) in CKD patients and in HS. Plasma IL-6 and TNF-α were higher in CKD patients than in HS (P < 0.001). IL-6 was similarly increased in patients with mild, moderate and severe CKD and largely independent of the GFR (r = -0.03, P = NS). TNF-α was inversely related to GFR, which was the first factor in rank (β = -0.37, P = 0.001) explaining the variability in TNF-α in CKD. SAT messenger RNA (mRNA) levels of IL-6, TNF-α, IL- β and IL-8 were similar in CKD patients and in HS. Plasma and SAT mRNA levels of IL-6 and TNF-α levels were largely unrelated. CONCLUSIONS Plasma IL-6 rises early in CKD and does not show any further increase at more severe stages of CKD, whereas TNF-α is inversely associated with the GFR indicating a substantial difference in the dynamics of the relationship between these cytokines and renal function. Cytokines are not overexpressed in SAT in these patients, and circulating IL-6 and TNF-α are dissociated from the corresponding mRNA levels in SAT, both in CKD patients and in HS.

A genetic marker of uric acid level, carotid atherosclerosis, and arterial stiffness: a family-based study.

BACKGROUND Hyperuricemia associates with atherosclerosis complications, but it is uncertain whether this relationship is causal in nature. The urate transporter GLUT9 (encoded by the SLC2A9 gene) is a major genetic determinant of serum uric acid level in humans. Because polymorphisms are distributed randomly at mating (Mendelian randomization), studies based on GLUT9 polymorphisms may provide unconfounded assessment of the nature of the link between uric acid and atherosclerosis. STUDY DESIGN Cross-sectional study. SETTING & PARTICIPANTS Family-based study including 449 individuals in 107 families in a genetically homogeneous population in Southern Italy. FACTOR Serum uric acid level, rs734553 allele, and age. OUTCOME Ultrasound biomarkers of atherosclerosis (intima-media thickness [IMT] and internal diameter) and pulse wave velocity (PWV). RESULTS Serum uric acid level was dose-dependently associated with the T allele of rs734553, a polymorphism in SLC2A9 (P=8×10(-6)). Serum uric acid level was a strong modifier of the relationship between age and IMT in fully adjusted analyses (β=0.33; P=0.01), whereas no such relationship was found for internal diameter (β=-0.15; P=0.3) or PWV (β=0.10; P=0.6). The T allele coherently associated with carotid IMT, internal diameter, and PWV and emerged as an even stronger modifier of the age-IMT and age-internal diameter relationships in both crude and fully adjusted (β=0.40 [P<0.001] and β=0.48 [P=0.003], respectively) analyses. LIMITATIONS This is a hypothesis-generating study. CONCLUSIONS Results in this family-based study implicate uric acid as an important modifier of the age-dependent risk for atherosclerosis. Trials testing uric acid-lowering interventions are needed to prove this hypothesis.

eNOS and Caveolin-1 Gene Polymorphisms Interaction and Intima Media Thickness: A Proof of Concept Study in ESRD Patients

BACKGROUND Caveolae are a prominent microdomain in endothelial cells and appropriate localization in caveolae is fundamental for endothelial nitric oxide synthase (eNOS) activity. Since the Glu298Asp variant in the eNOS gene alters caveolar localization of the corresponding enzyme, we tested the interaction between this variant and the rs4730751 polymorphism of the caveolin-1 (CAV-1) gene as related to arterial remodeling in end-stage renal disease (ESRD) patients. METHODS One hundred and thirty-three ethnically homogeneous ESRD patients underwent carotid ultrasonographic studies to measure intima-media thickness (IMT) and carotid cross-sectional area (CSA). Genotyping was performed by high-throughput allelic discrimination assays on real-time PCR. RESULTS Arterial remodeling was associated to the number of G alleles of CAV-1 polymorphism, GG homozygotes displaying an IMT and a CSA that were, respectively, 16% and 21% higher than those in patients without the risk allele (P < 0.012). In multiple linear regression analyses including the CAV-1 and the eNOS polymorphisms and adjusting for classical risk factors and risk factors peculiar to ESRD both polymorphisms were independent correlates of IMT (CAV-1: β = 0.20, P = 0.01; eNOS β = 0.25, P = 0.001) and CSA (CAV-1: β = 0.20, P = 0.01: eNOS β = 0.13, P = 0.09). Furthermore, strong interactions emerged between the two polymorphisms for explaining the variability in IMT (P = 0.001) and in CSA (P = 0.038) in these patients. CONCLUSION Overall these findings form preliminary evidence that disturbed interaction between CAV-1 and eNOS may be of relevance for arterial disease in ESRD and perhaps in other human diseases.