Miguel Cendoroglo

Effect of two-chambered bicarbonate lactate-buffered peritoneal dialysis fluids on peripheral blood mononuclear cell and polymorphonuclear cell function in vitro☆☆☆

Low pH, high osmolality, increasing glucose concentration, and glucose degradation products (GDP) formed during heat sterilization of conventional peritoneal dialysis (PD) fluids have been shown to have a detrimental effect on cells involved in peritoneal host defense. The two-chambered PD fluid bag in which glucose at pH approximately 3 is separated from a bicarbonate (25 mmol/L)-lactate (15 mmol/L) buffer during heat sterilization permits PD fluids with lower GDP to be delivered to the patient at neutral pH. To establish the possible benefit of two-chambered bag PD fluids on peripheral blood mononuclear cell (PBMC) and polymorphonuclear (PMN) cell function, we compared conventional 1.5% Dianeal (1.5%D) with 1.5% two-chambered bag bicarbonate-lactate (1.5%D-B), and conventional 4.25% Dianeal (4.25%D) with 4.25% two-chambered bag bicarbonate-lactate (4.25%D-B). Furthermore, to study the effect of the sterilization process on PBMC and PMN function, we compared filter-sterilized 4.25%D (4.25%D-F) with 4.25%D and 4.25%D-B. PBMC were harvested by Ficoll-Hypaque separation, and 2.5 x 10(6) cells in RPMI were incubated with an equal volume of the test fluids for 4 hours, pelleted, and resuspended in RPMI containing 10 ng endotoxin for a further 20 hours. Tumor necrosis factor alpha (TNF-alpha) production by endotoxin-stimulated PBMC was not significantly different (P = 0.10) between 1.5%D-B and 1.5%D, but was significantly higher (P = 0.01) with 4.25%D-B compared with 4.25%D. PBMC exposed to filter-sterilized fluid (4.25%D-F) showed significantly higher endotoxin-stimulated TNF-alpha production compared with 4.25%D (P = 0.02), but was not significantly different from 4.25%D-B (P = 0.40). PMN were harvested by Ficoll-Hypaque separation and 10 x 10(6) cells incubated with test fluids for 30 minutes. After incubation, phagocytosis (phagocytosis index) was determined by the uptake of 14C-labeled Staphylococcus aureus, oxidative burst by reduction of ferricytochrome C to ferrocytochrome C on stimulation with PMA, and enzyme release by measurement of endotoxin-stimulated bactericidal/permeability increasing protein (BPI). Bicarbonate-lactate two-chambered fluids of similar osmolality and glucose concentration conferred a significant improvement in phagocytosis (P = 0.02 for 1.5%D-B and P < 0.001 for 4.25%D-B). Oxidative burst and BPI release were significantly higher in 4.25%D-B compared with 4.25%D (P < 0.001). Filter-sterilized 4.25%D-F conferred a significant improvement in phagocytosis and oxidative burst compared with 4.25%D (P < 0.001) or 4.25%D-B (P < 0.001). Furthermore, conventional 4.25%D was associated with significantly lower BPI release compared with 4.25%D-F (P = 0.01). GDPs acetaldehyde and 5-HMF were analyzed in 4.25%D-B, 4.25%D, and 4.25%D-F. Acetaldehyde was below the lower limit (0.79 ppm) of the standard curve in 4.25%D-B and 4.25%D-F fluids but was detected (3.76 to 5.12 ppm) in all of the 4.25%D fluids. Relative levels of 5-HMF in the 4.25%D-B (0.032 to 0.041 Abs @ 284 nm) and 4.25%D (0.031 to 0.036 Abs @ 284 nm) were similar. The lowest levels (0.001 Abs @ 284 nm) were observed in the filter-sterilized 4.25%D-F. The beneficial effects of two-chambered bicarbonate lactate-buffered PD fluids on PBMC and PMN function are probably related to reduction of GDP from heat sterilization of glucose in a separate chamber at a lower pH. This improvement in biocompatibility could have a beneficial affect on peritoneal defenses.

Shiga toxin induces superoxide production in polymorphonuclear cells with subsequent impairment of phagocytosis and responsiveness to phorbol esters

The role of inflammatory cells in the pathogenesis of hemolytic-uremic syndrome induced by Shiga toxin (Stx)-producing Escherichia coli remains unclear. The hypothesis that Stx has direct effects on polymorphonuclear cell (PMN) viability and function was examined by measuring apoptosis, necrosis, phagocytosis, and spontaneous and phorbol myristate acetate (PMA)-stimulated production of reactive oxygen intermediates. PMN from 6 healthy persons were exposed to medium, Stx1 (0.01-100 ng/mL), or heat-inactivated Stx1 or Stx1 B subunit (100 ng/mL). Stx1 induced a prominent dose-dependent respiratory burst from PMN at doses as low as 0.01 ng/mL; they were less responsive to PMA stimulation and had reduced ability for phagocytosis. This dysfunction was not due to cell death, as the magnitude of apoptosis and necrosis of PMN treated with Stx1 (100 ng/mL) for 20 h was identical to that of medium control. These results suggest that Stx has direct effects on PMN that could contribute to tissue injury early in the disease.

The use of regional citrate anticoagulation for continuous venovenous hemodiafiltration in acute kidney injury.

Objective:Continuous renal replacement therapy is commonly used in the treatment of acute kidney injury. Although the optimal anticoagulation system is not well defined, citrate has emerged as the most promising method. We evaluated the data of 143 patients with acute kidney injury subjected to citrate-based continuous venovenous hemodiafiltration. Design:Retrospective cohort study. Setting:Intensive care unit of tertiary care private hospital. Patients:Patients with acute kidney injury treated from February 2004 to July 2006. Interventions:None. Measurements and Main Results:The main cause of acute kidney injury was sepsis (58%). The mean dialysis dose was 36.6 mL/kg/hr allowing for excellent metabolic control (last tests: creatinine, 1.1 mg/dL; urea, 46 mg/dL). No significant bleeding, severe electrolyte, or calcium disorders were observed. Of the 418 filters used, almost 28,000 hrs of treatment, hemofilter patency was 68% at 72 hrs. Hospital mortality was 59%, and 22% of survivors were dialysis-dependent at the time of discharge. Within our sample, we identified 21 patients with liver failure (mean prothrombin time index, 21% vs. 67%, p < 0.001). This group presented with a lesser median systemic ionized calcium level (1.06 vs. 1.12 mmol/L, p < 0.001) and similar mean total calcium level (8.5 vs. 8.6 mg/dL, not significant), compared with patients without liver failure. These subjects also showed acidemia (median pH, 7.31 vs. 7.40, p < 0.001); however, they exhibited higher levels of lactate (median 29 vs. 16 mg/dL, p < 0.001), chloride (mean 109 vs. 107 mEq/L, p = 0.045) and had a trend to higher mortality rate (76% vs. 56%). Conclusions:Besides a trend toward higher mortality rate observed in the group with liver failure, we found that citrate-based continuous venovenous hemodiafiltration allowed an effective dialysis dose and reasonable filter patency.

Coronary Artery Calcification, Systemic Inflammation Markers and Mineral Metabolism in a Peritoneal Dialysis Population

Aims: To assess the prevalence of coronary artery calcification (CAC) in peritoneal dialysis (PD) patients and to determine whether comorbidities such as inflammation, dyslipidemia and mineral metabolism disorders correlate with its development. Methods: Forty-nine PD patients (45% male; median age, 52 years) were submitted to multislice computed tomography. Inflammatory markers, anti-oxidized LDL antibody, calcium–phosphate balance and lipid profiles were assessed. Results: Twenty-nine patients (59.2%) presented CAC (median calcium score, 234.7 Agatston units). Patients with CAC were older than those without, more frequently presented a history of coronary artery disease or hypertension and had lower HDL cholesterol levels, as well as presenting higher levels of osteoprotegerin and LDL oxidation. The logistic regression revealed that the independent determinants of CAC were age (odds ratio = 1.12; p = 0.006) and number of prescribed anti-hypertensive drugs (odds ratio = 2.38; p = 0.048). When the population was stratified by calcium score quartile, soluble Fas levels were significantly higher in patients with severe calcification. In patients younger than 45, CAC correlated positively with phosphorus levels (r = 0.52; p = 0.04). Conclusion: In PD patients, CAC is highly prevalent. Our results indicate that conditions such as inflammation and mineral disturbances are associated with its development.

Modulation of Neutrophil Apoptosis by Uremic Plasma during Hemodialysis

During hemodialysis (HD), blood-membrane interactions lead to activation of several circulating cells and plasma proteins. The resultant activation and/or release of mediators can modulate the structure, function and survival of circulating neutrophils. Little is known of plasma factors that influence apoptosis of neutrophils in hemodialyzed patients. Hence, we investigated the effect of uremic plasma obtained during HD on the survival of neutrophils obtained from healthy volunteers. Neutrophils harvested from healthy volunteers were incubated in ultrafiltered culture medium supplemented with either 50% heterologous normal plasma obtained from healthy volunteers (n = 15) or 50% uremic plasma collected from long-term HD patients dialyzed with cuprophan (CU) (n = 8), cellulose triacetate (CTA) (n = 8) or polysulfone (PS) (n = 8) dialyzers. Plasma samples were drawn predialysis, 15 min after starting dialysis, and postdialysis. After 24-hour incubation, neutrophil aliquots were processed for quantification of apoptosis by flow cytometry, using propidium iodide DNA staining. In addition, tumor necrosis factor α (TNFα) and interleukin-10 (IL-10) were measured in normal and predialysis uremic plasma samples. Neutrophils from healthy volunteers exposed to heterologous normal plasma samples exhibited 10.3 ± 1.2% apoptosis. In contrast, the proportion of apoptosis was significantly higher among neutrophils exposed to predialysis (28.5 ± 2.3%, p < 0.0001), 15 min (23.0 ± 2.4%, p < 0.0001), or postdialysis uremic plasma samples (25.7 ± 2.3%, p < 0.0001). Compared to neutrophils exposed to predialysis uremic plasma samples, a significantly lower proportion of apoptosis was observed in neutrophils exposed to the 15-min plasma samples among patients dialyzed with CU (26.4 ± 2.9 vs. 18.2 ± 3.5%; p < 0.001) but not with CTA or PS dialyzers. Futher, CU membranes induced the greatest percentage decrease in neutrophil apoptosis at 15 min. There was a direct correlation between neutrophil apoptosis and plasma levels of TNFα (r = 0.424, p = 0.02) and IL-10 (r = 0.744, p < 0.0001). The results of the study suggest that normal neutrophils exposed to uremic plasma undergo accelerated in vitro apoptosis compared to those incubated with normal plasma. Further, during HD, the apoptosis-inducing activity of uremic plasma is modulated by the use of dialyzers with different degrees of biocompatibility. The identification of soluble factors that are responsible for the increased apoptosis-inducing activity of uremic plasma needs to be further investigated.

New Polyether Sulfone Dialyzers Attenuate Passage of Cytokine- Inducing Substances from Pseudomonas aeruginosa Contaminated Dialysate

The use of bicarbonate dialysate and high-flux and reprocessed dialyzers has raised concerns about the reverse transfer of dialysate contaminants into the blood compartment. This in vitro study was performed to investigate the reverse transfer of soluble Pseudomonas aeruginosa bacterial products across a polyether sulfone (PES), a newly developed synthetic polymer dialyzer. In vitro dialysis was carried out at 37°C in a closed countercurrent recirculating loop dialysis circuit with a new PES dialyzer. An equal mixture of heparinized whole blood (from healthy volunteers) with pyrogen-free tissue culture medium was circulated in the blood compartment, and bicarbonate dialysate was circulated in the dialysate compartment. After 15 min of dialysis, the dialysate was challenged sequentially with 10–4, 10–3, and 10–2 dilutions of a P. aeruginosa culture supernatant. 1-ml samples were drawn from the blood compartment 5 and 15 min after each challenge and incubated upright at 37°C. At the end of 24 h, Triton X-100 was added, in order to extract total interleukin (IL) 6 and IL-8 production by the whole-blood mixture. These cytokines were measured by electrochemiluminescence assays. At dilutions of 10–4 and 10–3, the reverse transfer of soluble bacterial products across the dialyzer was negligible. Five and 15 min after contaminating the dialysate with the highest concentration (10–2 dilution), the increase in IL-6 production was 239 ± 170% (p = 0.06) and 886 ± 444% (p = 0.02), respectively. However, comparing the IL-6-inducing potency of the 10–2 bacterial supernatant dilution to the spontaneous IL-6 production in the blood compartment during dialysis with the same dilution of dialysate contaminant, there was a dramatic reduction in IL-6 production by 94 and 89% at 5 and 15 min, respectively. Similarly, 5 and 15 min after contaminating the dialysate with the 10–2 dilution, the increase in IL-8 production was 357 ± 147% (p = 0.07) and 630 ± 229% (p = 0.04), respectively. However, comparing the IL-8-inducing potency of the 10–2 bacterial supernatant dilution to the spontaneous IL-8 production in the blood compartment during dialysis with the same dilution of dialysate contaminant, there was a dramatic reduction in IL-8 production by 93 and 92% at 5 and 15 min, respectively. These results demonstrate that PES dialyzers markedly attenuate passage of cytokine-inducing substances from contaminated dialysate, using a method that detects the entire cytokine synthetic output in the blood compartment.

Effect of phosphate binders on oxidative stress and inflammation markers in hemodialysis patients

It has been suggested that phosphate binders may reduce the inflammatory state of hemodialysis (HD) patients. However, it is not clear whether it has any effect on oxidative stress. The objective of this study was to evaluate the effect of sevelamer hydrochloride (SH) and calcium acetate (CA) on oxidative stress and inflammation markers in HD patients. Hemodialysis patients were randomly assigned to therapy with SH (n=17) or CA (n=14) for 1 year. Before the initiation of therapy (baseline) and at 12 months, we measured in vitro reactive oxygen species (ROS) production by stimulated and unstimulated polymorphonuclear neutrophils and serum levels of tumor necrosis factor α, interleukin‐10, C‐reactive protein, and albumin. There was a significant reduction of spontaneous ROS production in both groups after 12 months of therapy. There was a significant decrease of Staphylococcus aureus stimulated ROS production in the SH group. There was a significant increase in albumin serum levels only in the SH group. In the SH group, there was also a decrease in the serum levels of tumor necrosis factor α and C‐reactive protein. Our results suggest that compared with CA treatment, SH may lead to a reduction in oxidative stress and inflammation. Therefore, it is possible that phosphate binders exert pleiotropic effects on oxidative stress and inflammation, which could contribute toward decreasing endothelial injury in patients in HD.

Correlates of Urea Kinetic Modeling during Hemodialysis in Patients with Acute Renal Failure

The current guidelines on dialysis adequacy in acute renal failure (ARF) are loosely defined and have been extrapolated from patients with end-stage renal disease. The objectives of this study were (1) to compare three methods of urea kinetic modeling measurement in patients with ARF receiving intermittent hemodialysis, (2) to compare prescribed to delivered dose of dialysis, and (3) to explore the factors that are associated with dialysis delivery. ‘Single-pool’ urea kinetic modeling was assessed by the Ureakin® software and the second-generation equation which uses a logarithmic estimate of spKt/V. ‘Equilibrated’ Kt/V (eKt/V) was calculated using the rate adjustment equation. The prescribed dose was derived using the manufacturer’s specifications of the dialyzer clearance, prescribed time, actual delivered blood and dialysate flow, and estimates of volume of urea distribution. A total of 78 consecutive spKt/V measurements were obtained in 24 patients. The mean urea reduction ratio was 51 ± 1%. The delivered spKt/V was significantly lower than that prescribed (0.87 ± 0.03 or 0.83 ± 0.03 vs. 1.28 ± 0.05; p = 0.0001). The equilibrated Kt/V was markedly lower than the delivered spKt/V (0.73 ± 0.03 vs. 0.83 ± 0.03; p = 0.0001). Univariate analyses demonstrated that female gender, low body mass index, low predialysis weight, use of cellulose acetate dialyzers, and increased prescribed time were associated with increased odds of prescribed spKt/V ≧1.2. Similarly, old age, increased delivered time, and high cytokine production were associated with increased odds of delivered spKt/V ≧1.2. In summary, while the impact of delivered intermittent hemodialysis on the survival of patients with ARF remains to be determined, these results indicate that dialysis delivery is suboptimal in ARF, and empiric dosing should strongly consider factors related to lean body mass, including age and gender.

Tumour necrosis factor-α plus interleukin-10 low producer phenotype predicts acute kidney injury and death in intensive care unit patients

Genetic polymorphism studies of cytokines may provide an insight into the understanding of acute kidney injury (AKI) and death in intensive care unit (ICU) patients. The aim of this study was to investigate whether the genetic polymorphisms of −308 G < A tumour necrosis factor (TNF)‐α, −174 G > C interleukin (IL)‐6 and −1082 G > A IL‐10 may predispose ICU patients to the development of AKI and/or death. In a prospective nested case–control study, 303 ICU patients and 244 healthy individuals were evaluated. The study group included ICU patients who developed AKI (n = 139) and 164 ICU patients without AKI. The GG genotype of TNF‐α (low producer phenotype) was significantly lower in the with AKI than without AKI groups and healthy individuals (55 versus 62 versus 73%, respectively; P = 0·01). When genotypes were stratified into four categories of TNF‐α/IL‐10 combinations, it was observed that low TNF‐α plus low IL‐10 producer phenotypes were more prevalent in patients with AKI, renal replacement therapy and death (P < 0·05). In logistic regression analysis, low TNF‐α producer plus low IL‐10 producer phenotypes remained as independent risk factors for AKI and/or death [odds ratio (OR) = 2·37, 95% confidence interval (CI): 1·16–4·84; P = 0·02] and for renal replacement therapy (RRT) and/or death (OR = 3·82, 95% CI: 1·19–12·23; P = 0·02). In this study, the combination of low TNF‐α plus low IL‐10 producer phenotypes was an independent risk factor to AKI and/or death and RRT and/or death in critically ill patients. Our results should be validated in a larger prospective study with long‐term follow‐up to emphasize the combination of these genotypes as potential risk factors to AKI in critically ill patients.

Effects of Spermidine and p-Cresol on Polymorphonuclear Cell Apoptosis and Function

Polymorphonuclear leukocytes (PMNs) from chronic kidney disease (CKD) patients display accelerated apoptosis and dysfunction, which may predispose CKD patients to infections. In this study, we investigated the effect of spermidine and p-cresol on apoptosis and function on PMN from healthy subjects. We measured the effect of spermidine and p-cresol on apoptosis, ROS production unstimulated and stimulated (S. aureus and PMA) and expression of CD95, caspase 3, and CD11b on PMN. After incubation with p-cresol and spermidine, we did not observe any changes in apoptosis, viability or expression of caspase 3 and CD95 in PMN from healthy subjects. PMN incubated for 10 minutes with spermidine demonstrated a significant reduction in spontaneous, S. aureus and PMA-stimulated ROS production. p-cresol induced a decrease in PMA-stimulated ROS production. Spermidine and p-cresol also induced a decrease in the expression of CD11b on PMN. Spermidine and p-cresol decreased the expression of CD11b and oxidative burst of PMN from healthy subjects and had no effect on PMN apoptosis and viability.