Unbalanced circulating Humanin levels and cardiovascular risk in chronic hemodialysis patients: a pilot, prospective study.

BACKGROUND: Mortality and cardiovascular (CV) risk prediction in individuals with end-stage kidney disease (ESKD) on chronic hemodialysis (HD) remains challenging due to the multitude of implicated factors. In a multicenter ESKD-HD cohort, we tested the prognostic yield of the assessment of circulating Humanin, a small mitochondrial-derived peptide involved in CV protection, on CV events and mortality. METHODS: We conducted a prospective, observational, pilot study on 94 prevalent HD patients. The prognostic capacity of circulating Humanin levels was tested on a primary composite (all-cause mortality + non-fatal CV events) and a secondary exploratory endpoint (all-cause mortality alone). RESULTS: Baseline Humanin level was comparable in patients reaching the primary or secondary endpoint as compared to others (p = 0.69 and 0.76, respectively). Unadjusted followed by multivariable Cox regression analyses adjusted for age, left ventricular mass index (LVMi), E/e', pulse pressure and diabetes mellitus indicated a non-linear relationship between Humanin levels and the composite outcome with the highest Hazard Ratio (HR) associated with very low (< 450.7 pg/mL; HR ranging from 4.25 to 2.49) and very high (> 759.5 pg/mL; HR ranging from 5.84 to 4.50) Humanin values. Restricted cubic splines fitting univariate and multivariate Cox regression analyses visually confirmed a curvilinear trend with an increasing risk observed for lower and higher Humanin values around the median, respectively. A similar, u-shaped association was also evidenced with the secondary endpoint. CONCLUSIONS: Altered Humanin levels may impart prognostic information in ESKD-HD patients at risk of death or CV events. Future investigations are needed to confirm whether Humanin measurement could improve CV and mortality risk prediction beyond traditional risk models.

The mitochondrial-derived peptide MOTS-c may refine mortality and cardiovascular risk prediction in chronic hemodialysis patients: a multicenter cohort study.

Introduction Uremic patients exhibit remarkably increased rates of mortality and cardiovascular events, but risk prediction in this setting remains difficult. Systemic mitochondrial dysfunction is pervasive in ESKD and may contribute to CV complications. We tested the clinical significance of circulating MOTS-c, a small mitochondrial-derived peptide, as a biomarker for improving mortality and CV risk prediction in hemodialysis (HD) patients. Methods We conducted a prospective, observational, multicenter study on 94 prevalent HD patients. The study endpoint was a composite of all-cause mortality and non-fatal CV events. The diagnostic and prognostic capacity of predictive models based on cohort-related risk factors were tested before and after the inclusion of MOTS-c. Results MOTS-c levels were higher in HD patients than in controls (p<0.001) and even more elevated (p=0.01) in the 53 individuals experiencing the combined endpoint during follow-up (median duration: 26.5 mo.). MOTS-c was independently associated with the endpoint at either multivariate logistic (OR 1.020; 95%CI 1.011-1.109; p=0.03) or Cox-regression analyses (HR 1.004; 95%CI 1.000-1.025; p=0.05) and the addition of this biomarker to prognostic models including the other cohort-related risk predictors (age, LVMi, E/e', diabetes, pulse pressure) significantly improved the calibration, risk variability explanation, discrimination (ROC-AUC from 0.727 to 0.743; C-index from 0.658 to 0.700) and, particularly, the overall reclassification capacity (NRI 15.87%; p=0.01). Conclusions In HD patients, the mitochondrial-derived peptide MOTS-c may impart significant information to refine CV risk prediction, beyond cohort-related risk factors. Future investigations are needed to generalize these findings in larger and more heterogeneous cohorts.

Urinary Post-Translationally Modified Fetuin-A (uPTM-FetA) in Chronic Kidney Disease Patients with and without Diabetic Kidney Disease.

Background and Objectives: A novel post-translational modification (PTM) fragment derived from the cleavage of Fetuin-A (PTM-FetA) has recently emerged as a sensitive biomarker for kidney damage in diabetic patients, but evidence in other chronic renal diseases is lacking. In this pilot study, we aimed at evaluating the clinical significance of urinary PTM-FetA (uPTM-FetA) in a mixed cohort of patients with non-advanced chronic kidney disease (CKD) secondary to diabetic kidney disease (DKD) or other causes. Materials and Methods: We enrolled 47 adult patients with CKD (mean CKD-Epi 40.10 ± 16.5 mL/min/1.73 m2) due to DKD (n = 34) or other etiology (n = 13). uPTM-FetA was measured in the urine using a commercially available ELISA kit. Fifteen healthy individuals served as controls. Results: Collectively, all CKD patients displayed remarkably higher levels of uPTM-FetA than controls (0.84 [0.10-1.15] vs. 29.68 [2.50-55.16] ng/mL p = 0.0005), but values were lower in non-DKD than in DKD patients (1.66 [0.09-4.19] vs. 13.9 [0.01-45.02] ng/mL; p = 0.01). uPTM-FetA showed a great diagnostic capacity at ROC analyses to identify the presence of CKD (AUC 0.776; p < 0.001) and, within CKD patients, to discriminate the diabetic and non-diabetic etiology (AUC 0.673; p = 0.02). At multivariate correlation analyses, proteinuria (ß = 0.442; p = 0.02) and BMI (ß = -0.334; p = 0.04) were the sole independent predictors of uPTM-FetA in this study population. Conclusions: uPTM-FetA could be a novel sensitive biomarker at the crossroad of chronic renal damage and metabolic dysfunction. Additionally, this biomarker could also represent a non-invasive, complementary tool for discriminating among different CKD etiologies (DKD vs. non-DKD) in difficult cases or when renal biopsy is not available.

Incidental thyroid carcinoma in an endemic goiter area in Italy: histopathological features and predictors of a common finding.

PURPOSE: The occurrence and histopathological features of incidental thyroid carcinoma (ITC) vary considerably among populations from different geographical regions. The aim of this study is to assess the prevalence and histopathological characteristics of ITC in patients who underwent thyroid surgery for apparently benign thyroid diseases in an endemic goiter area in Italy. METHODS: A total of 649 consecutive patients (531 females and 118 males; mean age, 52.9 ± 11.0 years), who underwent thyroid surgery at the Endocrine Surgery Unit of the tertiary care "Renato Dulbecco" University Hospital (Catanzaro, Italy) in the period between years 2017 and 2022, were included in this retrospective study. A comprehensive histopathological examination was performed on surgically excised thyroid tissue. Logistic regression analysis was employed to identify potential predictors of ITC. RESULTS: The histopathological examination revealed the presence of ITC in 81 patients, accounting for 12.5% of the total study population. The female to male ratio was found to be 6.4 to 1. Among the patients with ITC, 72 had papillary carcinoma (PTC), with 53 of these tumors being microcarcinomas (microPTC). Additionally, 5 patients had follicular thyroid carcinoma, 2 patients had low-risk follicular cell-derived thyroid neoplasms, 1 patient had an oncocytic carcinoma, and 1 patient had a medullary thyroid carcinoma. Logistic regression analysis demonstrated a significant association between female sex and incidental microPTC. CONCLUSIONS: These findings provide further evidence of the common occurrence of ITC, typically in the form of microPTC, among individuals who undergo thyroid surgery for apparently benign thyroid diseases.

Circulating miRNA 122-5p Expression Predicts Mortality and Cardiovascular Events in Chronic Hemodialysis Patients: A Multicentric, Pilot, Prospective Study.

BACKGROUND: Despite patients undergoing chronic hemodialysis (HD) being notoriously prone to adverse cardiovascular (CV) events, risk prediction in this population remains challenging. miRNA 122-5p, a short, non-coding RNA predominantly involved in lipid and carbohydrate metabolism, has recently been related to the onset and progression of CV disease. METHODS: We run a pilot, multicenter, longitudinal, observational study to evaluate the clinical significance and prognostic usefulness of circulating miRNA 122-5p in a multicentric cohort of 74 individuals on maintenance HD. RESULTS: Patients displayed lower circulating miRNA 122-5p as compared to healthy controls (p = 0.004). At correlation analyses, ALT (ß = 0.333; p = 0.02), E/e' (ß = 0.265; p = 0.02) and CRP (ß = -0.219; p = 0.041) were independent predictors of miRNA 122-5p levels. During a median follow-up of 22 months (range of 1-24), 30 subjects (40.5%) experienced a composite endpoint of all-cause mortality and fatal/non-fatal CV events. Baseline circulating miRNA 122-5p was higher in these subjects (p = 0.01) and it predicted a significantly higher risk of endpoint occurrence (Kaplan-Meier crude HR 3.192; 95% CI 1.529-6.663; p = 0.002; Cox regression adjusted HR 1.115; 95% CI 1.009-1.232; p = 0.03). CONCLUSIONS: Altered miRNA 122-5p levels in HD patients may reflect hepatic and CV damage and may impart important prognostic information for improving CV risk prediction in this particular setting.

Liver Damage and Impaired Coagulation in COVID-19 Patients: A Case Series.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has generated an unprecedented challenge for healthcare systems worldwide. Currently, the scientific community wonders if liver injury in patients suffering from severe forms is a direct consequence of the virus or secondary manifestations of systemic inflammation. The liver plays an essential role in the development of the inflammatory storm typical of this disease, and its involvement is associated with worse clinical outcomes and a higher risk of morbidity and mortality from Coronavirus disease 2019 (COVID-19). METHODS: Ten patients suffering from severe COVID-19 disease who died between January 2020 and December 2021 were included in the present analysis. These subjects underwent a post mortem examination with a focused evaluation of the hepatic injury. Also, several laboratory parameters have been evaluated, with a primary focus on prothrombin time, partial thromboplastin time, fibrinogen, antithrombin III, and D-dimers to detect coagulative changes. RESULTS: The main cause of death was represented by pulmonary thromboembolism events (50%). The analysis of coagulation laboratory parameters and liver biomarkers revealed a statistically significant rise in aPTT and ALP, and a decrease in albumin, when comparing the blood value at admission and death. We also found high levels of D-dimers in most of the subjects at the time of hospitalization. Interestingly, the post mortem analysis of the liver showed ample morphologic variability, with several disease features. In detail, the liver histology revealed the following: the presence of a variable degree of micro- and macrovacuolar steatosis, inflammation (also, hepato-cholangitis), and variable fibrosis. Of mention, we were also able to detect organized fibrinous material. CONCLUSIONS: Our results indicate that in subjects with a severe form of COVID-19, liver disease is related to changes in coagulative and fibrinolytic pathways. In particular, we noted low fibrinogen levels and high D-dimer levels with histological liver findings. Our data suggest that fibrinogen and D-dimers may be used as prognostic markers to detect the severity of liver disease in patients with COVID-19. Finally, we underline the crucial role of coagulation balance in subjects with severe forms of COVID-19.

Urinary Marinobufagenin in Patients with Non-Advanced Chronic Kidney Disease: A Cross-Sectional Study.

Background and Objectives: The global prevalence of chronic kidney disease (CKD) is on the rise, posing important challenges for healthcare systems. Thus, the search for new factors potentially involved in the pathogenesis, progression and complications of early CKD remains urgent. Marinobufagenin (MBG) is a natriuretic endogenous cardiotonic steroid, and increased circulating levels of it may accelerate kidney damage. In this study, we explored the possible clinical significance of measuring urinary marinobufagenin (uMBG) in patients with non-advanced CKD. Materials and Methods: One hundred and eight adult CKD patients (mean age 71.6 ± 10 years, 70.4% male; mean eGFR 40.54 ± 17 mL/min/1.73 m2) were enrolled in this cross-sectional study. uMBG was measured together with a series of clinical, anthropometric, laboratory and instrumental analyses. Twenty-five healthy matched subjects served as controls for the uMBG measurement. Results: The uMBG values were lower in the patients with CKD as compared to those of the controls (0.37 [IQR: 0.25-0.45] vs. 0.64 [0.46-0.78] nmol/L. p = 0.004), and a significant trend in eGFR levels was noticed across the decreasing uMBG tertiles (p = 0.03). Regarding the correlation analyses, the uMBG values remained robustly associated with the eGFR in multivariate models employing either uMBG or eGFR as the dependent variable (ß = 0.248; p = 0.01 and ß = 0.139; p = 0.04, respectively). Besides the eGFR, the independent predictors of uMBG values in this population were the use of statins (ß = -0.326; p = 0.001), the presence of diabetes (ß = 0.243; p = 0.009) and urine sodium (ß = 0.204; p = 0.01). Conclusions: Reduced uMBG excretion may reflect impaired renal clearance, which may contribute to the detrimental effects attributed to this hormone due to systemic accumulation. Future studies are needed to clarify the biological mechanisms placing uMBG at the crossroad of sodium intake and the presence of diabetes in CKD-suffering individuals and to verify whether a statin treatment may somewhat limit the detrimental effects of MBG in the presence of impaired renal function.

Marinobufagenin, Left Ventricular Hypertrophy and Residual Renal Function in Kidney Transplant Recipients.

BACKGROUND: Left ventricular hypertrophy (LVH), which is a pervasive complication of end-stage kidney disease (ESKD), persists in some uremic individuals even after kidney transplantation (Ktx), contributing to worsening CV outcomes. Marinobufagenin (MBG), an endogenous steroid cardiotonic hormone endowed with natriuretic and vasoconstrictive properties, is an acknowledged trigger of uremic cardiomyopathy. However, its clinical significance in the setting of Ktx remains undefined. METHODS: In a cohort of chronic Ktx recipients (n = 40), we assessed circulating MBG together with a thorough clinical and echocardiographic examination. Forty matched haemodialysis (HD) patients and thirty healthy subjects served as controls for MBG measurements. Patients were then prospectively followed up to 12 months and the occurrence of an established cardio-renal endpoint (death, CV events, renal events, graft rejection) was recorded. RESULTS: Median MBG plasma levels were lower in Ktx as compared with HD patients (p = 0.02), but higher as compared with healthy controls (p = 0.0005). Urinary sodium (ß = 0.423; p = 0.01) and eGFR (ß = -0.324; p = 0.02) were the sole independent predictors of MBG in this cohort, while a strong correlation with left ventricular mass index (LVMi), found in univariate analyses (R = 0.543; p = 0.0007), gained significance only in multivariate models not including eGFR. Logistic regression analyses indicated MBG as a significant predictor of the combined endpoint (OR 2.38 [1.10-5.12] per each 1 nmoL/L increase; p = 0.01), as well as eGFR, LVMi, serum phosphate and proteinuria. CONCLUSIONS: Ktx recipients display altered MBG levels which are influenced by sodium balance, renal impairment and the severity of LVH. Thus, MBG might represent an important missing link between reduced graft function and pathological cardiac remodelling and may hold important prognostic value for improving cardio-renal risk assessment.

A small circulating miRNAs signature predicts mortality and adverse cardiovascular outcomes in chronic hemodialysis patients.

Background: Chronic hemodialysis (HD) patients exhibit severe morpho-functional cardiac alterations, putting them at a high risk of death and adverse cardiovascular (CV) outcomes. Despite the fact that an unbalanced expression of various microRNAs (miRNAs) has been related to pathological cardiac remodeling and worse CV outcomes, scarce evidence exists on their role in this setting. Methods: We evaluated circulating levels of a selected miRNAs panel (30a-5p, 23a-3p, 451a and let7d-5p) in 74 chronic HD patients together with a thorough clinical and echocardiography assessment. Individuals were then prospectively followed (median 22 months). The primary endpoint was a composite of all-cause and CV mortality and non-fatal CV events. Results: Circulating levels of all miRNAs were lower in HD patients as compared with healthy controls and independently correlated to the severity of cardiac dysfunction. miRNA 30a-5p, 23a-3p and 451a expression was even lower in 30 subjects (40.5%) reaching the composite endpoint (P < .001), while no differences were reported for let7d-5p. The predictive value of these miRNAs was supported by univariate followed by multivariate Cox regression analyses [hazard ratio (HR) ranging from 0.943 to 0.995; P = .05 to .02] while Kaplan-Meier analyses confirmed a faster progression to the endpoint in individuals displaying miRNA levels below an optimal receiver operating characteristic-derived cut-off value (P ranging from .001 to <.0001; crude HRs 7.95 to 8.61). Conclusions: Lower circulating levels of miRNA 30-5p, 23a-3p and 451a in HD patients may reflect cardiac abnormalities and predict a higher risk of worse clinical outcomes in the short mid-term. Future studies on larger HD populations are needed to generalize these findings.

Maternal Preconception Body Mass Index Overtakes Age as a Risk Factor for Gestational Diabetes Mellitus.

Introduction-The purpose of this study was to determine the relative impact of modifiable and non-modifiable risk factors in the development of gestational diabetes mellitus (GDM), with a particular focus on maternal preconception body mass index (BMI) and age, two important determinants of insulin resistance. Understanding the factors that contribute most to the current escalation of GDM rates in pregnant women could help to inform prevention and intervention strategies, particularly in areas where this female endocrine disorder has an elevated prevalence. Methods-A retrospective, contemporary, large population of singleton pregnant women from southern Italy who underwent 75 g OGTT for GDM screening was enrolled at the Endocrinology Unit, "Pugliese Ciaccio" Hospital, Catanzaro. Relevant clinical data were collected, and the characteristics of women diagnosed with GDM or with normal glucose tolerance were compared. The effect estimates of maternal preconception BMI and age as risk factors for GDM development were calculated through correlation and logistic regression analysis by adjusting for potential confounders. Results-Out of the 3856 women enrolled, 885 (23.0%) were diagnosed with GDM as per IADPSG criteria. Advanced maternal age (≥35 years), gravidity, reproductive history of spontaneous abortion(s), previous GDM, and thyroid and thrombophilic diseases, all emerged as non-modifiable risk factors of GDM, whereas preconception overweight or obesity was the sole potentially modifiable risk factor among those investigated. Maternal preconception BMI, but not age, had a moderate positive association with fasting glucose levels at the time of 75 g OGTT (Pearson coefficient: 0.245, p < 0.001). Abnormalities in fasting glucose drove the majority (60%) of the GDM diagnoses in this study. Maternal preconception obesity almost tripled the risk of developing GDM, but even being overweight resulted in a more pronounced increased risk of developing GDM than advanced maternal age (adjusted OR for preconception overweight: 1.63, 95% CI 1.320-2.019; adjusted OR for advanced maternal age: 1.45, 95% CI 1.184-1.776). Conclusions-Excess body weight prior to conception leads to more detrimental metabolic effects than advanced maternal age in pregnant women with GDM. Thus, in areas in which GDM is particularly common, such as southern Italy, measures aiming to counteracting maternal preconception overweight and obesity may be efficient in reducing GDM prevalence.

Does NUCB2/Nesfatin-1 Influence Eating Behaviors in Obese Patients with Binge Eating Disorder? Toward a Neurobiological Pathway.

Nesfatin-1 is a new anorexigenic neuropeptide involved in the regulation of hunger/satiety, eating, and affective disorders. We aimed to investigate nesfatin-1 secretion in vitro, in murine adipose cells, and in human adipose fat samples, as well as to assess the link between circulating nesfatin-1 levels, NUCB2 and Fat Mass and Obesity Gene (FTO) polymorphisms, BMI, Eating Disorders (EDs), and pathological behaviors. Nesfatin-1 secretion was evaluated both in normoxic fully differentiated 3T3-L1 mouse adipocytes and after incubation under hypoxic conditions for 24 h. Omental Visceral Adipose tissue (VAT) specimens of 11 obese subjects, and nesfatin-1 serum levels' evaluation, eating behaviors, NUCB2 rs757081, and FTO rs9939609 polymorphisms of 71 outpatients seeking treatment for EDs with different Body Mass Index (BMI) were studied. Significantly higher levels of nesfatin-1 were detected in hypoxic 3T3-L1 cultured adipocytes compared to normoxic ones. Nesfatin-1 was highly detectable in the VAT of obese compared to normal-weight subjects. Nesfatin-1 serum levels did not vary according to BMI, sex, and EDs diagnosis, but correlations with grazing; emotional, sweet, and binge eating; hyperphagia; social eating; childhood obesity were evident. Obese subjects with CG genotype NUCB2 rs757081 and AT genotype FTO rs9939609 polymorphisms had higher nesfatin-1 levels. It could represent a new biomarker of EDs comorbidity among obese patients.

Circulating Omentin-1, Sustained Inflammation and Hyperphosphatemia at the Interface of Subclinical Atherosclerosis in Chronic Kidney Disease Patients on Chronic Renal Replacement Therapy.

Background and Objectives: Subclinical atherosclerosis, reflected by abnormal carotid intima-media thickness (cIMT), is pervasive among chronic kidney disease patients on chronic renal replacement therapy (RRT), being mostly influenced by uremia-related rather than traditional risk factors. Materials and Methods: In this pilot study, we measured circulating levels of Omentin-1, a recently discovered adipokine with strong anti-atherogenic properties, in a heterogeneous cohort of 77 asymptomatic RRT individuals (40 chronic kidney transplant recipients, Ktx; and 37 chronic hemodialysis patients, HD) and in 30 age-matched controls. Results: Omentin-1 was increased in RRT individuals as compared with controls (p = 0.03). When stratifying for renal replacement modality, we found Ktx patients to have significantly lower Omentin-1 than HD patients (p = 0.01). Lower Omentin-1 levels were also found among RRT individuals with pathological cIMT (168.7 [51.1-457.8] vs. 474.9 [197.2-1432.1]; p = 0.004). Our multivariate correlations analysis revealed Omentin-1 as the most robust independent predictor of carotid atherosclerosis (ß-0.687; p = 0.03), even more than total cholesterol, diastolic BP and age, and this adipokine was at the crossroad of a complex interplay with sustained inflammation (high CRP and ferritin) and hyperphosphatemia in predicting higher cIMT values. Conclusion: The findings reported extend to renal patients with advanced disease, with the possible involvement of Omentin-1 in the pathogenesis of atherosclerosis. This may set the stage for future interventional studies of Omentin-1 replacement to retard atherosclerosis progression, as it is currently being investigated in other disease settings.

Marinobufagenin, left ventricular geometry and cardiac dysfunction in end-stage kidney disease patients.

PURPOSE: Left ventricular hypertrophy (LVH) is remarkably prevalent among end-stage kidney disease (ESKD) on chronic dialysis and has a strong prognostic value for adverse outcomes. In experimental models, the endogenous cardiotonic steroid Marinobufagenin (MBG) promotes cardiac hypertrophy and accelerates uremic cardiomyopathy. In this study, we investigated the possible relationships between MBG, LV geometry and cardiac dysfunction in a clinical setting of ESKD. METHODS: Plasmatic MBG was measured in 46 prevalent ESKD patients (n = 30 HD, n = 16 PD) together with a thorough laboratory, clinical, bioimpedance and echocardiography assessment. Different patterns of LV geometry were defined by left ventricular mass index (LVMi) and ventricular morphology. Diastolic dysfunction was diagnosed by the ASE/EACVI criteria. RESULTS: MBG levels were significantly higher in ESKD patients than in healthy controls (p = 0.001) and more elevated in PD than in HD (p = 0.02). At multivariate analyses, E/e' (ß = 0.38; p = 0.009) and LVMi (ß = 0.42; p = 0.02) remained the sole independent predictors of MBG. A statistically significant trend in MBG levels (p = 0.01) was noticed across different patterns of LV geometry, with the highest values found in eccentric LVH. MBG levels were higher in the presence of diastolic dysfunction (p = 0.01) and this substance displayed a remarkable diagnostic capacity in distinguish patients with normal LV geometry, LV hypertrophy and, particularly, eccentric LVH (AUC 0.888; p < 0.0001) and diastolic dysfunction (AUC 0.79; p = 0.001). CONCLUSIONS: Deranged plasma MBG levels in ESKD patients on chronic dialysis reflect alterations in LV structure and function. MBG may, thus, candidate as a novel biomarker for improving cardiac assessment in this high-risk population.

Circulating Omentin-1 levels and altered iron balance in chronic haemodialysis patients.

BACKGROUND: Iron deficiency is highly prevalent among patients undergoing chronic haemodialysis (HD) but its correct identification is often problematic as common biomarkers of iron status, such as transferrin saturation (TSAT) and ferritin, can be altered by inflammation or malnutrition. METHODS: In this pilot multicentre study, we aimed at evaluating circulating levels of Omentin-1, a novel fat depot-specific adipokine that is also involved in iron regulation, in a cohort of 85 chronic HD patients with relation to their iron status. RESULTS: Omentin-1 levels in HD were statistically higher than in healthy controls (P = 0.03) and there was a significant, growing trend in all iron parameters across Omentin-1 tertiles (P < 0.001). Compared with patients with optimal iron status, Omentin-1 levels were lower in subjects categorized according to TSAT ≤20% or serum ferritin ≤200 µg/L (both P < 0.001) and even more reduced in 19 patients (22%) simultaneously displaying low levels of both markers (P < 0.001). In this latter group, Omentin-1 levels increased in parallel to all other iron markers after iron correction by i.v. supplementation. At multivariate regression analyses, ferritin (ß = 0.71; P < 0.001) and TSAT (ß = 0.32; P = 0.03) remained the sole independent predictors of Omentin-1 levels. This biomarker also showed a remarkable diagnostic capacity at receiver operating characteristic analyses in identifying iron-depleted HD patients according to a criterion of TSAT ≤20% [area under the curve (AUC) 0.827], ferritin ≤200 µg/L (AUC 0.863) or low levels of both parameters (AUC 0.907). CONCLUSIONS: Findings obtained indicate that Omentin-1 is somewhat involved in iron balance regulation and might be a candidate biomarker for diagnosing and managing altered iron conditions in HD patients.

Altered Frequencies and Functions of Innate Lymphoid Cells in Melanoma Patients Are Modulated by Immune Checkpoints Inhibitors.

Monoclonal antibodies targeting immune checkpoints improved clinical outcome of patients with malignant melanoma. However, the mechanisms are not fully elucidated. Since immune check-point receptors are also expressed by helper innate lymphoid cells (ILCs), we investigated the capability of immune checkpoints inhibitors to modulate ILCs in metastatic melanoma patients as well as melanoma cells effects on ILC functions. Here, we demonstrated that, compared to healthy donors, patients showed a higher frequency of total peripheral ILCs, lower percentages of CD117+ ILC2s and CD117+ ILCs as well as higher frequencies of CD117- ILCs. Functionally, melanoma patients also displayed an impaired TNFα secretion by CD117- ILCs and CD117+ ILCs. Nivolumab therapy reduced the frequency of total peripheral ILCs but increased the percentage of CD117- ILC2s and enhanced the capability of ILC2s and CD117+ ILCs to secrete IL-13 and TNFα, respectively. Before Nivolumab therapy, high CCL2 serum levels were associated with longer Overall Survival and Progression Free Survival. After two months of treatment, CD117- ILC2s frequency as well as serum concentrations of IL-6, CXCL8 and VEGF negatively correlated with both the parameters. Moreover, melanoma cells boosted TNFα production in all ILC subsets and increased the number of IL-13 producing ILC2s in vitro. Our work shows for the first time that PD-1 blockade is able to affect ILCs proportions and functions in melanoma patients and that a specific subpopulation is associated with the therapy response.

Decreased Cathepsin-K Mirrors the Severity of Subclinical Atherosclerosis in Kidney Transplant Recipients.

Background: In kidney transplantation (Ktx) recipients, cardiovascular (CV) disease remains the leading cause of death. Abnormal carotid intima-media thickness (IMT) represents a valid indicator of incipient atherosclerosis also in this setting. Cathepsin-K (CatK) is a cysteine protease involved in vascular remodelling, as well as in progressive atherosclerosis. In this study we evaluated clinical predictors of CatK in Ktx recipients, with a particular focus on its possible relationships with subclinical atherosclerosis. Methods: Circulating CatK was measured in 40 stable Ktx recipients together with several laboratory, clinical and echocardiography parameters. 30 healthy subjects and 30 hemodialysis (HD) patients served as controls for CatK values. Carotid IMT was measured in Ktx and these subjects were then categorized according to age-gender reference cut-offs of normal IMT. Results: CatK levels were similar in Ktx recipients and healthy subjects but significantly reduced as compared to HD (p = 0.0001). In Ktx, at multivariate analyses CatK was associated with the LV end-diastolic volume (LVEDVi) ( ß = 0.514; p = 0.05), Ktx vintage ( ß = -0.333; p = 0.05) and mean IMT ( ß = -0.545; p = 0.05); this latter robust inverse association was confirmed also in another multivariate model with IMT as the dependent variable. Logistic regression analyses confirmed the beneficial meaning of CatK increase towards subclinical atherosclerosis [Odds Ratio (OR) 0.761; 95% Confidence Interval (CI) 0.569-0.918, p = 0.04]. At Receiver Operating Characteristics (ROC) analyses, CatK held a remarkable discriminatory power in identifying Ktx patients with abnormally increased IMT [Area Under the Curve (AUC) 0.763; 95% CI 0.601-0.926; p = 0.001]). Conclusions: In Ktx recipients, reduced CatK levels reflect the time-dependent improvement in the uremic milieu, cardiac adaptations and, above all, the severity of subclinical atherosclerosis. CatK measurement in Ktx may therefore hold significance for improving early CV risk stratification.

Altered circulating marinobufagenin levels and recurrent intradialytic hypotensive episodes in chronic hemodialysis patients: a pilot, prospective study.

Intradialytic hypotension (IDH) is a sudden and often serious complication of chronic hemodialysis (HD). In this prospective study, we aimed at evaluating the clinical predictors of IDH in a homogeneous cohort of chronic HD patients, with a particular focus on marinobufagenin (MBG), an endogenous cardiotonic steroid which alterations have previously been involved in various cardiovascular disorders. MBG levels in HD patients were significantly higher than in controls (p = 0.03), remained unchanged throughout a single HD session and were not correlated with the absolute or partial fluid loss achieved. During a 30-day follow-up, 19 patients (65.5%) experienced at least one IDH (73 total episodes). An inverse correlation was found between baseline MBG and the number of IDH (R = -0.55; p = 0.001). HD patients experiencing IDH presented remarkably lower baseline MBG as compared to others (p = 0.008) with a statistically significant trend during HD (p = 0.02). At Kaplan-Meier analyses, HD patients with lower MBG manifested a four-to-six fold increased risk of IDH during follow-up (crude Hazard Ratio ranging from 4.37 to 6.68). At Cox regression analyses, MBG measurement at different time points resulted the strongest time-dependent predictors of IDH among all the variables considered (HR ranging from 0.068 to 0.155; p: 0.002 to <0.0001). Findings obtained suggest that differently altered MBG in chronic HD patients may reflect a diverse vascular and hemodynamic tolerance to HD stress, eventually leading to recurrent IDH episodes. Further studies are needed to confirm the prognostic capacity of MBG for identifying HD patients at high risk of IDH, particularly those with apparently optimal fluid status.

Icariin Protects H9c2 Rat Cardiomyoblasts from Doxorubicin-Induced Cardiotoxicity: Role of Caveolin-1 Upregulation and Enhanced Autophagic Response.

Doxorubicin (Doxo) is a widely used antineoplastic drug which often induces cardiomyopathy, leading to congestive heart failure through the intramyocardial production of reactive oxygen species (ROS). Icariin (Ica) is a flavonoid isolated from Epimedii Herba (Berberidaceae). Some reports on the pharmacological activity of Ica explained its antioxidant and cardioprotective effects. The aim of our study was to assess the protective activities of Ica against Doxo-detrimental effects on rat heart-tissue derived embryonic cardiac myoblasts (H9c2 cells) and to identify, at least in part, the molecular mechanisms involved. Our results showed that pretreatment of H9c2 cells with 1 µM and 5 µM of Ica, prior to Doxo exposure, resulted in an improvement in cell viability, a reduction in ROS generation, the prevention of mitochondrial dysfunction and mPTP opening. Furthermore, for the first time, we identified one feasible molecular mechanism through which Ica could exerts its cardioprotective effects. Indeed, our data showed a significant reduction in Caveolin-1(Cav-1) expression levels and a specific inhibitory effect on phosphodiesterase 5 (PDE5a) activity, improving mitochondrial function compared to Doxo-treated cells. Besides, Ica significantly prevented apoptotic cell death and downregulated the main pro-autophagic marker Beclin-1 and LC3 lipidation rate, restoring physiological levels of activation of the protective autophagic process. These results suggest that Ica might have beneficial cardioprotective effects in attenuating cardiotoxicity in patients requiring anthracycline chemotherapy through the inhibition of oxidative stress and, in particular, through the modulation of Cav-1 expression levels and the involvement of PDE5a activity, thereby leading to cardiac cell survival.

Insulin Resistance and Cancer: In Search for a Causal Link.

Insulin resistance (IR) is a condition which refers to individuals whose cells and tissues become insensitive to the peptide hormone, insulin. Over the recent years, a wealth of data has made it clear that a synergistic relationship exists between IR, type 2 diabetes mellitus, and cancer. Although the underlying mechanism(s) for this association remain unclear, it is well established that hyperinsulinemia, a hallmark of IR, may play a role in tumorigenesis. On the other hand, IR is strongly associated with visceral adiposity dysfunction and systemic inflammation, two conditions which favor the establishment of a pro-tumorigenic environment. Similarly, epigenetic modifications, such as DNA methylation, histone modifications, and non-coding RNA, in IR states, have been often associated with tumorigenesis in numerous types of human cancer. In addition to these observations, it is also broadly accepted that gut microbiota may play an intriguing role in the development of IR-related diseases, including type 2 diabetes and cancer, whereas potential chemopreventive properties have been attributed to some of the most commonly used antidiabetic medications. Herein we provide a concise overview of the most recent literature in this field and discuss how different but interrelated molecular pathways may impact on tumor development.

Methods to Study Protein-Binding to Pseudogene Transcripts.

One of the most commonly described biological feature of processed pseudogenes is the ability to influence the expression of their parental coding genes. As evidenced in several studies, the high sequence similarity between these RNA pairs sets up a certain level of competition for posttranscriptional regulators, including, among others, RNA-binding proteins (RBPs). RBPs may affect, positively or negatively, the stability of bound mRNAs, so that, if an overexpressed pseudogene competes with its homologous coding gene, the downstream protein synthesis would change, with potential pathological consequences. Given these premises, a rigorous and comprehensive understanding of interactions between pseudogene-parental gene RNA pairs and RBPs could provide further insights into the biological bases of complex diseases, such as cancer, cardiovascular disease, and type 2 diabetes, identifying novel predictive and/or prognostic biomarkers.Herein, we detail easily adaptable protocols of plasmid-based molecular cloning and RNA-electrophoretic mobility shift assay (EMSA) used in our laboratory for determining the interaction between a cytoplasmatic stabilizing protein (αCP1) and the pseudogene-parental gene RNA pair HMGA1-p /HMGA1. We also offer a general overview of RNA immunoprecipitation procedures and present novel bioinformatic tools for predicting RBPs binding sites on pseudogene transcripts.