Chromosomal structural rearrangements consist of anomalies in genomic architecture that may or may not be associated with genetic material gain and loss. Evaluating the precise breakpoint is crucial from a diagnostic point of view, highlighting possible gene disruption and addressing to appropriate genotype–phenotype association. Structural rearrangements can either occur randomly within the genome or present with a recurrence, mainly due to peculiar genomic features of the surrounding regions. We report about three non-related individuals, harboring chromosomal structural rearrangements interrupting SETBP1 , leading to gene haploinsufficiency. Two out of them resulted negative to Chromosomal Microarray Analysis (CMA), being the rearrangement balanced at a microarray resolution. The third one, presenting with a complex three-chromosome rearrangement, had been previously diagnosed with SETBP1 haploinsufficiency due to a partial gene deletion at one of the chromosomal breakpoints. We thoroughly characterized the rearrangements by means of Optical Genome Mapping (OGM) and Whole Genome Sequencing (WGS), providing details about the involved sequences and the underlying mechanisms. We propose structural variants as a recurrent event in SETBP1 haploinsufficiency, which may be overlooked by laboratory routine genomic analyses (CMA and Whole Exome Sequencing) or only partially determined when associated with genomic losses at breakpoints. We finally introduce a possible role of SETBP1 in a Noonan-like phenotype.

Indole-3-acetic acid is a protein-bound indolic uremic toxin deriving from tryptophan metabolism. Increased levels are associated with higher thrombotic risk and both cardiovascular and all-cause mortality. An emerging biomarker of cardiovascular disease is the monocyte-to-high-density lipoprotein ratio (MHR). The main purpose of this study was to investigate the association of indole-3-acetic acid with MHR and other markers of cardiovascular risk in patients with chronic kidney disease (CKD). We enrolled 61 non-dialysis CKD patients and 6 dialysis patients. Indole-3-acetic acid levels were measured with ELISA technique. In the whole cohort of 67 patients, indole-3-acetic acid was directly related to Ca × P (ρ = 0.256; P = 0.0365) and MHR (ρ = 0.321; P = 0.0082). In the 40 patients with previous cardiovascular events, indole-3-acetic acid correlated with uric acid (r = 0.3952; P = 0.0116) and MHR (ρ = 0.380; P = 0.0157). MHR was related with fibrinogen (ρ = 0.426; P = 0.0010), arterial hypertension (ρ = 0.274; P = 0.0251), C-reactive protein (ρ = 0.332; P = 0.0061), gender (ρ = − 0.375; P = 0.0017; 0 = male, 1 = female), and CKD stage (ρ = 0.260; P = 0.0337). A multiple regression analysis suggested that indole-3-acetic acid might be an independent predictor of MHR. This study shows a significant association between indole-3-acetic acid and MHR. Prospective studies are required to evaluate if decreasing indole-3-acetic acid concentrations may reduce MHR levels and cardiovascular events and improve clinical outcomes.

Aging is a physiological process that induces several changes in cells and tissues, resulting in a greater risk of disease and death. Though this process has been extensively studied both in vivo and in vitro, including human erythrocytes which are an appropriate model to study aging, the involved pathways have not been fully elucidated. D‐Galactose (D‐Gal) has been widely used in vitro to model the characteristics of premature aging. Nevertheless, there is few information regarding its effect on Band 3 protein (B3p) in human erythrocytes. Band 3 protein is an anion exchanger, essential to maintain homeostasis of erythrocytes and, in turn, of the whole organism.

CONCLUSION: An enriched but dysbiotic gut microbiota was presented in ESKD patients, in whichthe bacteria that were present increase amino acid metabolism linked to the production of protein-bound uremic toxins. BACKGROUND AIMS: Obesity is associated with lower circulating levels of N- terminal pro-BNP (NT-proBNP) both in the general population and in patients with heart failure. In asymptomatic patients with CKD, NT-proBNP levels are generally elevated. The prevalence of obesity is markedly increasing among patients with CKD. However, whether obesity NT-proBNP levels and thresholds for increased risk of clinical unclear. METHOD: We examined the associations between NT-proBNP and obesity in 408 stage 3–5 CKD patients (268 men and 140 women; mean age 65 6 13 years) free of heart failure at baseline, who were categorized into 3 groups according to body mass index (BMI; kg/m 2 ) as normal ( < 24.0), overweight (24.0–27.9), and obese ( (cid:2) 28.0). Multivariate Cox proportional hazards models were used to compare the risk associations of NT-proBNP with the composite of ESKD (needing chronic dialysis or kidney transplantation) or all-cause mortality across BMI categories. RESULTS: NT-proBNP levels were significantly lower in obese patients than in normal weight and overweight subjects ( P < 0.001). Within each CKD stage, NT-proBNP levels decreased with increasing BMI. In a multivariate analysis, NT-proBNP was inversely associated with obesity, as well as BMI and its fat and lean body mass components (all P < 0.001), independent of demographics, comorbidities, and severity of CKD. During a median follow-up of 2.2 years, 117 patients progressed to ESKD and 46 patients died. Overall, risk thrombotic both all-cause An emerging biomarker the monocyte to high-density lipoprotein (HDL) ratio (MHR). Its prognostic value related to the ability of monocytes several cytokines involved in inflammation and atherogenesis to the protective role of HDL removal of cholesterol from peripheral tissues of both monocyte progenitor cell proliferation and differentiation and monocyte activation. In this single-centre cross-sectional observational study, we investigated the potential association of IAA with MHR and other markers of cardiovascular risk in a cohort of patients with CKD and evaluated the effect of a single midweek dialysis session with AFB (Acetate-free Biofiltration) technique on IAA serum concentrations. METHOD: We enrolled 61 non-dialysis CKD adult patients and 6 dialysis patients treated with AFB technique. IAA levels were measured using an enzyme-linked immunosorbent assay (ELISA) kit (Cat. number abx150354; Abbexa Ltd, Cambridge, UK). Post-dialysis IAA levels were corrected for haemoconcentration. RESULTS: In the whole cohort of 67 patients, IAA was directly related to creatinine ( q = 0.247; P = 0.0441), potassium (r = 0.2871; P = 0.0185), Ca x P product ( q = 0.256; P = 0.0365) and MHR ( q = 0.321; P = 0.0082). After adjustment for creatinine, correlation between IAA potassium significant = 0.1968; P = 0.1133). Stratifying noticed neuronal progenitor differentiating the progenitor cells with differentiating ability The aim of the study is to investigate the IS mediated cell damage on differentiating neuronal progenitor cells METHOD: The study was divided into 2 parts: clinical investigation and in vitro study. The clinical investigation collected 36 participants (12 health controls and 24 ESRD patients with cognitive impairment by MMSE < =24). The in vitro study used the human neuroblastoma cell line SH-SY5Y cells with differentiating protocol by retinoic acid (RA, with concentration 10%). Indoxyl sulfate was given at concentration at 0, 25, 50 and 100 l M at initiation of retinoic acid given and the 7 days after retinoic acid was given. Microscopic morphology, MTT assay, annexin V-PI dual stain,

Zoledronic acid (Zol) is a widely used intravenous aminobisphosphonate to treat both benign and malignant skeletal diseases, and bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a serious side effect whose pathophysiology remains poorly understood. Vascular Endothelial Growth Factor (VEGF) has been recognized to mediate BRONJ in cancer patients undergoing Zol treatment, however data on VEGF are lacking in patients with osteoporosis. Increasing evidences demonstrate that vitamin D influences VEGF levels. The aim of this study was to investigate the influence of Zol on VEGF levels and the possible role for vitamin D on the Zol mediated changes of VEGF concentration in women with postmenopausal osteoporosis. Twenty-eight postmenopausal women with osteoporosis were enrolled and randomized into two groups to receive Zol (5 mg) or placebo. At baseline, at day-3 and day-30 VEGF serum levels were measured; bone turnover markers, 25-hydroxyvitamin D [25(OH)D] and serum calcium were evaluated at baseline. In Zol-treated women, VEGF increased significantly on day-3, and then decreased on day-30. In the Zol-treated women, the percent change of VEGF levels between baseline and day-30 (−18% at day-30 vs. baseline, p = 0.01) was significantly associated with serum 25(OH)D values (r = 0.29, p = 0.028). At a stepwise multiple regression analysis, after correcting for age, BMI, time since menopause, femoral neck BMD, osteocalcin, C-terminal telopeptide of type 1 collagen, and baseline VEGF levels, 25(OH)D levels were independently associated with VEGF change (β = 1.7, SE = 0.71, p = 0.03). For the first time, we detected early modifications of circulating VEGF in postmenopausal women receiving Zol for osteoporosis, identifying a vitamin D-dependent modulation of these changes.

Background: Zoledronate (Zol) is an aminobisphosphonate commonly used to treat osteoporosis and other benign and malignant skeletal diseases. Exposure to bisphosphonates has been previously associated with the risk of osteonecrosis of the jaw (BRONJ), a rare but serious side effect. In cancer patients, the Vascular Endothelial Growth Factor (VEGF) has been advocated to take part to BRONJ pathogenesis via interfering with angiogenesis. No information is currently available on the VEGF concentrations after Zol administration for osteoporosis. Objectives: To explore change of VEGF concentrations after Zol administration in postmenopausal women with osteoporosis. Methods: A total of twenty-eight postmenopausal women with osteopenia or osteoporosis and at least one prevalent vertebral fracture were recruited and randomized in two groups. Eighteen women received a single i.v. dose of Zol 5 mg, while the other ten served as controls. Serum samples were collected at baseline, after 3 and 30 days, for repeated measurements of VEGF, and measurement of bone turn over markers and 25-hydroxyvitamin D (25(OH)D). Results: VEGF levels increased significantly after 3 days in women receiving Zol; then levels decreased after 30 days compared with VEGF concentrations at both day-3 and baseline (-18% at day-30 vs. baseline, p=0.01). 25(OH)D level, a surrogate of vitamin D status, was associated with VEGF change at the end of the study (r=0.29, p=0.028), and this association was maintained also after correcting for age, BMI, time since menopause, femoral neck BMD, osteocalcin, C-terminal telopeptide of collagen type 1 and baseline levels of VEGF (β=1.7, SE=0.71, p=0.03). Conclusion: Zol administration induced a reduction of circulating VEGF in postmenopausal women treated for osteoporosis, and vitamin D status has been showed to modulate this change. Further studies in this setting of women are needed to define whether VEGF modifications may predict the risk of BRONJ. Disclosure of Interests: None declared.

The recurrent 2q13 deletion syndrome is a rare genetic disorder associated with developmental delay, cardiac and urogenital malformations, and minor facial anomalies. Congenital heart defects (CHDs) are the most frequent malformations associated with del2q13. Experimental studies in zebrafish suggest that two genes mapping within the 2q13 critical region (FBLN7 and TMEM87B) could confer susceptibility to congenital heart defects in affected individuals. We reviewed the cardiac characteristics in four patients with 2q13 deletion admitted to our hospitals, and in published patients. Two of our patients had congenital heart defects, consisting in partial anomalous pulmonary venous connection, ostium secundum atrial septal defect ostium secundum, and small muscular ventricular septal defect in one of them, and aortic valve insufficiency with partial fusion of two commissures (incomplete bicuspid aortic valve) and mitral valve insufficiency due to trivial mitral valve prolapse in the other. The anatomic types of CHD in del2q13 syndrome are highly variable and distributed widely, including laterality defects, complex atrioventricular septal defect, septal anomalies, and cardiomyopathies. Cardiac evaluation should be part of the clinical workup at diagnosis of 2q13 deletion.

Magnesium (Mg) is critically involved in the pathophysiology of multiple human diseases; nevertheless, Mg disorders are often poorly considered in the clinical practice. To update the prevalence and incidence of hypomagnesemia and hypermagnesemia in a real-life scenario, which better represents clinical practice, we analyzed data from 12,696 patients whose Mg serum levels were measured from January 1, 2015, through December 31, 2017 at our University Hospital. Hypomagnesemia and hypermagnesemia were defined by Mg concentrations <1.5 mg/dL (0.6 mmol/L) and >3.8 mg/dL (1.5 mmol/L), in accordance with the reference values for magnesemia of our laboratory (1.5-3.8 mg/dL). The prevalence of hypomagnesemia and hypermagnesemia was 8.43% (n=1071) and 1.78% (n=226), respectively. Hypomagnesemia occurred more frequently in females compared with males [53.3% (n=560) versus 47.7% (n=511), χ2=4.03, p<0.045]; the highest prevalence of hypomagnesemia was found in patients over 65 yr. [59.01% (n=632)], when compared with the other age groups [59.01% (n=632) versus 9.52% (n=102) in patients aged 0-18 yr. and 31.46% (n=337) in patients between 19 and 65 yr., χ2=592.64; p<0.0001)]. Incidence of hypomagnesemia decreased over time in subjects over 65 yr. (r=-0.99; p=0.07). Geriatrics, oncology, and intensive care division showed the highest incidences of hypomagnesemia. Mg disorders and remarkably hypomagnesemia are quite common in the clinical practice, particularly in older hospitalized patients. Thus, they should be routinely checked and corrected.

To verify whether myo-inositol plus α-lactalbumin may reduce insulin resistance and excessive fetal growth in women with gestational diabetes mellitus. In a 12-month period, 120 women with a diagnosis of gestational diabetes mellitus were consecutively enrolled with an allocation of 1:1 in each group and randomly treated with myo-inositol plus α-lactalbumin plus folic acid (treated group) or folic acid (control group) for 2 months. Primary outcome was the variation of insulin resistance through the study evaluated by HOMA-IR. Secondary outcome was the evaluation, through the study, of fetal growth by ultrasound measurements of abdominal circumference centiles and estimated fat thickness. Some clinical outcomes were also considered. After 2 months, in the treated group, a significant reduction in insulin resistance (HOMA values 3.1 ± 1.4 vs 6.1 ± 3.4, p = 0.0002) and fetal growth was shown (Abdominal circumference centiles 54.9 ± 23.5 vs 67.5 ± 22.6, P = 0.006). Among clinical outcomes, a significant decrease in the rate of women who needed insulin (6.7% vs 20.3%, p = 0.03) and of pre-term birth (0 vs 15.2%, p = 0.007) was evidenced. A combination of myo-inositol and α-lactalbumin may reduce insulin resistance and excessive fetal growth. Clinical trial registration : ClinicalTrials.gov, http://www.clinicaltrials.gov , NCT 03763669, first posted date 04/12/2018; last posted date December 06/12/2018.

Abstract Background and Aims Indole-3-acetic acid (IAA, also called auxin) is a protein-bound indolic uremic toxin deriving from tryptophan metabolism by the intestinal bacteria. Previous studies have shown that increased IAA is associated with enhanced tissue factor synthesis in endothelial and peripheral blood mononuclear cells, oxidative stress and endothelial inflammation with resulting higher risk of thrombotic events and both cardiovascular and all-cause mortality. An emerging biomarker of cardiovascular disease is the monocyte to high-density lipoprotein (HDL) ratio (MHR). Its prognostic value is related to the ability of monocytes to release several cytokines involved in inflammation and atherogenesis and to the protective role of HDL through removal of cholesterol from peripheral tissues and suppression of both monocyte progenitor cell proliferation and differentiation and monocyte activation. In this single-centre cross-sectional observational study, we investigated the potential association of IAA with MHR and other markers of cardiovascular risk in a cohort of patients with CKD and evaluated the effect of a single midweek dialysis session with AFB (Acetate-free Biofiltration) technique on IAA serum concentrations. Method We enrolled 61 non-dialysis CKD adult patients and 6 dialysis patients treated with AFB technique. IAA levels were measured using an enzyme-linked immunosorbent assay (ELISA) kit (Cat. number abx150354; Abbexa Ltd, Cambridge, UK). Post-dialysis IAA levels were corrected for haemoconcentration. Results In the whole cohort of 67 patients, IAA was directly related to creatinine (ρ = 0.247; P = 0.0441), potassium (r = 0.2871; P = 0.0185), Ca x P product (ρ = 0.256; P = 0.0365) and MHR (ρ = 0.321; P = 0.0082). After adjustment for creatinine, the correlation between IAA and potassium became not significant (r = 0.1968; P = 0.1133). Stratifying patients according to the history of cardiovascular disease, in the 40 patients with previous cardiovascular events IAA levels correlated significantly with uric acid (r = 0.3952; P = 0.0116) and MHR (ρ = 0.380; P = 0.0157). In the remaining 27 patients without history of cardiovascular disease, IAA only correlated with potassium (r = 0.3912; P=0.0481) and, though borderline significantly, with creatinine (ρ = 0.349; P = 0.0805). To assess whether IAA would independently predict MHR values, we evaluated potential correlations of MHR with risk factors for cardiovascular disease. MHR was related with fibrinogen (ρ = 0.426; P = 0.0010), arterial hypertension (ρ = 0.274; P = 0.0251), C-reactive protein (ρ = 0.332; P = 0.0061), gender (ρ = -0.375; P = 0.0017; 0 = male, 1 = female), and CKD stage (ρ = 0.260; P = 0.0337). A multiple regression analysis identified IAA as an independent predictor of MHR. Lastly, IAA levels were higher in dialysis patients compared to non-dialysis CKD patients (97.44 ± 21.58 versus 65.08 ± 24.38 ng/ml respectively; P = 0.0026) and it was significantly removed by a single AFB session (97.44 ± 21.58 versus 54.59 ± 21.74 ng/ml; P = 0.0028) with a reduction ratio of 43.80 ± 17.47%. Conclusion This study shows a statistically significant association between IAA and MHR. Based on previous experimental studies, such relationship could be explained by the activation of the transcription factor aryl hydrocarbon receptor. Indeed, IAA is a potent ligand of aryl hydrocarbon receptor and the latter has proinflammatory and proatherogenic activities and can reduce HDL levels. Moreover, AFB efficiently removes IAA during a single dialysis session. Prospective studies with appropriate sample size and sufficiently long period of observation are required to evaluate if decreasing IAA levels, through targeted therapeutic strategies in non dialysis CKD patients or by optimization of dialysis techniques and prescriptions in patients receiving renal replacement therapy, may reduce MHR levels and cardiovascular events and improve clinical outcomes and survival.

Background: Systemic sclerosis (SSc) is characterized by early vasculopathy and fibrosis in the skin, lungs, and other tissues. Vascular manifestations of SSc include Raynaud’s phenomenon, digital ulcers, and pulmonary artery hypertension (PAH). PAH is the second most common cause of mortality in SSc. Circulating CD34+ cells associated with cardiovascular health status in several conditions, including chronic immune-inflammatory disease. CD34+ cell numbers have been found inconstantly reduced in SSc. Endocan, a proteoglycan expressed by endothelial cells, was recently suggested as a marker of vascular stress. We tested the relationships among CD34+ cells, endocan, inflammatory markers, vitamin D levels, and clinical parameters in SSc patients with PAH. METHODS: Standard echocardiography was performed. Vitamin D levels, CD34+ cells, inflammatory markers, endocan plasma levels were determined in 36 female SSc patients (24 diffuse/12 limited) and 36 matched controls (HC). RESULTS: We found no difference in CD34+ and vitamin D levels in SSc as compared to controls; ESR, CRP, fibrinogen, endocan, sPAP were higher in SSc with respect to controls. We found a correlation between endocan and: CD34+ cells (r: −0.540, p = 0.002), pulmonary arterial pressure (sPAP) (r: 0.565, p < 0.001), tricuspid annular plane excursion (TAPSE) (r: −0.311, p < 0.01), and E/A ratio (r: −0.487, p < 0.001), but not with ejection fraction (r: −0.057, p = 0.785) in SSc. CD34+ cells correlate with fibrinogen (r: −0.619, p < 0.001), sPAP (r: −0.404, p = 0.011), E/A (r: 0.470, p < 0.005 in SSc. CONCLUSION: CD34+ cell number was significantly correlated with endocan levels and with sPAP in SSc; endocan and CD34+ progenitor cells might be suggested as a potential marker of disease status.

Purpose Multiple factors influence intrauterine growth and lead to low birth sizes. The impact of genetic alterations on both pre- and post-natal growth is still largely unknown. The aim of this study was to investigate the prevalence of CNVs in an Italian cohort of SGA children with persistent short stature and complex clinical phenotype. rhGH treatment efficacy was evaluated according to the different genotypes. Subjects and methods Twenty-four SGA children (10F/14M) with persistent short stature associated with dysmorphic features and/or developmental delay underwent CNV evaluation. Results CNVs were present in 14/24 (58%) SGA children. Six patients had a microdeletion involving the following regions: 3q24q25.1, 8p21.2p12, 15q26, 19q13.11, 20q11.21q12, 22q11.2. In three females, the same microdeletion involving 17p13.3 region was identified. In two different patients, two microduplications involving 10q21.3 and Xp11.3 region were observed. A further female patient showed both an 11q12.1 and an Xq27.1 microduplication, inherited from her mother and from her father, respectively. In a boy, the presence of a 12p13.33 microdeletion and a 19q13.43 microduplication was found. GH treatment efficacy, expressed by height gain and height velocity in the first 12 months of therapy, was similar in subjects with and without CNVs. Conclusions These results show that pathogenic CNVs are common in SGA children with short stature associated with additional clinical features. Interestingly, the involvement of 17p13.3 region occurs with a relative high frequency, suggesting that genes located in this region could play a key role in pre- and post-natal growth. rhGH therapy has similar efficacy in the short term whether CNVs are present or not.

Background: An association between bone health and consumption of citrus fruits have been previously reported; however, the effect of lemon juice on bone metabolism have not been explored yet. Objectives: To investigate bone metabolic changes in postmenopausal women assuming lemon juice. Methods: Participants were postmenopausal osteoporotic women without history of clinical fractures who agreed to enrich their diet with lemon juice (Acti Lemon, Polenghi) over a 2-month period. The daily juice dose of 30 ml we suggested was equivalent to one Sicilian organic lemon. Surrogate markers of bone formation as procollagen type 1 N-propeptide (P1NP) and of bone resorption as C-terminal telopeptide of type I collagen (CTX), but also some regulators of bone metabolism as RANK-L, OPG, RANK-L/OPG ratio and sclerostin were assessed at baseline and then at 1 and 2 months after lemon juice administration. Controls were represented by a placebo group of age-matched osteoporotic postmenopausal women. Results: 47 participants [mean age 60.2 ± 4.1 yr.] completed the study, without reporting any adverse events. Lemon juice was well tolerated. Over the observation period modifications of bone metabolism occurred: we detected a decreased RANK-L/OPG ratio and increased CTX levels at all time points vs. baseline. Particularly, change at month-1 of sclerostin (versus baseline) has been positively associated with change at month-1 and month-2 of CTX (r=0.46, p=0.01 and r=0.43, p=0.01, respectively). Change at month-1 of OPG was positively associated with change at month-1 of P1NP (r=0.49, p=0.006). Change at month-1 of RANKL/OPG has been related with variation at day 30 of P1NP (r=-0.44, p=0.013). Variation of P1NP at month-1 was related with sclerostin variation at day 30 (r=-0.56, p=0.02) and month-2 vs. baseline value (r=0.44, p=0.017) and with sclerostin variation between month-1 and month-2 (r=0.69, p<0.001). Variation of P1NP between month-1 and month-2 was associated with RANKL change at month-1 (r=-0.35, p=0.05), with sclerostin change at month-1 (r=-0.49, p=0.008) and with sclerostin change between month-1 and month-2 (r=0.41, p=0.028). At a multiple regression analysis the change of P1NP between month-1 and month-2 was independently predicted by the change of sclerostin at month-1 (ß=-1.5, SE 0.5, p=0.006), after correcting for age, BMI and change of RANKL and CTX levels at month-1. No significant modifications raised from controls. Conclusion: Drinking lemon juice may boost bone metabolic changes involving both bone resorption and bone formation. Disclosure of Interests: None declared

Objective: The impact of acute inflammation, revealed by C-reactive protein (CRP) plasma levels, has been studied on the erythrocytes anion exchanger Band 3 protein.Methods: Anion exchange capability through Band 3 protein, lipid peroxidation, -SH membrane groups and intracellular GSH levels have been measured on erythrocytes from patients with CRP >8 mg/L.Results: Under acute inflammation, a significant increase in anion exchange capability, increased lipid peroxidation, decreased-SH groups and GSH content were observed. Serum CRP levels recovery (after one week) was associated to -SH groups and GSH recovery, but not to anion exchange capability restoration. After 2 months, a total recovery of all parameters was observed.Conclusion: Band 3 protein anion exchange capability is affected by acute inflammation; the accelerated rate of anion exchange may be mainly due to lipid peroxidation, rather than to -SH groups oxidation; erythrocytes renewal could be needed to have a total recover of their function.

Abstract Background and Aims Kynurenine pathway of tryptophan metabolism is involved in the pathophysiology of chronic kidney disease (CKD) and diabetes mellitus (DM), mainly through the inflammation-induced activity of indoleamine 2,3-dioxygenase (IDO). Renin-angiotensin-aldosterone system inhibitors (RAASis) [angiotensin converting enzyme inhibitors (ACEis) and angiotensin II receptor antagonists (ARBs)] are recommended in these conditions to decrease proteinuria, slow CKD progression and reduce cardiovascular risk. The interactions between RAAS and the kynurenine pathway and the potential effects of RAASis have been reported in few experimental models but whether these drugs influence kynurenine levels in humans is unknown. We performed a single-centre cross-sectional observational study to evaluate tryptophan and kynurenine serum levels and IDO activity in CKD patients with and without type 2 DM, their correlations with markers of renal dysfunction, and their relationship with RAAS-inhibiting therapy. Method We enrolled 72 consecutive adult patients with CKD, of which 38 had DM, who were admitted to the Unit of Nephrology and Dialysis of our Hospital. Of them, 55 were receiving RAASis whereas 17 were not. Tryptophan was assessed by HPLC (high-performance liquid chromatography) analysis with an UltiMateTM 3000 chromatograph (Thermo Fisher Scientific, 168 Third Avenue Waltham, MA, USA); kynurenine was measured using an enzyme-linked immunosorbent assay (ELISA) kit (Catalogue n. K 7728; Immundiagnostik AG, Bensheim, Germany); IDO activity was calculated with the formula (kynurenine/tryptophan) x 100. Results Patients receiving RAASis and patients not under therapy only differed for frequency of arterial hypertension (100% vs 76.47%; P=0.002) and kynurenine levels, the latter being significantly lower in the treated group compared to the untreated one (1.56 ± 0.79 vs 2.16 ± 1.51 µmol/l; P=0.0378). Kynurenine did not correlate with estimated glomerular filtration rate (eGFR), proteinuria or albuminuria in the whole study cohort. Conversely, in patients not receiving RAASis it was inversely related to eGFR (r=-0.4862; P=0.0478) and directly related to proteinuria (ρ=0.493; P=0.0444) and albuminuria (ρ=0.542; P=0.0247); moreover, it varied across classes of albuminuria, being lower in patients with normoalbuminuria (0.98 ± 0.55 µmol/l) and higher in those with micro- (2.63 ± 1.53 µmol/l) and macroalbuminuria (3.09 ± 1.61 µmol/l) at the ANOVA test (F=4.008; P=0.042). In the same group, the relationship between IDO activity and eGFR was significant (ρ=-0.554; P=0.0210) and IDO activity differed across classes of albuminuria (F=3.702; P=0.05). Moreover, IDO activity was higher in patients with history of cardiovascular disease compared to those with no such history [10.22 (8.39 to 22.92) vs 7.57 (4.76 to 8.09) %; P=0.0343] whereas tryptophan had an opposite behaviour (17.00 ± 2.06 vs 22.00 ± 3.78 µmol/l; P=0.0036). Conclusion We observed lower kynurenine levels in CKD patients treated with RAASis compared to untreated patients, independently of the presence of DM, and a significant association between kynurenine and markers of renal damage only in the group not receiving RAASis. We could hypothesize that kynurenine may play a role in the pathophysiology of renal damage, as already suggested, and that drugs interfering with RAAS activation may act also by reducing kynurenine levels, in addition to their already known effects at the renal level. Indeed, kynurenine synthesis results to be higher in states of inflammation and RAAS has pro-inflammatory and pro-fibrotic actions; accordingly, RAASis could reduce the enzymatic activity responsible for kynurenine increase and this could be one of the mechanisms mediating the beneficial effects of RAAS inhibition on CKD progression. This may be relevant because kynurenine has shown pro-thrombotic effects and is emerging as a potential new biomarker of CKD.

d-Galactose (d-Gal), when abnormally accumulated in the plasma, results in oxidative stress production, and may alter the homeostasis of erythrocytes, which are particularly exposed to oxidants driven by the blood stream. In the present investigation, the effect of d-Gal (0.1 and 10 mM, for 3 and 24 h incubation), known to induce oxidative stress, has been assayed on human erythrocytes by determining the rate constant of SO42− uptake through the anion exchanger Band 3 protein (B3p), essential to erythrocytes homeostasis. Moreover, lipid peroxidation, membrane sulfhydryl groups oxidation, glycated hemoglobin (% A1c), methemoglobin levels (% MetHb), and expression levels of B3p have been verified. Our results show that d-Gal reduces anion exchange capability of B3p, involving neither lipid peroxidation, nor oxidation of sulfhydryl membrane groups, nor MetHb formation, nor altered expression levels of B3p. d-Gal-induced %A1c, known to crosslink with B3p, could be responsible for rate of anion exchange alteration. The present findings confirm that erythrocytes are a suitable model to study the impact of high sugar concentrations on cell homeostasis; show the first in vitro effect of d-Gal on B3p, contributing to the understanding of mechanisms underlying an in vitro model of aging; demonstrate that the first impact of d-Gal on B3p is mediated by early Hb glycation, rather than by oxidative stress, which may be involved on a later stage, possibly adding more knowledge about the consequences of d-Gal accumulation.

Abstract Electrolytes disturbances are commonly observed in clinical practice. However, magnesium (Mg) disorders are often poorly considered. Our primary aim was to update the prevalence and the incidence of hypomagnesemia and hypermagnesemia in a real-life scenario. The secondary aim was to investigate the association of Mg disturbances with age and gender. Data from patients whose Mg serum levels were measured between January 2015 and December 2017 at our University Hospital were recorded. Hypomagnesemia was defined by Mg levels <1.5 mg/dL and hypermagnesemia by levels> 3.8 mg/dL. Hypo and hypermagnesemia cases were considered according to age, gender, year of observation and setting of subjects including outpatients and inpatients. In particular, inpatients were recruited from a total of 25 Hospital Divisions (i.e. Cardiology, Endocrinology, Geriatrics, Hematology, Intensive Care, Internal Medicine, Infectious Disease, Nephrology, Neonatal Intensive Care, Neurology, Neuropsychiatry, Neuroradiology, Oncology, Pulmonology, Pediatrics, Psychiatry, Rheumatology and Surgical Area that included 8 Units). Over the observation period, we recognized 12,696 patients whose Mg levels were checked. Prevalences of hypomagnesemia and hypermagnesemia were 8.43% (n=1071) and 1.78% (n=226) respectively. Hypomagnesemia was encountered more frequently in female [53.3% (n=560)] rather than in male patients [47.7 % (n=511)] (χ2= 4.03, p<0.045) and was significantly influenced by age: the higher prevalence of hypomagnesemia was found in patients over 65 yr. [59.01% (n=632)], whereas a lower prevalence was detected in the other age groups, specifically in 9.52 % (n=102) of patients aged 0-18 yr. and in 31.46 % (n=337) of patients between 19 and 65 yr. (χ2= 592.64; p<0.0001). Incidence of hypomagnesemia decreased over time with a borderline significance only in subjects over 65 yr. (r=-0.99; p=0.07). Geriatrics, Oncology and Intensive Care Division showed the highest incidences of hypomagnesemia. The hypermagnesemia prevalence was observed higher in outpatients in comparison with inpatients from any other Hospital Division and the hypermagnesemia incidence did not significantly change over time (r=0.96; p=0.16). Mg disorders, mainly hypomagnesemia, are quite common in clinical practice particularly in older hospitalized patients. Among the other electrolytes’ disturbances, Mg disorders, because of life-threatening significances, may be checked and corrected.

Hyperglycemia is considered a threat for cell homeostasis, as it is associated to oxidative stress (OS). As erythrocytes are continuously exposed to OS, this study was conceived to verify the impact of either diabetic conditions attested to by glycated hemoglobin (Hb) levels (>6.5% or higher) or treatment with high glucose (15–35 mM, for 24 h) on erythrocyte homeostasis. To this aim, anion exchange capability through the Band 3 protein (B3p) was monitored by the rate constant for SO42− uptake. Thiobarbituric acid reactive species (TBARS), membrane sulfhydryl groups mostly belonging to B3p, glutathione reduced (GSH) levels, and B3p expression levels were also evaluated. The rate constant for SO42− uptake (0.063 ± 0.001 min−1, 16 min in healthy volunteers) was accelerated in erythrocytes from diabetic volunteers (0.113 ± 0.001 min−1, 9 min) and after exposure to high glucose (0.129 ± 0.001in−1, 7 min), but only in diabetic volunteers was there an increase in TBARS levels and oxidation of membrane sulfhydryl groups, and a decrease in both GSH and B3p expression levels was observed. A combined effect due to the glycated Hb and OS may explain what was observed in diabetic erythrocytes, while in in vitro hyperglycemia, early OS could explain B3p anion exchange capability alterations as proven by the use of melatonin. Finally, measurement of B3p anion exchange capability is a suitable tool to monitor the impact of hyperglycemia on erythrocytes homeostasis, being the first line of high glucose impact before Hb glycation. Melatonin may be useful to counteract hyperglycemia-induced OS at the B3p level.