Platelet thrombus formation in patients with end-stage renal disease before and after hemodialysis as measured by the total thrombus-formation analysis system.

BACKGROUND: Patients with end-stage renal disease (ESRD) receiving hemodialysis (HD) often experience bleeding. However, mechanisms behind this bleeding tendency are incompletely understood but may involve platelet dysfunction. We, therefore, studied platelet-dependent thrombus formation in flowing whole blood inside a microchip coated with collagen, and its association with circulating von Willebrand factor (VWF). METHODS: Blood samples were obtained in 22 patients before and after HD. The area under the 10 min flow pressure curve in a microchip (AUC10) reflecting total platelet thrombogenicity was measured, using the Total Thrombus-formation Analysis System (T-TAS01). AUC10 < 260 indicates platelet dysfunction. VWF activity and antigen in plasma were also assayed. RESULTS: VWF levels were moderately elevated and increased further after HD (P < 0.01 or lower). In contrast, AUC10 before and after HD was < 260 in 17/22 patients and < 130 in 15/22 patients, with no statistically significant difference in pre- vs post-HD measurements, indicating reduced platelet thrombogenicity, but with some variability as 5/22 patients showed normal platelet responsiveness. AUC10 and VWF activity or antigen levels in plasma were not correlated, either before or after HD. CONCLUSIONS: Most ESRD patients display moderate-to-severe platelet dysfunction as assessed by shear-induced platelet-dependent thrombus formation with T-TAS01. HD does not influence platelet function despite HD-induced elevations in VWF. T-TAS01 should be further evaluated as a tool in the assessment of bleeding risk in patients on HD.

Combined Effect of Inter- and Intrapatient Variability in Tacrolimus Exposure on Graft Impairment Within a 3-Year Period Following Kidney Transplantation: A Single-Center Experience.

BACKGROUND AND OBJECTIVE: Tacrolimus is a cornerstone of the most immunosuppressive protocols after kidney transplantation, but its use is complicated by notable interpatient and intrapatient variability (IPV). The goal of this study was to evaluate whether or not tacrolimus IPV, or average dose-adjusted trough concentration (C0/D), during 6-12 months post-transplantation might have contributed to graft function decline in a 3-year period following kidney transplantation. After primary evaluation of individual effects of tacrolimus IPV and C0/D, the study aimed to estimate the combined effect of tacrolimus IPV and C0/D on composite endpoint (consisting of graft failure, chronic allograft dysfunction, chronic rejection, and doubling of serum creatinine concentration) in the period between 13 and 36 months after kidney transplantation. In addition, the goal was to analyze the impact of genetics on interpatient variability in tacrolimus exposure in the early and late post-transplantation periods. METHODS: The study enrolled 104 Caucasian patients and included 2541 patient examinations up to 36 months after kidney transplantation. All patients were genotyped on CYP3A5 6986A>G and ABCB1 3435C>T gene polymorphism. Patients were divided into groups based on the tacrolimus IPV tertiles and the median value of average C0/D during 6-12 months post-transplantation. RESULTS: The results showed a more pronounced decline in estimated glomerular filtration rate values within the high IPV tertile group (p = 0.018), as well as within the low C0/D group (p = 0.013) in a 3-year period after kidney transplantation. The carriers of CYP3A5*1/*3 genotype had lower C0/D compared to the CYP3A5*3/*3 carriers during the entire study period, while the results for ABCB1 were inconsistent when considering tacrolimus C0/D. Patients with high IPV/low C0/D had significantly reduced graft survival compared to the other tacrolimus IPV/C0/D combination groups (i.e., high IPV/high C0/D, low IPV/low C0/D, low IPV/high C0/D) with the hazard ratio of 3.14 in Cox analysis for reaching the composite endpoint. CONCLUSION: The findings of this study suggest that combined assessment of tacrolimus IPV and tacrolimus C0/D may categorize patients towards risk of graft deterioration in the long-term post-transplantation period.

Protein Carbonyl Content Is a Predictive Biomarker of Eccentric Left Ventricular Hypertrophy in Hemodialysis Patients.

High prevalence of left ventricular hypertrophy (LVH) and elevated oxidative stress are associated with poor outcomes in chronic hemodialysis patients. Abnormal left ventriculаr geomеtry and different geometric patterns play an important role as well. Our study analyzed the role of oxidative stress on myocardial remodeling in these patients. Plasma malondialdehyde (MDA), protein carbonyl (PC) content, and total antioxidative capacity (TAC) were investigated in 104 hemodialysis patients together with transthoracic echocardiography. Compared to patients with normal ventricular geometry, patients with LVH had increased MDA and PC plasma concentration. Multivariate analysis demonstrated that protein carbonyls, as biomarkers of oxidative protein modification, were an independent predictor of eccentric hypertrophy (eLVH), including higher LV end-diastolic diameter and LV end-diastolic volume, (ß = 0.32 and ß = 0.28, p < 0.001 for both). The incidence of eLVH increased progressively from the lowest to the highest baseline PC tertile (p < 0.001 for the trend) and the subjects in the former group showed a 76% greater risk of developing eLVH compared to their counterparts. After further adjustment for the potential mediators, PCs carried eLVH odds (95% confidence interval (CI)) of 1.256 (0.998-1.514), per standard deviation increase. High plasma protein carbonyls levels are a significant independent predictor of eccentric LVH in chronic hemodialysis patients.

Investigation of CYP 3A5 and ABCB1 gene polymorphisms in the long-term following renal transplantation: Effects on tacrolimus exposure and kidney function.

The clinical use of tacrolimus (Tac) is complicated by the large inter-individual variability in its pharmacokinetics as well as by chronic adverse effects on renal function. The main goal of this study was to evaluate the potential influence of cytochrome P450 3A5 (CYP 3A5) and ATP-binding cassette transporter B1 (ABCB1) gene polymorphisms on Tac dose requirements and dose-adjusted concentrations in different long-term periods following renal transplantation. Another aim was to investigate whether these polymorphisms affect renal function in late post-transplant period. A total of 91 renal transplant recipients were enrolled for genotyping analysis, and 53 of these entered into a pharmacokinetic-pharmacogenetic study. Allele-specific polymerase chain reaction was used for CYP 3A5 and ABCB1 polymorphism determination. Pharmacokinetic data (dose, trough concentration and dose-adjusted concentration of Tac) and renal function parameters [creatinine (Cre) clearance and serum Cre level] were analyzed in relation to patient genotype at 6, 12 and 24 months after transplantation. Also, linear regression analysis was performed to evaluate the effect of CYP 3A5 and ABCB1 genotypes on Tac exposure and renal function up to 24 months post-transplant. Individuals carrying the CYP 3A5*1/*3 genotype had higher Tac dose requirements than CYP 3A5*3/*3 carriers at 6, 12 and 24 months after renal transplantation. The results revealed that ABCB1 polymorphism did not influence Tac dose requirements independently. Regression analysis showed that CYP 3A5 influenced the Tac dose-adjusted concentration as well as renal function up to 24 months post-transplant. These findings confirmed that CYP 3A5 polymorphism represents the most important determinant of Tac dose and exposure in the late period following renal transplantation. Furthermore, the obtained results indicate that the decline in renal function may be more pronounced in patients with CYP 3A5*1 in the long-term period after renal transplantation.

Potential role of tacrolimus in erythrocytes' antioxidative capacity in long-term period after renal transplantation.

The main goal of this study was to evaluate the influence of tacrolimus daily dose (TDD) as well as cytochrome P450 (CYP) 3A5 6986A>G and ABCB1 3435C>T polymorphisms on the erythrocytes' oxidative stress parameters in long-term period after renal transplantation (Tx). Secondly, we investigated whether tacrolimus and/or oxidative injury might have affected renal function or it was independent from both. In order to evaluate erythrocytes' oxidative stress status in 72 renal transplant recipients and 62 healthy volunteers, we measured the levels of thiobarbituric acid reactive substances (TBARS) and reduced glutathione (GSH) and the activities of superoxide dismutase (SOD), glutathione peroxidase (GPX) and glutathione reductase (GR) as well. Also, we performed allele-specific PCR to determine CYP 3A5 and ABCB1 polymorphisms. Erythrocytes' TBARS positively correlated with SOD, GPX and negatively with GFR. Tested polymorphisms affected TDD, but not oxidative stress parameters. TDD positively correlated with GSH and negatively with GFR. Additionally, tacrolimus dose-adjusted trough concentrations positively correlated with GFR and negatively with GPX and GSH. Furthermore, regression analysis showed that TBARS and TDD independently and negatively affected GFR in long term period after Tx. Our findings suggest that tacrolimus may increase erythrocytes' antioxidative capacity. Regardless, it may be involved in renal function decline in a long-term period after Tx, which seems to be independent from oxidative stress mediated reduction in renal function.

Variability of mycophenolic acid elimination in the renal transplant recipients ­ population pharmacokinetic approach.

The aim of this study was to develop a population pharmacokinetic (PK) model for clearance of mycophenolic acid (MPA) in adult renal transplant recipients, to quantify the PK parameters and the influence of covariates on the MPA pharmacokinetic parameters. Parameters associated with plasma concentrations of MPA at steady-state were analyzed in 70 renal transplant recipients (mean age 42.97 years; mean total body weight 75.33 kg) using nonlinear mixed-effect modeling (NONMEM). Characteristics of patients screened for influence on the pharmacokinetic parameters were gender, age, body weight, time after transplantation, whether the patient was diagnosed as having diabetes mellitus, organ source (living or deceased donor), biochemical parameters and co-therapy (tacrolimus, cyclosporine, prednisolone, omeprazole, bisoprolol, carvedilol, nifedipine). A validation set of 25 renal transplant recipients was used to estimate the predictive performance of population pharmacokinetic model. Typical mean value of MPA oral clearance, estimated by base model (without covariates) was 0.741 L h(-1). During population modeling, the full model showed that clearance of the MPA was significantly influenced by age, total daily dose of MPA, creatinine clearance, albumin level, status and gender of a donor, and the nifedipine and tacrolimus co-therapy. In the final model, clearance of MPA was reported to be significantly influenced by age, total daily dose of MPA and thenifedipine co-therapy. The derived model describes adequately MPA clearance in terms of characteristics of our patients, offering basis for individual pharmacotherapy approach.

Pharmacogenetics may Influence Tacrolimus Daily Dose, but not Urinary Tubular Damage Markers in the Long-Term Period after Renal Transplantation.

BACKGROUND: The primary goal of this study was to evaluate the influence of cytochrome P450 (CYP) 3A5 (6986A>G) and ABCB1 (3435C>T) polymorphisms on tacrolimus (TAC) dosage regimen and exposure. Second, we evaluated the influence of TAC dosage regimen and the tested polymorphisms on renal oxidative injury, as well as the urinary activities of tubular ectoenzymes in a long-term period after transplantation. Also, we aimed to determine the association between renal oxidative stress and tubular damage markers in the renal transplant patients. METHODS: The study included 72 patients who were on TAC based immunosuppression. Allele-specific PCR was used for polymorphism determination. We measured the urinary thiobarbituric acid reactive substances (TBARS) and reactive carbonyl derivates (RCD) in order to evaluate oxidative injury, as well as the urinary activities of ectoenzymes (N-acetyl-ß-D-glucosaminidase, aminopeptidase N and dipeptidyl peptidase IV) to evaluate tubular damage. RESULTS: The carriers of CYP 3A5*1 allele required statistically higher daily doses of TAC than CYP *3/*3 carriers, as well as the carriers of C allele of ABCB1 gene compared to those with TT genotype. Also, there were no differences in TBARS, RCD and the activities of ectoenzymes between the patients' genotypes. Our results showed significant correlations between urinary TBARS and RCD and the ectoenzymes' activities. CONCLUSIONS: Our findings suggest that CYP 3A5 and ABCB1 3435 polymorphism may affect TAC daily doses, but not the drug's tubular toxicity. Furthermore, tubular damage may be associated with increased renal oxidative stress.

Monitoring the toxic effects of Pb, Cd and Cu on hematological parameters of Wistar rats and potential protective role of lipoic acid and glutathione.

Heavy metal pollution is a serious environmental and health problem. The negative effects of heavy metals that can enter human body can be reduced by the addition of some supplements. In this study, the effects of lead (Pb), cadmium (Cd) and copper (Cu) on the hematological parameters in Wistar rats in the absence and presence of lipoic acid and glutathione were analyzed. Pb, Cd and Cu intoxication significantly affected the hematological parameters of treated animals. The main effects in the case of Pb and Cd intoxication were decreased values of erythrocytes, hemoglobin and hematocrit (up to 30% and 20% for these two metals, respectively) compared with the control group. Cu intoxication caused decrease in hematocrit, thrombocytes, mean cell volume values (c.a. 15%) and slight decrease in the erythrocyte number, while the value of hemoglobin increased (c.a. 7%). The treatment with lipoic acid and glutathione reduced the toxic effects of these metals in all cases.

Gender differences in oxidative and nitrosative stress parameters in kidney transplant patients on tacrolimus-based immunosuppression.

BACKGROUND: Cardiovascular (CV) morbidity and mortality rates are still higher after kidney transplantation than in general population. It is known that oxidative and nitrosative stress may contribute to the progress of CV disease in a post-transplant period, but still gender aspect has not been elucidated completely. The aim of this study was to analyze the gender differences in the oxidative and nitrosative stress parameters, as well as asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) levels among kidney transplant patients on tacrolimus-based immunosuppression. METHODS: Our research included 35 patients (20 men and 15 women) with renal transplant and 25 healthy volunteers. Patients were on chronic immunosuppressive regimen, which included tacrolimus, mycophenolate mofetil and prednisone. In order to estimate oxidative and nitrosative stress, we determined plasma levels of thiobarbituric acid-reactive substances (TBARS), activity of catalase (CAT), levels of total (protein and non-protein) sulfhydryl (SH) groups, advanced oxidation protein products (AOPP), ADMA and SDMA, as well as nitrite/nitrate (NOx) ratio. RESULTS: TBARS, CAT and SH in plasma were significantly higher in male patients than in female patients (p < 0.05, p < 0.01 and p < 0.05, respectively). There were no gender-dependent differences in AOPP, ADMA, SDMA and NOx in kidney transplant patients. Correlation analysis, Pearson and Spearman, showed significant correlations between tested oxidative and nitrosative stress parameters in male kidney transplant patients. Alternatively, in female patients, there were no significant correlations between tested parameters. CONCLUSION: Our findings show that men might be more prone to oxidative damage than women. ADMA, the proven marker of CV morbidity and mortality, may be more significant in male kidney transplant patients concerning oxidative stress control of its level and function.

Endothelial dysfunction, inflammation and malnutrition markers as predictors of mortality in dialysis patients: multimarker approach.

BACKGROUNDS: Cardiovascular disease is reported to be major cause of mortality in dialysis patients. Multimarker approach is new approach in risk stratification. Creating a common predictive value, many different pathophysiological components are covered. The aim of this study was to examine the combined predictive value of markers of endothelial dysfunction (ADMA), inflammation (CRP, SAA) and malnutrition (albumin) in dialysis patients. METHODS: In this prospective 3-year follow-up study, 153 prevalent patients (105 males and 48 females) on hemodialysis were included. ADMA were measured by HPLC; CRP and SAA were measured using immunonephelometric assays. Albumins were measured by the use of standard methods on the automated analyzer. The patients were stratified into five groups based on the presence of 1, 2, and 3 or more risk markers, respectively, namely high ADMA (≥0.49 µmol/L), high CRP (≥6.0 mg/L), high SAA (≥7.6 mg/L) and low albumin (<30.3 g/L). RESULTS: The patients with 1, 2, 3 or more risk markers had an adjusted hazard ratio (HR) of 2.419 (0.728-8.034), 6.720 (2.100-21.503), 10.455 (3.193-24.227), respectively, for mortality, compared to those without risk markers. The patients with 1, 2, 3 or more risk markers had an adjusted HR of 1.838 (0.307-11.006), 9.924 (2.052-28.003), 14.823 (0.2.962-34.189), respectively, for cardiovascular mortality than those without risk markers. CONCLUSIONS: The results of this study demonstrate that the common predictive value of several markers is higher than individual predictive value of examined risk factors. Patients with multiple risk factors had higher mortality. Multimarker approach provides an opportunity for better risk stratification in dialysis patients.

How the duration period of erythropoietin treatment influences the oxidative status of hemodialysis patients.

BACKGROUND: End-stage renal disease is a state of enhanced oxidative stress (OS) and hemodialysis (HD) and renal anemia further augment this disbalance. Anemia correction with erythropoietin (EPO) may improve oxidative status. However, there is no evidence of time dependent effects of EPO therapy on redox status of HD patients. OBJECTIVE: The aim of this study was to evaluate whether the duration of EPO treatment may affect OS parameters in uremic patients. PATIENTS AND METHODS: 104 HD patients and 29 healthy volunteers were included. Patients were divided into 3 groups according to the duration of EPO treatment. Forth group consisted of HD patients without EPO treatment. Plasma and erythrocyte malondialdehyde (MDA, MDA(rbc)), reactive carbonyl groups (RCG), plasma sulfhydryl (-SH) groups and total antioxidative capacity (TAC) levels were evaluated. RESULTS: HD patients both with and without EPO treatment, showed a significant increase in all oxidative parameters without significance between EPO treated and -untreated group. The decrease in MDA and MDA(rbc) levels coincided with the duration of EPO treatment. A negative correlation was observed between the duration of EPO treatment and serum MDA (r=-0.309, p=0.003). Increasing periods of EPO treatment were associated with decrease in RCG, without significance between EPO groups. Increase in TAC accompanied increasing durations of EPO treatment, with EPO treatment for more than 24 months causing the most striking changes (p<0.05). There were no significant differences in -SH levels between EPO subgroups. CONCLUSION: Our results suggest that long term administration of EPO attenuated the lipid peroxidation process and restored the levels of antioxidants.

Protective effects of glutathione and lipoic acid against cadmium-induced oxidative stress in rat's kidney.

Cadmium is a widespread, toxic industrial pollutant. The proximal tubule of the mammalian kidney is a major target of Cd-induced toxicity. We analyzed the effects of cadmium exposure on the model system of experimental animals, the thiobarbituric acid (TBA)-reactive substance (TBARS) level, and the activity of xanthine oxidase (XO) and catalase in kidney of rats, with and without glutathione and lipoic acid (LA). The experimental animals were classified into six groups, regarding cadmium, glutathione, and LA intake. The concentration of TBARSs in the homogenate was determined by spectrophotometric method according to Nabavi et al. The specific activity of XO was determined spectrophotometrically by the method of Aygul et al. Catalase activity in tissues was determined by spectrophotometric method according to Nabavi et al. The increased level of TBARS and the increased activity of XO in kidney tissue in cadmium poisoning are statistically significant compared to control (p < 0.001). Glutathione and LA applied along with cadmium lowered TBARS concentration and reduced XO activity (p < 0.001). Catalase activity in the kidney tissue was increased in the group, which was administered cadmium (p < 0.001). In conclusion, glutathione and LA, as physiological antioxidants applied with cadmium, have reduced the level of lipid peroxide and the activity of XO, and can be used as protectors in conditions of cadmium poisoning.

Influence of dialysis modality and membrane flux on quality of life in hemodialysis patients.

BACKGROUND: The quality of life in patients undergoing hemodialysis is significantly disturbed. There are data that hemodiafiltration (HDF) may be more effective than conventional hemodialysis in the removal of uremic toxins and may reduce frequency and severity of intradialytic and postdialysis adverse symptoms in patients. Also, some researchers suggest advantages of using high-flux membranes compared with low-flux. OBJECTIVE: The aim of this study was to examine whether hemodialysis modality and membrane flux, independent of membrane biocompatibility, make differences in quality of life in patients. METHODS: In our cross-sectional study, we evaluated 124 patients who were divided, based on therapy, into three groups: online HDF, high-flux hemodialysis, and low-flux hemodialysis. Data were collected using the Short Form-36 questionnaire combined with special questionnaire, which included demographic and clinically related questions. RESULTS: Health-related quality of life was better in patients on HDF compared with patients on hemodialysis, especially compared with low-flux hemodialysis patients in most of the scales and in both dimensions: physical component scale and mental component scale. There were no statistically significant differences in Short Form-36 domains between high-flux hemodialysis and low-flux hemodialysis. CONCLUSION: Our data suggest the potential advantages of HDF with regard to influence on quality of life, which is sufficient to justify further research in prospective and longitudinal study design.