d-Galactose induced early aging in human erythrocytes: Role of band 3 protein.

Aging, a time-dependent multifaceted process, affects both cell structure and function and involves oxidative stress as well as glycation. The present investigation focuses on the role of the band 3 protein (B3p), an anion exchanger essential to red cells homeostasis, in a d-galactose ( d-Gal)-induced aging model. Anion exchange capability, measured by the rate constant of SO4²- uptake through B3p, levels of lipid peroxidation, oxidation of membrane sulfhydryl groups, B3p expression, methemoglobin, glycated hemoglobin (Hb), and the reduced glutathione/oxidized glutathione ratio were determined after exposure of human erythrocytes to 25, 35, 50, and 100 mmol/L d-Gal for 24 h. Our results show that: (i) in vitro application of d-Gal is useful to model early aging in human erythrocytes; (ii) assessment of B3p ion transport function is a sensitive tool to monitor aging development; (iii) d-Gal leads to Hb glycation and produces substantial changes on the endogenous antioxidant system; (iv) the impact of aging on B3p function proceeds through steps, first involving Hb glycation and then oxidative events at the membrane level. These findings offer a useful tool to understand the mechanisms of aging in human erythrocytes and propose B3p as a possible target for new therapeutic strategies to counteract age-related disturbances.

Erythropoiesis and chronic kidney disease-related anemia: From physiology to new therapeutic advancements.

Erythropoiesis is triggered by hypoxia and is strictly regulated by hormones, growth factors, cytokines, and vitamins to ensure an adequate oxygen delivery to all body cells. Abnormalities in one or more of these factors may induce different kinds of anemia requiring different treatments. A key player in red blood cell production is erythropoietin. It is a glycoprotein hormone, mainly produced by the kidneys, that promotes erythroid progenitor cell survival and differentiation in the bone marrow and regulates iron metabolism. A deficit in erythropoietin synthesis is the main cause of the normochromic normocytic anemia frequently observed in patients with progressive chronic kidney disease. The present review summarizes the most recent findings about each step of the erythropoietic process, going from the renal oxygen sensing system to the cascade of events induced by erythropoietin through its own receptor in the bone marrow. The paper also describes the new class of drugs designed to stabilize the hypoxia-inducible factor by inhibiting prolyl hydroxylase, with a discussion about their metabolism, disposition, efficacy, and safety. According to many trials, these drugs seem able to simulate tissue hypoxia and then stimulate erythropoiesis in patients affected by renal impairment. In conclusion, the in-depth investigation of all events involved in erythropoiesis is crucial to understand anemia pathophysiology and to identify new therapeutic strategies, in an attempt to overcome the potential side effects of the commonly used erythropoiesis-stimulating agents.

Decreased Endothelial Progenitor Cells (EPCs) and increased Natural Killer (NK) cells in peripheral blood as possible early markers of preeclampsia: a case-control analysis.

PURPOSE: Endothelial Progenitor Cells (EPCs) and Natural Killer (NK) cells were recently advocates in the pathogenesis of preeclampsia (PE), since they can be mobilized into the bloodstream and may orchestrate vascular endothelium function. The aim of our study was to evaluate in early pregnancy circulating EPCs and NK cells in peripheral blood in women who later developed PE compared to uncomplicated pregnancies. METHODS: We prospectively enrolled pregnant women at 9+0-11+6 weeks of gestation at the time of first-trimester integrated screening for trisomy 21, who underwent peripheral venous blood (20 mL) sample. We included only women who later developed PE (cases) and women with uncomplicated pregnancy (controls), matched for maternal age, parity, and Body Mass Index. In these groups, we evaluated the levels of CD16+CD45+CD56+ NK cells and CD34+CD133+VEGF-R2+ EPCs in peripheral blood samples previously stored. RESULTS: EPCs were significantly lower (p < 0.001), whereas NK cells were significantly higher (p < 0.001) in PE group compared to uncomplicated pregnancies during the first trimester. CONCLUSION: The evaluation of EPCs and NK cells in peripheral blood during the first trimester may be considered an effective screening for the early identification of women at risk of developing PE.

Sclerostin levels in uremic patients: a link between bone and vascular disease.

Sclerostin is a marker of low-turnover bone disease in end stage renal disease patients. The aim of this study was to evaluate serum sclerostin in uremic patients, analyzing its behavior during a single hemodialysis session. Twenty-one adult patients on intermittent hemodialysis treatment were enrolled. Acetate Free Bio-filtration (AFB) was the technique employed. Uremic patients were characterized by higher levels of serum sclerostin when compared with values observed in healthy subjects. Sclerostin assessed in pre-dialysis samples was 1.4 ± 1.02 ng/mL, whereas, in post dialysis samples, a reduction of sclerostin values was observed (0.8 ± 0.6 ng/mL; p: 0.008). Sclerostin correlated with parameters of dialysis adequacy, such as creatinine levels and Kt/V values, and it was significantly associated with atherosclerotic disease. Receiver operating characteristics analysis revealed a good diagnostic profile in identifying atherosclerotic disease. Sclerostin, a full dialyzable substance during AFB dialysis, is closely associated with atherosclerotic disease. Its reduction obtained through AFB could represent a defensive mechanism, improving vascular disease and renal osteodystrophy.

Does Vitamin D3 Have an Impact on Clinical and Biochemical Parameters Related to Third Molar Surgery.

The purpose of this study was to evaluate the clinical effect on the biochemical inflammatory markers of a single oral high dose of cholecalciferol in vitamin D-deficient patients undergoing the surgical removal of lower third molars.A randomized, split-mouth, single-blind study was conducted on 25 vitamin D-deficient patients ranging between 18 and 40 years of age requiring lower third molars extraction and referred at the Oral Surgery Unit of the School of Dentistry of the University of Messina.All patients, with vitamin D3 blood levels ≦30 ng/mL, underwent bilateral surgical removal. The first extraction (control group) being conducted with the administration of a placebo, the second one (test group) being conducted with the preliminary administration of 300,000 IU of cholecalciferol 4 days before the procedure.At each surgery, clinical indexes, such as pain, edema and any functional limitation have been recorded. Clinical and biochemical parameters were registered 4 days before, immediately after, 3 and 7 days after the surgical procedure. The data obtained were processed using paired t-test. The clinical outcome parameters showed a slight to moderate improvement between the control and the vitamin-D treatment group, with statistical significance being obtained regarding the edema at defined time points. Interleukin-1-beta, interleukin-6, and tumor necrosis factor-alpha values were significantly lower (P < 0.01) for the test group after the surgery. The increase of vitamin D serum levels showed an impact on the outcome of the third molar surgery, eliciting a reduced inflammatory response and leading to a more favorable clinical course.

Semaphorin 3A serum levels are influenced by haemodialysis: what clinical significance?

AIM: Semaphorin 3A urinary levels represent an early, predictive biomarker of acute kidney injury and positively correlate with albumin-to-creatinine ratio and serum creatinine in hypertensive patients with chronic kidney disease. Our purpose has been to evaluate semaphorin 3A serum levels in a cohort of haemodialysis (HD) patients, the influence of a single HD session on its concentrations, and the potential correlation with clinical and biochemical parameters. METHODS: We enrolled 18 patients receiving HD with Acetate-Free Biofiltration technique and 16 healthy subjects as controls. Peripheral venous blood samples were obtained from patients at different intervals: start of dialysis (pre-HD), middle, and end of the treatment (post-HD). We also collected dialysate samples by the Quantiscan monitoring system (Hospal, Bologna, Italy). RESULTS: Semaphorin 3A was significantly lower in HD patients at baseline compared to controls (median 19.50 (interquartile range 1.00-65.00) versus 97.50 (23.50-161.00) ng/mL, P = 0.0237). A statistically significant reduction was seen during a single HD session (from 19.50 (1.00-65.00) to 0.86 (0.82-4.21) ng/mL, P < 0.0001), with a reduction ratio of 65.92 ± 33.51%. The median concentration in dialysate was 54.00 (15.00-102.00) ng/mL. Pre-HD values were directly related to serum vitamin D (r = 0.872; P = 0.001) and inversely correlated with calcium levels (r = -0.426; P = 0.012) and calcium × phosphate product (r = -0.422; P = 0.0252). CONCLUSION: Semaphorin 3A removal during HD may be clinically relevant due to its involvement in different aspects of cell physiology and in bone remodelling. Semaphorin 3A both inhibits osteoclastic bone reabsorption and increases osteoblastic new bone formation, thus playing a dual osteoprotective role.

Toll-like receptor 3 and interleukin 1ß expression in CD34+ cells from patients with rheumatoid arthritis: association with inflammation and vascular involvement.

OBJECTIVES: Circulating proangiogenic haematopoietic cells (PHCs), including CD34+ cells, play an important role in endothelial homeostasis. Among PHCs, CD34+ cells are the largest cell population, thus, much of the regenerative/reparative potential of PHCs may be attributed to CD34+ cells. Our aim was to determine the association between inflammation and CD34+ cell number, intracellular levels of reactive oxygen species (ROS) and expression of Toll-like receptor 3 (TLR3) and interleukin 1ß (IL-1ß), arterial stiffness (AS) indices, and carotid intima-media thickness (cIMT) in patients affected by rheumatoid arthritis (RA). METHODS: CD34+ cells were isolated from 24 RA patients and 26 matched controls. ROS levels, TLR3 and IL-1ß expression were measured. C-reactive protein (CRP), fibrinogen, AS, and cIMT were also evaluated. RESULTS: CD34+ count was lower in RA patients as compared to controls. In CD34+ cells from RA patients, ROS, TLR3 and IL-1ß expressions were increased compared to controls. In RA patients, we found higher CRP and fibrinogen levels, and higher values of Pulse Wave Velocity (PWV) and Augmentation Index (AIx), both AS indices, and of cIMT. CD34+ cell numbers were inversely correlated with CRP, TLR3, IL-1ß, ROS, and AS indices. TLR3 levels were related to CRP, IL-1ß, fibrinogen and ROS. IL-1ß levels were correlated with expression of CRP, ROS, and PWV. CONCLUSIONS: Inflammatory status in RA is associated with an increased expression of TLR3 and of IL-1ß in CD34+ cells, which appear to affect cell number. These new findings suggest a perspective on accelerated atherosclerosis and vascular damage in RA.

Apelin, plasmatic osmolality and hypotension in dialyzed patients.

BACKGROUND/AIMS: To evaluate the balance between arginine-vasopressin (AVP) and apelin during hemodialysis and its role in hypotension onset and in the inflammation status. METHODS: We enrolled 50 patients chronically treated with hemodialysis. We assessed plasmatic osmolality, AVP, apelin, mean blood pressure (BP), high-sensitivity C-reactive protein (hsCRP) and ß(2)-microglobulin. RESULTS: Apelin rises during dialytic treatment (from 0.68 ± 0.34 to 1.89 ± 0.56 pg/ml, p < 0.0001), while plasmatic osmolality (from 325 ± 4.54 to 311 ± 1.20 mosm/kg H(2)O, p < 0.0001), AVP (from 4.28 ± 1.12 to 2.48 ± 0.50 pg/ml, p < 0.0001) and mean BP (from 124 ± 6 to 110 ± 7 mm Hg, p < 0.0001) decrease. At multivariate regression with respect to apelin, only mean BP remains (r = -0.95, p < 0.0001). We also correlated the AVP/apelin ratio with BP. Moreover, apelin is inversely related to hsCRP (r = -0.79, p < 0.0001). CONCLUSIONS: The AVP/apelin balance changes with plasmatic osmolality variations induced by hemodialytic sessions and could represent a physiopathological marker of arterial hypo- and hypertension. Finally, apelin appears inversely related to inflammation markers.

Impact of acute inflammation on Band 3 protein anion exchange capability in human erythrocytes.

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.

Indole-3-acetic acid correlates with monocyte-to-high-density lipoprotein (HDL) ratio (MHR) in chronic kidney disease patients.

PURPOSE: 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). METHODS: We enrolled 61 non-dialysis CKD patients and 6 dialysis patients. Indole-3-acetic acid levels were measured with ELISA technique. RESULTS: 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. CONCLUSION: 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.

Obestatin: a new element for mineral metabolism and inflammation in patients on hemodialysis.

BACKGROUND: Obestatin plays a key role in the process of energy balance maintenance with an anorectic effect. The main aim of the study was to evaluate obestatin in uremic patients to determine whether it is correlated with nutritional and inflammatory status. METHODS: We studied plasma obestatin in uremic patients (n = 50) undergoing hemodialysis therapy and in healthy subjects. Plasma obestatin was measured using an ELISA kit. RESULTS: Obestatin levels in uremic patients were lower than in healthy subjects (p < 0.0001). Patients with a body mass index (BMI) >23 had lower obestatin levels than those with a BMI <23 (p = 0.001). After multivariate analysis, direct correlations were maintained between obestatin and high-sensitivity C-reactive protein (ß = 0.68, p < 0.0001) and total alkaline phosphatases (ß = 0.30, p = 0.03), while inverse correlations were found with iron (ß = -0.32, p = 0.002) and calcium-phosphorous product (ß = -0.40, p = 0.001). CONCLUSIONS: Based on the present observational data, obestatin might be implicated in the inflammatory state and the disturbances of calcium/phosphate metabolism of hemodialysis patients. However, further studies are warranted to determine whether this hormone plays a key role in contributing to malnutrition and to the chronic inflammatory process.

Early Changes of VEGF Levels After Zoledronic Acid in Women With Postmenopausal Osteoporosis: A Potential Role of Vitamin D.

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 (ß = 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.

Myoinositol plus α-lactalbumin supplementation, insulin resistance and birth outcomes in women with gestational diabetes mellitus: a randomized, controlled study.

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 , NCT03763669, first posted date 04/12/2018; last posted date December 06/12/2018.

Rates of hypomagnesemia and hypermagnesemia in medical settings.

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.

Endocan and Circulating Progenitor Cells in Women with Systemic Sclerosis: Association with Inflammation and Pulmonary Hypertension.

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.

Bisphosphonates induce apoptosis of circulating endothelial cells in multiple myeloma patients and in subjects with bisphosphonate-induced osteonecrosis of the jaws.

Bisphosphonates (BPs) are the current standard of care for bone lesions in patients with multiple myeloma (MM) but they are associated with a number of side effects such as osteonecrosis of the jaw. The exact mechanisms of osteonecrosis are not elucidated, and its physiopathology is based on several hypotheses such as a decrease in bone remodeling or an inhibitory effect on angiogenesis. The aim of our study was to investigate the mechanism involved in the pathogenesis of osteonecrosis. We examined the apoptosis of circulating endothelial progenitor cells in MM subjects before and after BP treatment and in osteonecrosis patients using a flow-cytometric analysis. Our data showed an increase in endothelial cell apoptosis in MM patients after BP administration and in osteonecrosis subjects. Our study seems in agreement with the hypothesis that BPs can inhibit angiogenesis interfering with endothelial cell proliferation and survival, leading to loss of blood vessels and avascular necrosis.

Genomic damage in endothelial progenitor cells from uremic patients in hemodialysis.

INTRODUCTION: End stage renal disease (ESRD) is associated with a high incidence of cardiovascular disease and cancer. Patients undergoing hemodialysis show a reduced number and an impaired function of endothelial progenitor cells (EPCs), which in physiological conditions contribute to repair the vascular damage. In patients with ESRD, massive oxidative genome damage has been demonstrated but the role of HD in causing it is still a controversial issue. The aim of our study was to analyze the effects of a single HD session on the number of cells marked with CD34 (including sub-type cells known to be EPCs); we then evaluated the genomic damage in these cells using COMET assay. PATIENTS AND METHODS: We quantified CD34(+) cells in blood samples in 30 patients in hemodiafiltration treatment for 3.5 to 4 hours 3 times/week and in 30 healthy volunteers. In HD patients, blood samples were drawn at different time intervals: start of dialysis (T(0)), at the end of the treatment (T(end)) and 24 hours afterwards in the interdialytic day (T(inter)). Staining and analysis was performed using the ISHAGE (International Society of Hematotherapy and Graft Engineering) guidelines. EPCs count was conducted using a multiparameter flow cytometric lyse no-wash method. Genomic damage was evaluated by Comet assay. RESULTS: The number of CD34(+) cells in the HD patients at the beginning of the dialysis session (T(0)) was significantly lower than in healthy controls. HD patients showed a significant increase in CD34 number at the end of the session (T(end)) with respect to T(0). In the interdialytic period (T(int)), the number of CD34(+) cells was significantly reduced with respect to T(end). COMET assay performed on CD34(+) cells showed a higher basal level of genomic damage in HD patients than in controls; it increased in a statistically significant manner after the hemodialysis session, while in the interdialytic period it came back to T(0) level. CONCLUSIONS: Uremic status is characterized by lower levels of circulating EPCs, which increase after a single session of HD together with genomic damage to the CD34(+) cells.

High Glucose Concentrations Affect Band 3 Protein in Human Erythrocytes.

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.

d-Galactose Decreases Anion Exchange Capability through Band 3 Protein in Human Erythrocytes.

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.

RAS inhibition modulates kynurenine levels in a CKD population with and without type 2 diabetes mellitus.

Kynurenine pathway of tryptophan metabolism is involved in the pathophysiology of chronic kidney disease (CKD) and diabetes mellitus, mainly through the inflammation-induced activity of indoleamine 2,3-dioxygenase (IDO), and few studies have investigated its potential link with proteinuria. Renin-angiotensin system inhibitors (RASis) are recommended in these patients to decrease proteinuria, slow CKD progression and reduce cardiovascular risk, but whether these drugs influence kynurenine levels in humans is unknown. We evaluated serum tryptophan and kynurenine in patients suffering from CKD with or without type 2 diabetes mellitus, their correlations with markers of reduced kidney function, and their relationship with RAS-inhibiting therapy. Of 72 adult patients enrolled, 55 were receiving RASis, whereas 17 were not. Tryptophan was assessed by HPLC (high-performance liquid chromatography); kynurenine was measured using an enzyme-linked immunosorbent assay kit; IDO activity (%) was calculated with the formula (kynurenine/tryptophan) × 100. Kynurenine levels were significantly lower in the group under RASis compared to the untreated group (1.56 ± 0.79 vs 2.16 ± 1.51 µmol/l; P = 0.0378). In patients not receiving RASis, kynurenine was inversely related to estimated glomerular filtration rate (eGFR) (r = - 0.4862; P = 0.0478) and directly related to both proteinuria (ρ = 0.493; P = 0.0444) and albuminuria (ρ = 0.542; P = 0.0247). IDO activity was higher in patients with history of cardiovascular disease compared to patients with no such history, and it negatively correlated with eGFR (ρ = - 0.554; P = 0.0210) in the same group. These findings may contribute to explain the well-known beneficial effects of RAS inhibition in CKD population, especially considering that kynurenine is emerging as a potential new biomarker of CKD.