Role of kidney function on Nrf2 mRNA levels in type 2 diabetes.

INTRODUCTION: Diabetic kidney disease (DKD) is a major complication in patients with diabetes and the main contributor to the chronic kidney disease (CKD) global burden. Oxidative stress is a crucial factor in DKD pathogenesis but the role of the antioxidant nuclear factor erythroid 2-related factor 2 (Nrf2) and its molecular regulators has been poorly investigated in man. RESEARCH DESIGN AND METHODS: In this case-control study, we analyzed the roles of Nrf2, a transcription factor shielding cells from oxidative stress, its repressor Kelch-like ECH-associated protein 1 (Keap1) and six microRNAs (miRNAs) that potentially suppress Nrf2. We categorized 99 participants into 3 groups: 33 non-dialysis patients with type 2 diabetes with DKD, 33 patients with type 2 diabetes without DKD and 33 control subjects and quantified the gene expression (messenger RNA (mRNA)) levels of Nrf2, Keap1 and 6 miRNAs. Moreover, we studied the correlation between gene expression levels and clinical indicators of kidney health. RESULTS: In patients with diabetes with DKD, Nrf2 mRNA levels were significantly lower than in patients without DKD (p=0.01) and controls (p=0.02), whereas no difference in Nrf2 expression levels existed between patients without DKD and controls. Conversely, in patients with and without DKD, Keap1 expression levels were significantly higher than in controls. Of the six miRNAs studied, miRNA 30e-5p showed differential expression, being markedly reduced in patients with DKD (p=0.007). Nrf2 mRNA levels directly correlated with estimated glomerular filtration rate (eGFR) in patients with DKD (r=0.34, p=0.05) and in a formal mediation analysis the eGFR emerged as the first factor in rank for explaining the difference in Nrf2 mRNA levels between patients with and without DKD. CONCLUSIONS: The observed dysregulation in the Nrf2-Keap1 axis and the unique expression pattern of miRNA30e-5p in DKD underscore the need for more focused research in this domain that can help identify novel intervention strategies for DKD in patients with type 2 diabetes.

Role of Gut Microbiota in Overweight Susceptibility in an Adult Population in Italy.

Although the gut microbiota is known to affect body weight, its relationship with overweight/obesity is unclear. Our aim was to characterize microbiota composition in a cohort from the southernmost area of Italy. We investigated whether an altered gut microbiota could play an etiological role in the pathogenesis of overweight/obesity. A total of 163 healthy adults were enrolled. Microbiome analysis was performed via 16S rRNA gene sequencing. We found significant phylum variations between overweight (N = 88) and normal-weight (N = 75) subjects. Bacteroidetes and Proteobacteria were higher in overweight participants (p = 0.004; p = 0.03), and Firmicutes and Verrucomicrobia were lower (p = 0.02; p = 0.008) compared to normal-weight participants. Additionally, Akkermansia and Bifidobacterium (genus level) were significantly lower in the overweight group, as well as Akkermansia muciniphila at the species level. The Firmicutes/Bacteroidetes ratio (F/B ratio), an index of dysbiosis, was found to be inversely associated with BMI in linear and logistic regression models (p = 0.001; p = 0.005). The association remained statistically significant after adjustment for potential confounders. This cross-sectional study contributes to defining the gut microbiota composition in an adult population living in southern Italy. It confirms the relationship between overweight susceptibility and the dysbiosis status, highlighting the possible etiological role of the F/B ratio in disease susceptibility.

SMCHD1 and LRIF1 converge at the FSHD-associated D4Z4 repeat and LRIF1 promoter yet display different modes of action.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by the epigenetic derepression of the 4q-linked D4Z4 macrosatellite repeat resulting in inappropriate expression of the D4Z4 repeat-encoded DUX4 gene in skeletal muscle. In 5% of FSHD cases, D4Z4 chromatin relaxation is due to germline mutations in one of the chromatin modifiers SMCHD1, DNMT3B or LRIF1. The mechanism of SMCHD1- and LRIF1-mediated D4Z4 repression is not clear. We show that somatic loss-of-function of either SMCHD1 or LRIF1 does not result in D4Z4 chromatin changes and that SMCHD1 and LRIF1 form an auxiliary layer of D4Z4 repressive mechanisms. We uncover that SMCHD1, together with the long isoform of LRIF1, binds to the LRIF1 promoter and silences LRIF1 expression. The interdependency of SMCHD1 and LRIF1 binding differs between D4Z4 and the LRIF1 promoter, and both loci show different transcriptional responses to either early developmentally or somatically perturbed chromatin function of SMCHD1 and LRIF1.

Prevalence and detection rate of celiac disease in Italy: Results of a SIGENP multicenter screening in school-age children.

BACKGROUND: Celiac disease is a common lifelong disorder. Recent studies indicate that the number of clinically detected cases has increased over the last decades, however little is known about changes in the prevalence and the detection rate of celiac disease. AIM: To evaluate the current prevalence and detection rate of celiac disease in Italy by a multicenter, mass screening study on a large sample of school-age children. METHODS: children aged 5-11 years were screened at school by HLA-DQ2 and -DQ8 determination on a drop of blood in six Italian cities; total serum IgA and IgA anti-transglutaminase were determined in children showing HLA-DQ2 and/or -DQ8 positivity. Diagnosis of celiac disease was confirmed according to the European guidelines. RESULTS: 5994 children were eligible, 4438 participated and 1873 showed predisposing haplotypes (42.2%, 95% CI=40.7-43.7). The overall prevalence of celiac disease was 1.65% (95% CI, 1.34%-2.01%). Only 40% of celiac children had been diagnosed prior to the school screening. Symptoms evoking celiac disease were as common in celiac children as in controls. CONCLUSION: In this multicenter study the prevalence of celiac disease in school-age Italian children was one of the highest in the world. Determination of HLA predisposing genotypes is an easy and fast first-level screening test for celiac disease. Without a mass screening strategy, 60% of celiac patients remain currently undiagnosed in Italy.

Effectiveness of Apremilast in Real Life in Patients with Psoriasis: A Longitudinal Study.

Apremilast is an oral selective phosphodiesterase-4 inhibitor developed recently for psoriasis treatment. The aim of this study is to assess the real-life outcomes of use of apremilast in patients with psoriasis in everyday clinical practice. A total of 159 adult patients (90 males) with plaque psoriasis were included in the study. Fifty of the patients (31%) had psoriatic arthritis. All patients started apremilast at the time of enrolment. There was a marked improvement in Psoriasis Area and Severity Index, body surface area and Dermatology Life Quality Index scores across the follow-up period (12 months). The improvements in these scores were also consistent when the patients were stratified according to increasing body mass index. Only 10.6% of the patients discontinued apremilast, because of no response. In conclusion, apremilast is an effective and safe treatment in patients with psoriasis, and its effect is not influenced by body mass index.

Long-term blood pressure monitoring by office and 24-h ambulatory blood pressure in renal transplant patients: a longitudinal study.

BACKGROUND: Renal transplant patients have a high prevalence of nocturnal hypertension, and hypertension misclassification by office blood pressure (BP) is quite common in these patients. The potential impact of hypertension misclassification by office BP on hypertension management in this population has never been analysed. METHODS: We performed a longitudinal study in a cohort of 260 clinically stable renal transplant patients. In all, 785 paired office and 24-h ambulatory blood pressure monitoring (24-hABPM) measurements over a median follow-up of 3.9 years were available in the whole cohort. RESULTS: A total of 74% of patients had nocturnal hypertension (>120/70 mmHg). Average office BP and 24-hABPM remained quite stable over follow-up, as did the prevalence of nocturnal hypertension, which was 77% at the last observation. However, the global agreement between office BP and average 24 h, daytime and night-time BP was unsatisfactory (k-statistics 0.10-0.26). In 193 visits (25% of all visits) where office BP indicated the need of antihypertensive therapy institution or modification (BP >140/90 mmHg), 24-hABPM was actually normal (<130/80 mmHg), while in 94 visits (12%), 24-hABPM was in the hypertensive range while office BP was normal. Overall, in 37% of visits, office BP provided misleading therapeutic indications. CONCLUSIONS: Hypertension misclassification by office BP is a common phenomenon in stable renal transplant patients on long-term follow-up. Office BP may lead to inappropriate therapeutic decisions in over one-third of follow-up visits in these patients.

Mutations in the GLA Gene and LysoGb3: Is It Really Anderson-Fabry Disease?

Anderson-Fabry disease (FD) is a rare, progressive, multisystem storage disorder caused by the partial or total deficit of the lysosomal enzyme α-galactosidase A (α-Gal A). It is an X-linked, lysosomal enzymopathy due to mutations in the galactosidase alpha gene (GLA), encoding the α-Gal A. To date, more than 900 mutations in this gene have been described. In our laboratories, the study of genetic and enzymatic alterations related to FD was performed in about 17,000 subjects with a symptomatology referable to this disorder. The accumulation of globotriaosylsphingosine (LysoGb3) was determined in blood of positives. Exonic mutations in the GLA gene were detected in 471 patients (207 Probands and 264 relatives): 71.6% of mutations were associated with the classic phenotype, 19.8% were associated with the late-onset phenotype, and 8.6% of genetic variants were of unknown significance (GVUS). The accumulation of LysoGb3 was found in all male patients with a mutation responsible for classic or late-onset FD. LysoGb3 levels were consistent with the type of mutations and the symptomatology of patients. α-Gal A activity in these patients is absent or dramatically reduced. In recent years, confusion about the pathogenicity of some mutations led to an association between non-causative mutations and FD. Our study shows that the identification of FD patients is possible by associating clinical history, GLA gene analysis, α-Gal A assay, and blood accumulation of LysoGB3. In our experience, LysoGB3 can be considered a reliable marker, which is very useful to confirm the diagnosis of Fabry disease.

Systematic DNA Study for Fabry Disease in the End Stage Renal Disease Patients from a Southern Italy Area.

BACKGROUND/AIMS: Fabry disease (FD) is a lysosomal storage disorder characterized by pervasive renal involvement. However, this disease is underdiagnosed in patient with chronic kidney disease (CKD), including those with end stage renal disease (ESRD), so their investigation represents an unexploited opportunity for early diagnosis of the disease and for its identification in relatives of affected patients. METHODS: We investigated Fabry disease in a clinical and biological database including ESRD patients of unknown cause in a geographical area with 2 million residents. The study was based on state of art GLA gene sequencing and was extended to relatives of affected ESRD patients. RESULTS: Among ESRD patients qualified for enrollment into this study, a previously undiagnosed young man harboring the mutation p.I91T was identified. The study of the proband's family led to the identification of 8 additional cases. In another ESRD male patient, we identified the functional polymorphism p.D313Y. Furthermore, in 55 ESRD patients (24.2%) we found intronic polymorphisms of uncertain functional relevance in the non-coding regions of the GLA gene. CONCLUSION: A comprehensive survey of ESRD patients in a geographical area of 2 million residents identified one undiagnosed case of Fabry disease and led to the identification of 8 additional cases among his relatives. Screening protocols starting from the dialysis population and upstream extended to families of affected individuals may be an effective strategy to maximize the early identification of subjects with Fabry disease.

The sirtuin1 gene associates with left ventricular myocardial hypertrophy and remodeling in two chronic kidney disease cohorts: a longitudinal study.

BACKGROUND: Oxidative stress and inflammation are major drivers of myocardial hypertrophy in chronic kidney disease (CKD). The silent information regulator gene 1 (Sirt1) is a fundamental mediator of the response to oxidative stress and inflammation and promotes myocardial growth under stress conditions; therefore, it may contribute to myocardial hypertrophy and concentric remodeling of the left ventricle (LV) in CKD. METHODS: We investigated the cross-sectional and longitudinal relationship between three candidate polymorphisms in the Sirt1 gene and LV parameters in two cohorts of CKD patients including 235 stage G5D patients and 179 stages G1-5 patients, respectively. RESULTS: In both cohorts, the C allele of the Sirt1 rs7069102 polymorphism associated with the posterior wall thickness in separate and combined analyses (beta = 0.15, P = 2 × 10) but was unrelated with the LV volume and the LV mass index indicating a peculiar association of this allele with LV concentric remodeling. Accordingly, the same allele was linked with the LV mass-to-volume ratio in separate and combined (beta = 0.14, P = 2 × 10) analyses in the same cohorts. Furthermore, in longitudinal analyses patients harboring the C allele showed a more pronounced increase in LV mass-to-volume ratio over time than patients without such an allele (regression coefficient = 0.14, 95% confidence interval: 0.05-0.23; P = 3 × 10 in the combined analysis). CONCLUSION: The rs7069102 polymorphism in the Sirt1 gene is associated with LV concentric remodeling in two independent cohorts of stages G5D and G1-5 CKD patients. These results offer a genetic basis to the hypothesis that the Sirt1 gene plays a causal role in myocardial hypertrophy and LV concentric remodeling in these patients.

Office, standardized and 24-h ambulatory blood pressure and renal function loss in renal transplant patients.

OBJECTIVES: Hypertension is a risk factor for renal function loss in kidney transplant patients but there are still no longitudinal studies focusing on the relationship between ambulatory blood pressure (BP) monitoring (ABPM) and the glomerular filtration rate (GFR) evolution over time in these patients. METHODS: In a cohort of 260 renal transplant patients, we investigated the longitudinal relationship between repeated office BP measurements and simultaneous GFR measurements (on average 35 paired measurements per patient) and the relationship between baseline ABPM with the same outcome measure (by linear mixed models). Furthermore, we tested the prediction power of baseline ABPM and standardized BP measurements for a combined renal end point (GFR loss >30%, end-stage kidney disease or death) over a 3.7 years follow-up. RESULTS: Longitudinal office BP measurements were inversely related with simultaneous GFR measurements and the same was true both for baseline daytime and night-time BP. (all P < 0.001). Baseline 24-h ABPM [hazard ratio (5 mmHg):1.11; 95% confidence interval 1.03-1.19] and night-time SBP [hazard ratio (5 mmHg):1.10; 95% confidence interval 1.03-1.17] predicted the combined renal end point and the predictive model based on night-time SBP provided a data-fit superior than that by daytime SBP. CONCLUSION: In renal transplant patients, daytime and night-time SBP predict the risk of GFR loss overtime, and among the various BP metrics, night-time BP is the strongest indicator of the risk of renal function loss. Optimization of BP control and interventions targeting night-time BP may afford renal benefits in transplant patients, a hypothesis that remains to be tested in a clinical trial.

A polymorphism in a major antioxidant gene (Kelch-like ECH-associated protein 1) predicts incident cardiovascular events in chronic kidney disease patients: an exploratory study.

OBJECTIVE: Oxidative stress is considered a major pathway conducive to cardiovascular disease in chronic kidney disease (CKD) patients. However, observational studies and clinical trials testing this relationship are controversial. The Nuclear factor-erythroid-2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) system is a major system regulating antioxidant mechanisms in living organisms. Owing to the fact that genes are transmitted randomly (Mendelian randomization), genetic variants may provide unconfounded assessment of putative causal risk factors. METHODS: We have therefore explored the association of eight polymorphisms in the Nrf2 gene and three polymorphisms in the Keap1 gene (capturing over 80% of the genetic variance in the same genes) with cardiovascular events in a multicenter cohort study of 758 CKD patients. RESULTS: During the follow-up period, 117 patients had fatal and nonfatal cardiovascular events and 42 died. The hazard rate of fatal and nonfatal cardiovascular outcomes was about twice higher in patients with the AA or the CA genotype (dominant model) in the rs110857735 polymorphism of the Keap1 gene (hazard rate: 1.85, 95% CI: 1.20-2.84, P = 0.005) than in those with the CC genotype. Further analyses adjusting for Framingham risk factors and CKD-specific risk factors and a bootstrapping validation analysis did not modify the strength of this association. No association was registered between other Keap1 and Nrf2 polymorphisms and cardiovascular disease in the same cohort. CONCLUSION: In this exploratory study a gene-variant in Keap1, a major gene regulating the antioxidant response, predicts incident cardiovascular events in CKD patients. This finding is in keeping with the hypothesis implicating oxidative stress in cardiovascular disease in this population.

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 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 (ß=0.33; P=0.01), whereas no such relationship was found for internal diameter (ß=-0.15; P=0.3) or PWV (ß=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 (ß=0.40 [P<0.001] and ß=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.

Joint effect of insulin signaling genes on all-cause mortality.

OBJECTIVE: We have previously reported the combined effect of SNPs perturbing insulin signaling (ENPP1 K121Q, rs1044498; IRS1 G972R, rs1801278; TRIB3 Q84R, rs2295490) on insulin resistance (IR), type 2 diabetes (T2D) and cardiovascular events. We here investigated whether such a combined effect affects also all-cause mortality in a sample of 1851 Whites of European ancestry. METHODS: We investigated a first sample of 721 patients, 232 deaths, 3389 person-years (py). Replication was assessed in two samples of patients with T2D: the Gargano Mortality Study (GMS) of 714 patients, 127 deaths, 5426 py and the Joslin Kidney Study (JKS) comprising 416 patients, 214 deaths, 5325 py. RESULTS: In the first sample, individuals carrying 1 or ≥ 2 risk alleles had 33% (p = 0.06) and 51% (p = 0.02) increased risk of mortality, as compared with individuals with no risk alleles. A similar, though not significant, trend was obtained in the two replication samples only for subject carrying ≥ 2 risk alleles. In a pooled analysis, individuals carrying ≥ 2 risk alleles had higher mortality rate as compared to those carrying 0 risk alleles (HR = 1.34, 95%CI = 1.08-1.67; p = 0.008), and as compared to those carrying only one risk allele (HR = 1.41, 95%CI = 1.13-1.75; p = 0.002). This association was independent from several possible confounders including sex, age, BMI, hypertension and diabetes status. CONCLUSION: Our data suggest that variants affecting insulin signaling exert a joint effect on all-cause mortality and is consistent with a role of abnormal insulin signaling on mortality risk.

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 rs734553 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 rs734553 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. [Corrected]

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.

Joint effect of insulin signaling genes on cardiovascular events and on whole body and endothelial insulin resistance.

OBJECTIVE: Insulin resistance (IR) and cardiovascular disease (CVD) share a common soil. We investigated the combined role of single nucleotide polymorphisms (SNPs) affecting insulin signaling (ENPP1 K121Q, rs1044498; IRS1 G972R, rs1801278; TRIB3 Q84R, rs2295490) on CVD, age at myocardial infarction (MI), in vivo insulin sensitivity and in vitro insulin-stimulated nitric oxide synthase (NOS) activity. DESIGN AND SETTING: 1. We first studied, incident cardiovascular events (a composite endpoint comprising myocardial infarction-MI, stroke and cardiovascular death) in 733 patients (2186 person-years, 175 events). 2. In a replication attempt, age at MI was tested in 331 individuals. 3. OGTT-derived insulin sensitivity index (ISI) was assessed in 829 individuals with fasting glucose <126 mg/dl. 4. NOS activity was measured in 40 strains of human vein endothelial cells (HUVECs). RESULTS: 1. Risk variants jointly predicted cardiovascular events (HR = 1.181; p = 0.0009) and, when added to clinical risk factors, significantly improved survival C-statistics; they also allowed a significantly correct reclassification (by net reclassification index) in the whole sample (135/733 individuals) and, even more, in obese patients (116/204 individuals). 2. Risk variants were jointly associated with age at MI (p = 0.006). 3. A significant association was also observed with ISI (p = 0.02). 4. Finally, risk variants were jointly associated with insulin-stimulated NOS activity in HUVECs (p = 0.009). CONCLUSIONS: Insulin signaling genes variants jointly affect cardiovascular disease, very likely by promoting whole body and endothelium-specific insulin resistance. Further studies are needed to address whether their genotyping help identify very high-risk patients who need specific and/or more aggressive preventive strategies.

The fat-mass and obesity-associated gene (FTO) predicts mortality in chronic kidney disease of various severity.

BACKGROUND: Polymorphisms in the FTO (fat-mass and obesity-associated) gene have been associated with the body mass index, cancer, type 2 diabetes and hypertension. METHODS: We investigated the relationship between 17 tag single-nucleotide polymorphisms (SNPs) and all-cause mortality in three cohorts of dialysis patients (CREED-1, North Apulian and CREED-2 cohorts; n = 783) and in one cohort of stage 2-5 CKD patients (n = 757). RESULTS: We first explored the association between the 17 tag SNPs and all-cause mortality in the CREED-1 cohort and found that patients with the A allele of the FTO rs708259 polymorphism had an elevated risk of mortality (hazard ratio, HR: 1.52, 95% confidence interval (CI) 1.11-2.08; P = 0.008). Similarly, the A allele was associated with an increased risk of death also in the other two dialysis cohorts (North Apulian cohort, risk: +23%; CREED-2 cohort, risk: +21%). The elevated risk portended by this allele was even higher in the stage 2-5 CKD cohort (+97%). However, the risk of mortality associated with the A allele in the three confirmatory cohorts failed to achieve formal statistical significance. In a meta-analysis including the four cohorts (n = 1540; total deaths, n = 381), individuals with the A allele had a 42% excess risk of death (HR: 1.42, 95% CI 1.14-1.76, P = 0.002). CONCLUSION: The A allele of the FTO rs708259 polymorphism is an independent predictor of all-cause mortality in patients with CKD of various severity. These data support our hypothesis that the FTO gene may be a relevant genetic risk factor for mortality in this population.

Tissue inhibitor of metalloproteinases (TIMP-1), genetic markers of insulin resistance and cardiomyopathy in patients with kidney failure.

BACKGROUND: Left ventricular hypertrophy (LVH) is a major cardiovascular (CV) complication in patients with kidney failure, and an association between polymorphisms in the ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) gene, a genetic marker of insulin resistance, and LVH and Left ventricular (LV) concentric remodelling has been recently documented in these patients. Aims. Since myocardial fibrosis is a prominent feature in LVH induced by insulin resistance, we tested the hypothesis that the interaction between ENPP1 rs1974201 and rs9402349 polymorphisms and the tissue inhibitor of metalloproteinases (TIMP-1)--a pro-fibrotic protein which inhibits extracellular matrix degradation--is implicated in concentric LVH and diastolic dysfunction in a cohort of 223 dialysis patients. RESULTS: Both ENPP1 polymorphisms rs1974201 and rs9402349 were in Hardy-Weinberg equilibrium in dialysis patients. In an analysis stratified by ENPP1, rs1974201 polymorphism, circulating levels of TIMP-1 in GG patients were coherently associated with two markers of concentric remodelling [relative wall thickness (RWT) and LV mass-to-volume ratio] as well as with a marker of diastolic dysfunction (E/A ratio) (P ranging from 0.005 to 0.02), whereas no such associations existed in CC or CG patients. These observations suggest that the rs1974201 modifies the relationship between TIMP-1 and LV geometry and diastolic dysfunction. Accordingly, in a multiple regression model, an identical increase of TIMP-1 (100 ng/mL) was associated with an increase of 22% in RWT, 14% in LV mass-to-volume ratio and 29% in E/A ratio in GG patients but with almost no change (from -0.22 to -3.78%) in these echocardiographic indices in the remaining patients (P for the effect modification ≤ 0.024). The rs9402349 did not modify the relationship between TIMP-1 and LV geometry and function. CONCLUSIONS: In dialysis patients, the ENPP1 rs1974201 polymorphism modifies the association between TIMP-1 and LV geometry and diastolic function. These results are consistent with the hypothesis that insulin resistance is involved not only in LVH but also in myocardial fibrosis, an alteration of primary importance in the high risk of this population.

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: ß = 0.20, P = 0.01; eNOS ß = 0.25, P = 0.001) and CSA (CAV-1: ß = 0.20, P = 0.01: eNOS ß = 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.