P2X7 receptor/NLRP3 inflammasome complex and α-synuclein in peripheral blood mononuclear cells: a prospective study in neo-diagnosed, treatment-naïve Parkinson's disease.

BACKGROUND AND PURPOSE: Neuroinflammation and probably systemic inflammation, with abnormal α-synuclein deposition, participate in the development of Parkinson's disease (PD). The P2X7 receptor/NLRP3 inflammasome complex is upregulated in the brain of PD patients. By a prospective approach, the degree of systemic activation of such complex, and its regulatory mechanisms, were explored in treatment-naïve PD individuals. METHODS: The expression and functional activity of the inflammasome were measured in peripheral blood mononuclear cells of 25 newly diagnosed PD patients and 25 controls at baseline and after 12 months of pharmacological treatment, exploring the intracellular signalling involved and its epigenetic regulation. RESULTS: De novo PD patients were characterized by a systemic hyper-expression of the P2X7R/NLRP3 inflammasome platform, probably able to modulate lymphomonocyte α-synuclein, whose brain deposits represent the main pathogenetic factor of PD. A reduced c-Jun N-terminal kinase (JNK) phosphorylation might be the intracellular signalling mediating this effect. miR-7 and miR-30, implied in the pathogenesis of PD and in the post-transcriptional control of α-synuclein and NLRP3 expression, were also increased in PD. After 1 year of usual anti-Parkinson treatments, such inflammatory platform was significantly reduced. CONCLUSIONS: Mononuclear cells of newly diagnosed PD subjects display a hyper-expression of the P2X7R/NLRP3 inflammasome platform that seems to modulate cellular α-synuclein content and is reduced after PD treatment; an impaired JNK phosphorylation might be the intracellular signalling mediating this effect, undergoing an epigenetic regulation by miR-7 and miR-30.

Effect of Treatment of Periodontitis on Incretin Axis in Obese and Nonobese Individuals: A Cohort Study.

CONTEXT: Periodontitis confers an increased risk of developing type 2 diabetes and, in patients with obesity, it might interfere with the incretin axis. The effect of periodontal treatment on glucoregulatory hormones remains unknown. OBJECTIVE: To evaluate the effect of periodontal treatment on incretin axis in obese and lean nondiabetic individuals. SETTING: King's College Dental Hospital and Institute, London, UK. PARTICIPANTS AND METHODS: The metabolic profile of obese and normal-body-mass-index individuals affected by periodontitis was studied at baseline, 2, and 6 months after intensive periodontal treatment, by measuring plasma insulin, glucagon, glucagon-like peptide-1(GLP-1), and glucose-dependent insulinotropic polypeptide (GIP) and markers of systemic inflammation and oxidative stress. MAIN OUTCOME MEASURE(S): Circulating levels of incretins and inflammatory markers. RESULTS: At baseline, periodontal parameters were worse for obese than nonobese; this was accompanied by higher levels of circulating high-sensitivity C-reactive protein (hs-CRP), insulin, and GLP-1. The response to periodontal treatment was less favorable in the obese group, without significant variations of hs-CRP or malondialdehyde. Glucoregulatory hormones changed differently after treatment: while insulin and glucagon did not vary at 2 and 6 months, GLP-1 and GIP significantly increased at 6 months in both groups. In particular, GLP-1 increased more rapidly in obese participants, while the increase of GIP followed similar trends across visits in both groups. CONCLUSIONS: Nonsurgical treatment of periodontitis is associated with increased GLP-1 and GIP levels in nonobese and obese patients; changes in GLP-1 were more rapid in obese participants. This might have positive implications for the metabolic risk of these individuals.

Periodontitis affects glucoregulatory hormones in severely obese individuals.

OBJECTIVE: To evaluate the effect of periodontitis (PD) on glucoregulatory hormones in obesity, never explored so far, a cross-sectional study was conducted in 110 severely obese, non-diabetic individuals. METHODS: We collected clinical periodontal parameters, including probing pocket depth (PPD), bleeding on probing (BOP), clinical attachment level (CAL). Insulin, glucagon, GLP-1 and GIP were measured after 3 days of standardized diet. RESULTS: Forty-seven subjects had periodontitis (PD+) and 63 did not (PD-). PD+ showed 30.3% of gingival sites with PPD > 4 mm, 55.2% of BOP sites and a mean CAL loss of 4.1 mm. Compared with PD-, PD+ had higher glucagon (26.60 [25.22] vs 3.93 [7.50] ng/l, p < 0.0001) and GIP levels (10.56 [13.30] vs 6.43 [8.43] pmol/l, p < 0.001), while GLP-1 was reduced (11.78 [10.07] vs 23.34 [16.80] pmol/l, p < 0.0001). Insulin did not differ. In PD+, after adjustment for confounders, PPD was positively related to glucagon (ß = 0.424, p = 0.002) and inversely to GLP-1 (ß = -0.159, p = 0.044). CONCLUSIONS: We describe for the first time an impaired incretin axis coupled with a relative hyperglucagonemia in obese non-diabetic individuals with PD, that might contribute to deteriorate their glucose tolerance and partially explain the higher risk of diabetes observed in these patients.

Renal Resistive Index Predicts Post-Bariatric Surgery Renal Outcome in Nondiabetic Individuals with Severe Obesity.

OBJECTIVE: Bariatric surgery may ameliorate renal function through vascular mechanisms. This study tested surgery's ability to improve measured glomerular filtration rate (mGFR) and identified clinical, renal, and systemic vascular predictors of such improvement. METHODS: Twenty-five nondiabetic subjects with severe obesity were studied before and 1 year after Roux-en-Y gastric bypass, evaluating mGFR and renal plasma flow, basal renal resistive index (RI) and dynamic renal RI, renal visceral fat, and systemic vascular parameters, including flow-mediated dilation, aortic pulse wave velocity, and carotid intima media thickness and stiffness. RESULTS: After Roux-en-Y gastric bypass, BMI decreased by 31%. At follow-up, body surface area (BSA)-adjusted mGFR increased (from 86.9 ± 15.2 to 109.0 ± 18.2 mL/min/1.73 m2 , P <  0.001), whereas the absolute mGFR did not change. Renal plasma flow did not vary. RI decreased; flow-mediated dilation, pulse wave velocity, carotid intima media thickness, and carotid stiffness improved. mGFR changes after surgery (ΔmGFR/BSA) were associated with younger age and lower fasting glucose. Among vascular variables, an improved ΔmGFR/BSA was associated with smaller brachial artery diameter, lower intima media thickness, and lower RI; this latter association remained after adjusting for covariates. No measure of adiposity was associated with ΔmGFR. CONCLUSIONS: In subjects with obesity and normal renal function, bariatric surgery improves mGFR/BSA (although absolute mGFR is unchanged) and renal and systemic vascular function. Lower renal intravascular resistance can predict these improvements, maximizing them in relatively young individuals.

The level of physical training modulates cytokine levels through P2X7 receptor in healthy subjects.

INTRODUCTION: The P2X7 receptor-NLRP3 inflammasome complex (P2X7R-Infl) regulates inflammatory and immune responses. Physical exercise modulates heat-shock proteins (Hsps), influencing cytokine levels and oxidative stress; Hsp72 triggers P2X7R-Infl-dependent responses. SUBJECTS AND METHODS: We studied the effect of a single bout of maximal exercise on lymphomonocyte expression of P2X7R, NLRP3, caspase-1, NF-kB and Hsp72 and circulating levels of IL-1ß, IL-18 and MCP-1, all modulated by P2X7R-Infl, in healthy sedentary (SED), trained (ATH), endurance (END) male individuals. RESULTS: Baseline P2X7R, NLRP3 and Caspase-1 expression progressively increased from SED to ATH and END; NF-kß showed the same trend. Hsp72 did not differ among groups. Acute exercise strongly reduced P2X7R in all participants, irrespective of their degree of physical training. Inflammasome responses differed across groups: in SED, NLRP3 and Caspase-1 increased; in ATH, NLRP3 reduced and caspase-1 did not vary; in END, NLRP3 and Caspase-1 declined. Baseline IL-1ß, higher in END, was unmodified after exercise; IL-18 decreased; MCP-1 doubled in SED, did not vary in ATH, declined in END. In the whole study population, significant direct relationships emerged between P2X7R expression and IL-1ß, IL-18, MCP-1 levels, all P < .001; also Caspase-1 related with these markers. A multivariate analysis showed age, BMI and P2X7R as determinants of postexercise IL-1ß levels. CONCLUSION: Endurance show higher P2X7R-Infl expression and function vs SED and ATH; however, maximal exercise determines prevailing pro-inflammatory vs anti-inflammatory responses in untrained and trained participants, respectively, highlighting a likely cause-effect relationship between degree of physical activity and P2X7R-Infl-mediated responses.

Deficiency of the Purinergic Receptor 2X7 Attenuates Nonalcoholic Steatohepatitis Induced by High-Fat Diet: Possible Role of the NLRP3 Inflammasome.

Molecular mechanisms driving transition from simple steatosis to nonalcoholic steatohepatitis (NASH), a critical step in the progression of nonalcoholic fatty liver disease (NAFLD) to cirrhosis, are poorly defined. This study aimed at investigating the role of the purinergic receptor 2X7 (PR2X7), through the NLRP3 inflammasome, in the development of NASH. To this end, mice knockout for the Pr2x7 gene (Pr2x7-/-) and coeval wild-type (WT) mice were fed a high-fat diet (HFD) or normal-fat diet for 16 weeks. NAFLD grade and stage were lower in Pr2x7-/- than WT mice, and only 1/7 Pr2x7-/- animals showed evidence of NASH, as compared with 4/7 WT mice. Molecular markers of inflammation, oxidative stress, and fibrosis were markedly increased in WT-HFD mice, whereas no or significantly reduced increments were detected in Pr2x7-/- animals, which showed also decreased modulation of genes of lipid metabolism. Deletion of Pr2x7 gene was associated with blunted or abolished activation of NLRP3 inflammasome and expression of its components, which were induced in liver sinusoidal endothelial cells challenged with appropriate stimuli. These data show that Pr2x7 gene deletion protects mice from HFD-induced NASH, possibly through blunted activation of NLRP3 inflammasome, suggesting that PR2X7 and NLRP3 may represent novel therapeutic targets.

P2X7 Receptor and APOE Polymorphisms and Survival from Heart Failure: A Prospective Study in Frail Patients in a Geriatric Unit.

Heart failure (HF) is one of the most frequent cause of hospitalization in elderly and often coexists with concurrent geriatric syndromes, like cognitive disturbances; various pathophysiological mechanisms are shared by HF and cognitive decline, notably a substrate of low-grade inflammation. We investigated whether SNPs in the purinergic receptor (P2X7R) and apolipoprotein (APO) E genes, both involved in a series of inflammatory responses, are associated to HF or cognitive impairment and are able to predict post-discharge mortality in the elderly. We prospectively analyzed 198 patients (age 85 ± 8 years, predominantly females) admitted to a Geriatric unit for acute HF, whose diagnosis was based on clinical signs, brain natriuretic peptide (BNP) values and ecocardiography in uncertain diagnosis (BNP values between 100 and 400 pg/mL); cognitive performance was assesed by Short Portable Mental Status Questionnaire (SPMSQ). In all the participants, SNPs rs208294 and rs3751143 for P2X7R gene and rs429558 and rs7412 for APOE gene were assessed. Information on all-cause mortality was adjudicated by medical records review 36 months after discharge. We found no relationship between P2X7R and APOE polymorphisms and 36-month post-discharge mortality; a better outcome for overall survival was observed in patients with BNP values below the median (281 pg/mL) (p=0.002) persisting after adjustment for renal function and age, and in those with cognitive impairment (p<0.001). Patients harboring APOE-ε4 genotype showed higher BNP concentrations than noncarriers (1289.9 ± 226.9 vs 580.5 ± 90.2 pg/mL respectively,p=0.004), whereas none of the studied SNPs were associated to impairment in cognitive performance. In conclusion, neither P2X7R or APOE genotype seem to predict long-term mortality in elderly patients. Interestingly, APOE-ε4 genotype was associated to higher BNP values, suggesting a putative interaction between genetic and biochemical markers in identifying people at risk for HF.

Determinants of glomerular filtration rate following bariatric surgery in individuals with severe, otherwise uncomplicated, obesity: an observational, prospective study.

AIMS: Obesity-induced nephropathy is an established clinical entity arising from a "maladaptive" response to lipid accumulation at the nephron level. Bariatric surgery positively affects renal function, reducing or increasing glomerular filtration rate (GFR) in subjects with hyperfiltration and renal impairment, respectively. The effect of this surgery in patients with normal estimated GFR (eGFR) is less clear. METHODS: A complete clinical and biochemical assessment of 135 severely obese, otherwise healthy subjects, was obtained before Roux-en-Y gastric bypass (RYGB). All subjects underwent an OGTT with plasma glucose and insulin determinations. Follow-up data were recorded at 6, 12, 24 and 48 months after intervention. RESULTS: Baseline eGFR was 98.2 ± 13.6 ml/min/1.73 m2; hyperfiltration (>120 ml/min/1.73 m2) was present in 7% of the cohort. No eGFR variation over the follow-up emerged, except at the last visit (-3.6 ± 1.4 ml/min/1.73 m2 at month 48, p = 0.01 vs baseline). In the univariate analysis, the renal performance at 48 months was inversely related to baseline eGFR (r = -0.17, p = 0.04) and plasma triglycerides (r = -0.04, p = 0.05). Fasting and OGTT-derived variables did not impact eGFR. By multiple regression analysis, eGFR time course was independently predicted only by baseline eGFR (p = 0.03). Interestingly, patients having a baseline eGFR >100 ml/min/1.73 m2 (median value) showed, after 48 months, an average loss of -8.3 ± 2.2 ml/min/1.73 m2, while those with eGFR <100 exhibited a slight increase (+1.8 ± 2.3 ml/min/1.73 m2, p < 0.01). CONCLUSIONS: Long-term data confirm the safety of RYGB on renal function. Interestingly, a subtle hyperfiltration, i.e., occurring in high-normal range of eGFR, is attenuated by surgical procedure. Lastly, high serum triglycerides may track an unfavorable renal outcome.

Heterozygosity for the rs696217 SNP in the Preproghrelin Gene Predicts Weight Loss After Bariatric Surgery in Severely Obese Individuals.

BACKGROUND: Several patients encompass a scarce weight loss after Roux-en-Y gastric bypass (RYGB). As such event is not related to surgical complications, finding markers able to identify "well responders" and to predict weight loss outcome is clinically relevant. Ghrelin regulates appetite and energy balance. Common single nucleotide polymorphisms (SNPs) in its encoding genes have been associated with body weight regulation. Other peptides involved in satiety modulation, like the CD40/CD40L complex, are less explored. METHODS: One hundred, otherwise healthy, obese subjects (aged 45 ± 11 years, 65 females, BMI 48.0 ± 0.7 kg/m2) were sequentially enrolled in years 2014-2015. SNPs rs2241766 for adiponectin gene, rs490683 for ghrelin receptor, rs696217 and rs27647 for the preproghrelin/ghrelin gene, and rs1126535 for the CD40L gene were determined on DNA extracted from circulating lymphomonocytes. Patients were reevaluated at 6 (n = 100), 26 (n = 91), and 52 weeks (n = 79) after RYGB. RESULTS: Subjects carrying the rs696217 T allele encompassed a significantly greater reduction in BMI 52 weeks after surgery (GG vs GT 30.5 ± 1.1 vs 38.1 ± 2.1 %; p < 0.001). Carrying the rs1126535 C allele in the CD40L gene was associated with a significantly lower BMI reduction at week 52 (TT vs CT 33.2 ± 1.1 vs 28.1 ± 2.3 %, p = 0.049). rs490683 and rs27647 SNPs of ghrelin and rs2241766 for adiponectin gene did not show any difference between carriers and non-carriers of the mutant allele. CONCLUSION: Carrying a G to T substitution in rs696217 (preproghrelin gene) seems to mark a successful weight loss outcome; we also report for the first time that the rs1126535 C allele (CD40L gene) may predict a worse response to bariatric surgery.

Metabolic and Hormonal Determinants of Glomerular Filtration Rate and Renal Hemodynamics in Severely Obese Individuals.

OBJECTIVE: Renal function is often compromised in severe obesity. A true measurement of glomerular filtration rate (GFR) is unusual, and how estimation formulae (EstForm) perform in such individuals is unclear. We characterized renal function and hemodynamics in severely obese individuals, assessing the reliability of EstForm. METHODS: We measured GFR (mGFR) by iohexol plasma clearance, renal plasma flow (RPF) by 123I-ortho-iodo-hippurate, basal and stimulated vascular renal indices, endothelium-dependent and -independent vasodilation using flow-mediated dilation (FMD) as well as metabolic and hormonal profile in morbid, otherwise healthy, obese subjects. RESULTS: Compared with mGFR, the better performing EstForm was CKD-EPI (5.3 ml/min/1.73 m2 bias by Bland-Altman analysis). mGFR was directly related with RPF, total and incremental glucose AUC, and inversely with PTH and h8 cortisol. Patients with mGFR below the median shown significantly higher PTH and lower vitamin D3. Basal or dynamic renal resistive index, FMD, pulse wave velocity were not related with mGFR. In an adjusted regression model, renal diameter and plasma flow remained related with mGFR (R2 = 0.67), accounting for 15% and 21% of mGFR variance, respectively. CONCLUSIONS: CKD-EPI formula should be preferred in morbid obesity; glucose increments during oral glucose tolerance test correlate with hyperfiltration; RPF and diameter are independent determinants of mGFR; slightly high PTH values, frequent in obesity, might influence mGFR.

Adaptation of ß-Cell and Endothelial Function to Carbohydrate Loading: Influence of Insulin Resistance.

High-carbohydrate diets have been associated with ß-cell strain, dyslipidemia, and endothelial dysfunction. We examined how ß-cell and endothelial function adapt to carbohydrate overloading and the influence of insulin resistance. On sequential days in randomized order, nondiabetic subjects (classified as insulin-sensitive [IS] [n = 64] or insulin-resistant [IR] [n = 79] by euglycemic clamp) received four mixed meals over 14 h with either standard (300 kcal) or double carbohydrate content. ß-Cell function was reconstructed by mathematical modeling; brachial artery flow-mediated dilation (FMD) was measured before and after each meal. Compared with IS, IR subjects showed higher glycemia and insulin hypersecretion due to greater ß-cell glucose and rate sensitivity; potentiation of insulin secretion, however, was impaired. Circulating free fatty acids (FFAs) were less suppressed in IR than IS subjects. Baseline FMD was reduced in IR, and postprandial FMD attenuation occurred after each meal, particularly with high carbohydrate, similarly in IR and IS. Throughout the two study days, higher FFA levels were significantly associated with lower (incretin-induced) potentiation and impaired FMD. In nondiabetic individuals, enhanced glucose sensitivity and potentiation upregulate the insulin secretory response to carbohydrate overloading. With insulin resistance, this adaptation is impaired. Defective suppression of endogenous FFA is one common link between impaired potentiation and vascular endothelial dysfunction.

Tumour necrosis factor-alpha participates on the endothelin-1/nitric oxide imbalance in small arteries from obese patients: role of perivascular adipose tissue.

AIMS: We assessed the impact of vascular and perivascular tumour necrosis factor-alpha (TNF-α) on the endothelin (ET)-1/nitric oxide (NO) system and the molecular pathways involved in small arteries from visceral fat of obese patients (Obese) and Controls. METHODS AND RESULTS: Isolated small arteries from 16 Obese and 14 Controls were evaluated on a pressurized micromyograph. Endogenous ET-1 activity was assessed by the ETA blocker BQ-123. TNF-α and NO were tested by anti-TNF-α infliximab (IFX) and N(ω)-nitro-l-arginine methylester (L-NAME). Gene and protein expression of TNF-α, ET-1, ETA, and ETB receptors were determined by RT-PCR and IHC on arterial wall and in isolated adipocytes. Obese showed a blunted L-NAME-induced vasoconstriction, which was potentiated by IFX, and an increased relaxation to BQ-123, unaffected by L-NAME but attenuated by IFX. Perivascular adipose tissue (PVAT) removal reversed these effects. Obese showed intravascular superoxide excess, which was decreased by apocynin (NAD(P)H oxidase inhibitor), L-NAME, and BQ-123 incubations, and abolished by IFX. An increased vascular expression of ET-1, ETA, and ETB receptors, and higher vascular/perivascular TNF-α and TNF-α receptor expression were also detected. The arterial expression and phosphorylation of c-Jun N-terminal kinase (JNK) were higher in Obese vs. Controls, and downregulated by IFX. CONCLUSIONS: In small arteries of Obese, PVAT-derived TNF-α excess, and an increased vascular expression of ET-1 and ETA receptor, contribute to the ET-1/NO system imbalance, by impairing tonic NO release. Reactive oxygen species excess, via NAD(P)H oxidase activation, induces the endothelial nitric oxide synthase uncoupling, which in turn generates superoxide and impairs NO production. The up-regulated JNK pathway represents a crucial molecular signalling involved in this process.

The complex P2X7 receptor/inflammasome in perivascular fat tissue of heavy smokers.

OBJECTIVE: Smoking is a recognized cardiovascular risk factor. Perivascular visceral adipose tissue (PVAT) is a source of inflammatory molecules, thus contributing to atherosclerosis progression. The P2X7 receptor (P2X7 R)-inflammasome complex, crucial in determining IL-1ß and IL-18 release, participates in this scenario. We evaluated whether smoking might affect the PVAT inflammatory phenotype and explored the putative role of the axis P2X7 R-inflammasome in this picture. SUBJECTS AND METHODS: TNFα, IL-6, RBP4, MCP-1, as well as P2X7 R and inflammasome components NLRP3, ASC, caspase-1 and IL-1ß and IL-18 expression was determined in adipocytes isolated by PVAT of healthy smokers (Smok) and nonsmokers (No-Smok) subjects. Plasma and culture medium levels of these cytokines were also determined. RESULTS: Perivascular adipose tissue of Smok had a higher expression of P2X7 R and inflammasome components; via P2X7 R activation, it released more IL-1ß and IL-18, whose serum levels were also higher in Smok than in No-Smok. Linear correlations of NLRP3 with P2X7 R and IL-18 expression and release emerged. Smok also had a higher PVAT expression of the chemotactic factor MCP-1. However, no difference was observed in the PVAT expression of genes more strictly related to insulin resistance, like TNFα, RBP4, IL-6; this was coupled with similar plasma levels of TNFα and RBP4 in the two groups. CONCLUSION: Smoking contributes to the pro-inflammatory status of the PVAT by enhancing expression and activity of the P2X7 R-inflammasome complex; the effect on adipocytokines more related to insulin resistance and metabolic abnormalities appears trivial.

P2X7 receptor polymorphisms do not influence endothelial function and vascular tone in neo-diagnosed, treatment-naive essential hypertensive patients.

OBJECTIVE: Endothelial dysfunction and arterial stiffness are early vascular alterations, linked to oxidative stress and inflammation, with prognostic significance in essential hypertensive patients. P2X7 receptors (P2X7R), responding to extracellular ATP, are encoded by a highly polymorphic gene and modulate inflammatory responses and cell growth, potentially playing a role in the control of vascular tone. This study evaluated the effects of P2X7R gene polymorphisms (single nucleotide polymorphisms, SNPs) on a detailed vascular hypertensive phenotype. METHODS: We determined by real-time PCR two SNPs of the P2X7R gene (489C>T and 1513A>C) in 134 newly diagnosed, treatment-naive essential hypertensive patients and 131 normotensive controls (CTL). Endothelium-dependent response was assessed as flow-mediated dilatation (FMD) of the brachial artery, arterial stiffness as aortic pulse wave velocity (PWV) and augmentation index (AIx) by tonometry. Markers of oxidative stress were also measured. RESULTS: FMD was lower (P < 0.05), whereas aortic PWV and AIx were higher (P < 0.01) in essential hypertensive patients than in CTL. The allelic distribution of the two P2X7R SNPs was similar in essential hypertensive patients and CTL, either concerning homozygosis or presence of the mutant alleles. No difference was observed for FMD, aortic PWV, AIx or markers of oxidative stress between carriers and noncarriers of the mutant alleles, either in essential hypertensive patients and in CTL. In the whole group, logistic regression showed that the mutant allele of 1513A>C was a main determinant of AIx (odds ratio 1.90; P = 0.03). CONCLUSION: P2X7R 489C>T and 1513A>C SNPs are not associated with altered endothelial function or arterial stiffness in untreated newly diagnosed essential hypertensive patients; a possible role in influencing peripheral wave reflection should be further addressed.

The purinergic 2X7 receptor participates in renal inflammation and injury induced by high-fat diet: possible role of NLRP3 inflammasome activation.

Renal disease associated with type 2 diabetes and the metabolic syndrome is characterized by a distinct inflammatory phenotype. The purinergic 2X7 receptor (P2X7 R) and the nucleotide-binding and oligomerization domain-like receptor containing a pyrin domain 3 (NLRP3) inflammasome have been separately shown to play a role in two models of non-metabolic chronic kidney disease. Moreover, the NLRP3 inflammasome has been implicated in chronic low-grade sterile inflammation characterizing metabolic disorders, though the mechanism(s) involved in inflammasome activation under these conditions are still unknown. We investigated the role of P2X7 R (through activation of the NLRP3 inflammasome) in renal inflammation and injury induced by a high-fat diet, an established model of the metabolic syndrome. On a high-fat diet, mice lacking P2X7 R developed attenuated renal functional and structural alterations as well as reduced inflammation, fibrosis, and oxidative/carbonyl stress, as compared with wild-type animals, in the absence of significant differences in metabolic parameters. This was associated with blunted up-regulation of the NLRP3 inflammasome components NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), pro-caspase 1, pro-interleukin (IL)-1ß, and pro-IL-18, as well as reduced inflammasome activation, as evidenced by decreased formation of mature caspase 1, whereas mature IL-1ß and IL-18 were not detected. Up-regulated expression of NLRP3 and pro-caspase 1, post-translational processing of pro-caspase-1, and release of IL-18 in response to lipopolysaccharide + 2'(3')-O-(4-benzoylbenzoyl)ATP were attenuated by P2X7 R silencing in cultured mouse podocytes. Protein and mRNA expression of P2X7 R, NLRP3, and ASC were also increased in kidneys from subjects with type 2 diabetes and the metabolic syndrome, showing histologically documented renal disease. These data provide evidence of a major role for the purinergic system, at least in part through activation of the NLRP3 inflammasome, in the process driving 'metabolic' renal inflammation and injury and identify P2X7 R and NLRP3 as novel therapeutic targets.

Adipocytokines mark insulin sensitivity in euthyroid Hashimoto's patients.

The relationship between inflammation, Hashimoto's thyroiditis (HT) and insulin resistance is still controversial. In this regard, a pretty complete evaluation of adipocytokines levels in patients with HT has not been performed so far. We assessed retinol binding protein-4 (RBP4), adipocyte-fatty acid binding protein (A-FABP), neutrophil gelatinase-associated lipocalin (NGAL) and tumor necrosis factor-α (TNFα) levels in 93 euthyroid HT patients and 51 healthy controls (CTL), also evaluating the possible correlation between adipocytokines levels and markers of insulin resistance. No significant differences between HT patients and CTL in fasting plasma glucose and insulin levels, and HOMA index were observed. HT patients had significantly higher RBP4, NGAL and A-FABP levels than CTL, while TNFα levels did not differ between the two groups. In HT patients, RBP4 was significantly related with fT3 and fT4 levels, while A-FABP with fT4 only. Moreover, in HT patients, either RBP4 or A-FABP was directly associated with plasma insulin and HOMA index. Circulating levels of these adipocytokines were not influenced by the presence of antithyroid peroxidase or antithyroglobulin autoantibodies or only one of them, neither by autoantibodies titer. In conclusion, euthyroid HT patients are characterized by a peculiar inflammatory response of the adipose tissue, apparently related to an early reduction in insulin sensitivity and to serum thyroid hormone levels, although within the normal range. These results suggest that HT patients with high RBP4 and A-FABP levels might deserve a particular attention, being potentially more exposed to develop insulin resistance and increased cardiovascular risk.

Adipocytokine levels mark endothelial function in normotensive individuals.

BACKGROUND: Endothelial dysfunction is an independent risk factor for cardiovascular events. Inflammatory mediators released by the adipose tissue can lead to local insulin resistance and endothelial dysfunction. This study addressed the relationship of adipocytokines with endothelial function and blood pressure. METHODS: In 92 newly diagnosed, drug-naïve essential hypertensive patients (HT, mean age 49 yrs) without organ damage and 66 normotensive subjects (NT, mean age 47 yrs), by an automated system, we measured endothelium-dependent and -independent vasodilation as brachial artery flow-mediated dilation before and after administration of glyceryl-trinitrate. Retinol binding protein-4 (RBP4) and resistin levels were determined by ELISA and RIA, respectively. Oxidative stress was evaluated by measuring serum malondyaldehyde (MDA). RESULTS: Flow-mediated dilation was significantly (p = 0.03) lower in HT (5.3 ± 2.6%) than NT (6.1 ± 3.1%), while response to glyceryl-trinitrate (7.5 ± 3.7% vs 7.9 ± 3.4%) was similar. RBP4 (60.6 ± 25.1 vs 61.3 ± 25.9 µg/ml), resistin (18.8 ± 5.3 vs 19.9 ± 6.1 ng/ml) and MDA levels (2.39 ± 1.26 vs 2.08 ± 1.17 nmol/ml) were not different in HT and NT.RBP4 (r = -0.25; p = 0.04) and resistin levels (r = -0.29; p = 0.03) were related to flow-mediated dilation in NT, but not in HT (r = -0.03 and r = -0.10, respectively). In NT, multivariate analysis including RBP4 and confounders showed that only BMI or waist circumference remained related to flow- mediated dilation. In the multivariate model including resistin and confounders, BMI, age and resistin were significantly related to flow-mediated dilation, while only age significant correlated with this parameter when BMI was replaced by waist circumference. CONCLUSIONS: Adipocytokine levels may be independent predictors of endothelial dysfunction in the peripheral circulation of healthy subjects, providing a pathophysiological link between inflammation from adipose tissue and early vascular alterations.

Adipocyte P2X7 receptors expression: a role in modulating inflammatory response in subjects with metabolic syndrome?

OBJECTIVE: P2X(7) receptor (P2X(7)R), upon its stimulation with extracellular ATP, modulates several inflammatory responses in different cell types. No information is available on its presence in human adipocytes and its potential involvement in the chronic inflammation associated with metabolic syndrome (MS). Therefore, we evaluated P2X(7)R presence and functional activity in adipocytes from visceral (VAT) and subcutaneous (SAT) adipose tissue of patients with MS and controls (CTL). METHODS: Adipocyte gene expression of TNFα, IL-6 and PAI-1 (by realtime-PCR) and their plasma concentrations (ELISA); P2X(7)R expression (realtime-PCR, Western blot and immunofluorescence); P2X(7)R functional activity (intracellular calcium fluxes by fluorimetry); cytokine release from adipocytes (ELISA). The inflammasome components were also determined. RESULTS: In VAT, TNFα, IL-6 and PAI-1 were more expressed in MS than in CTL. These differences were confirmed in SAT for IL-6 and PAI-1. Plasma IL-6, PAI-1 and TNFα levels were higher in MS. P2X(7)R mRNA and protein, identified in both VAT and SAT, were more abundant in MS than in CTL. Immunofluoresce confirmed the typical "ring-like" arrangement of P2X(7)R at the plasma membrane. Benzoyl-benzoyl-ATP raised intracellular calcium both in VAT and SAT, and induced IL-6, TNFα and PAI-1 release in both MS and CTL cells. This effect was partially inhibited by KN62, specific human P2X(7)R blocker, or by P2X(7)R gene silencing. The inflammasome was more activated in MS than in CTL adipocytes. CONCLUSION: Human adipocytes express functionally active P2X(7)R, which modulate the release of inflammatory cytokines, at least in part via inflammasome activation. Adipocytes from MS patients show an enhanced P2X(7)R expression, which might contribute to the subclinical inflammatory status characterizing these patients and conferring them an increased CV risk.

Angiotensin-II and rosuvastatin influence matrix remodeling in human mesangial cells via metalloproteinase modulation.

OBJECTIVE: Persistent inflammation and oxidative stress influence the progression of diabetic nephropathy. Metalloproteinases (MMPs) participate in extracellular matrix remodeling. Statins show favorable anti-inflammatory effects in chronic kidney disease. We evaluated the effect of rosuvastatin on inflammatory and pro-fibrotic responses due to exposure to different glucose or free fatty acid (FFA) concentrations. METHODS: Human mesangial cells (HMCs) grown at 5.5 (normal glucose) or 22 mmol/l (high glucose) glucose or exposed to FFA were treated with angiotensin-II in the presence or absence of rosuvastatin. We measured MMP-2, MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), and TIMP-2 expression and activity, and quantified the fibrotic factors transforming growth factor-ß1 (TGF-ß1), fibronectin, and collagen IV. RESULTS: At normal glucose, angiotensin-II induced a dose-dependent downregulation of MMP-2; rosuvastatin reversed this effect. On the contrary, TIMP-2 and MMP-9 were upregulated by angiotensin-II and downregulated by rosuvastatin; the effects on TIMP-1 were negligible. Some of the angiotensin-II effects were potentiated in the presence of high glucose and FFA; under both conditions, rosuvastatin was able to reverse these effects. MMP-2 and MMP-9 activity followed the same trend of expression, with rosuvastatin able to upregulate MMP-2 activity. The modulation of the MMP/TIMP system was paralleled by an increase in TGF-ß1, fibronectin, and collagen-IV; all were reduced by rosuvastatin treatment. Silencing the MMP-2 gene confirmed its role in modulating some of these angiotensin-II effects. CONCLUSION: Angiotensin-II induces a pro-fibrotic response in HMCs mainly via a dysregulation of the MMP-2/TIMP-2 pattern. This effect, partially amplified in the presence of high glucose and FFA, is reversed by rosuvastatin, suggesting another potential therapeutic application for this 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor.

Effect of a fatty meal on inflammatory markers in healthy volunteers with a family history of type 2 diabetes.

A family history of type 2 diabetes (T2D) confers a high risk of developing the disease, independent of that due to other common risk factors. Postprandial state is a pro-inflammatory condition associated with a transiently impaired endothelial function; an increased oxidative stress is considered as a mediator of such effects in T2D. We evaluated the short-term effect of a lipid meal on markers of early vascular damage in subjects at risk of developing T2D. A total of thirty-two healthy volunteers, divided according to the presence (FHD+) or absence (FHD - ) of a family history of T2D, underwent a fatty meal test. We measured the monocyte mRNA expressions of IL-6, IL-8 and IL-1ß, and IL-6, soluble CD40 ligand (sCD40L), vascular cellular adhesion molecule-1 (VCAM-1), intracellular adhesion molecule-1 (ICAM-1) and nitrotyrosine plasma concentrations at baseline and in the post-meal phase, relating them to the lipid profile and other biochemical parameters. The basal expression of the cytokines did not differ in FHD -  and FHD+ subjects; neither was it modified by the meal ingestion. IL-6 and sCD40L plasma levels, similar in the two groups in the fasting state, did not vary after the meal. VCAM-1 and ICAM-1 increased in FHD+ subjects but not in FHD -  subjects. Nitrotyrosine, similar between the FHD -  and FHD+ subjects at baseline, increased more in FHD+ subjects than in FHD -  subjects after the meal. In conclusion, the presence of a familial history of T2D confers an abnormal endothelial activation after an oral lipid meal, coupled with an increased oxidative stress, supporting the hypothesis of an early endothelial dysfunction already present in healthy individuals prone to develop T2D.