Anna Pisano

Transient Receptor Potential Ankyrin 1 Channel Localized to Non-Neuronal Airway Cells Promotes Non-Neurogenic Inflammation

Background The transient receptor potential ankyrin 1 (TRPA1) channel, localized to airway sensory nerves, has been proposed to mediate airway inflammation evoked by allergen and cigarette smoke (CS) in rodents, via a neurogenic mechanism. However the limited clinical evidence for the role of neurogenic inflammation in asthma or chronic obstructive pulmonary disease raises an alternative possibility that airway inflammation is promoted by non-neuronal TRPA1. Methodology/Principal Findings By using Real-Time PCR and calcium imaging, we found that cultured human airway cells, including fibroblasts, epithelial and smooth muscle cells express functional TRPA1 channels. By using immunohistochemistry, TRPA1 staining was observed in airway epithelial and smooth muscle cells in sections taken from human airways and lung, and from airways and lung of wild-type, but not TRPA1-deficient mice. In cultured human airway epithelial and smooth muscle cells and fibroblasts, acrolein and CS extract evoked IL-8 release, a response selectively reduced by TRPA1 antagonists. Capsaicin, agonist of the transient receptor potential vanilloid 1 (TRPV1), a channel co-expressed with TRPA1 by airway sensory nerves, and acrolein or CS (TRPA1 agonists), or the neuropeptide substance P (SP), which is released from sensory nerve terminals by capsaicin, acrolein or CS), produced neurogenic inflammation in mouse airways. However, only acrolein and CS, but not capsaicin or SP, released the keratinocyte chemoattractant (CXCL-1/KC, IL-8 analogue) in bronchoalveolar lavage (BAL) fluid of wild-type mice. This effect of TRPA1 agonists was attenuated by TRPA1 antagonism or in TRPA1-deficient mice, but not by pharmacological ablation of sensory nerves. Conclusions Our results demonstrate that, although either TRPV1 or TRPA1 activation causes airway neurogenic inflammation, solely TRPA1 activation orchestrates an additional inflammatory response which is not neurogenic. This finding suggests that non-neuronal TRPA1 in the airways is functional and potentially capable of contributing to inflammatory airway diseases.

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.

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.

INSULIN RESISTANCE IN CHRONIC KIDNEY DISEASE: A SYSTEMATIC REVIEW

Insulin resistance (IR) is an early metabolic alteration in chronic kidney disease (CKD) patients, being apparent when the glomerular filtration rate is still within the normal range and becoming almost universal in those who reach the end stage of kidney failure. The skeletal muscle represents the primary site of IR in CKD, and alterations at sites beyond the insulin receptor are recognized as the main defect underlying IR in this condition. Estimates of IR based on fasting insulin concentration are easier and faster but may not be adequate in patients with CKD because renal insufficiency reduces insulin catabolism. The hyperinsulinemic euglycemic clamp is the gold standard for the assessment of insulin sensitivity because this technique allows a direct measure of skeletal muscle sensitivity to insulin. The etiology of IR in CKD is multifactorial in nature and may be secondary to disturbances that are prominent in renal diseases, including physical inactivity, chronic inflammation, oxidative stress, vitamin D deficiency, metabolic acidosis, anemia, adipokine derangement, and altered gut microbiome. IR contributes to the progression of renal disease by worsening renal hemodynamics by various mechanisms, including activation of the sympathetic nervous system, sodium retention, and downregulation of the natriuretic peptide system. IR has been solidly associated with intermediate mechanisms leading to cardiovascular (CV) disease in CKD including left ventricular hypertrophy, vascular dysfunction, and atherosclerosis. However, it remains unclear whether IR is an independent predictor of mortality and CV complications in CKD. Because IR is a modifiable risk factor and its reduction may lower CV morbidity and mortality, unveiling the molecular mechanisms responsible for the pathogenesis of CKD-related insulin resistance is of importance for the identification of novel therapeutic targets aimed at reducing the high CV risk of this condition.

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.

Renal Biopsy in 2015--From Epidemiology to Evidence-Based Indications.

Background: Although the number of patients reaching end-stage kidney disease without a biopsy-proven diagnosis is increasing, the utility of renal biopsy is still an object of debate. We analyzed epidemiological data and the main indications for renal biopsy with a systematic, evidence-based review at current literature. Summary: There is a high discrepancy observed in biopsy rates and in the epidemiology of glomerular diseases worldwide, related to the different time frame of the analyzed reports, lack of data collection, the different reference source population and the heterogeneity of indications. The evidence-based analysis of indications showed that renal biopsy should be crucial in adults with nephrotic syndrome or urinary abnormalities as coexistent hematuria and proteinuria and in corticosteroid resistant-children with severe proteinuria. The knowledge of renal histology can change the clinical management in patients with acute kidney injury significantly, after the exclusion of pre-renal or obstructive causes of kidney damage. Scarce evidence indicates that renal biopsy can be useful in patients with advanced chronic kidney disease and its use should always be considered after weighing the benefits and potential risks. Renal biopsy should be crucial in patients with renal involvement due to systemic disease. In patients with diabetes with atypical features, renal biopsy may be fundamental to diagnose an unexpected parenchymal disease mislabeled as diabetic nephropathy. Finally, in elderly patients, the indications and the risks are not different from those in the general population. Key Message: Renal biopsy still remains a concrete approach for managing a substantial percentage of renal diseases.

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.