Hemodialysis membrane-induced activation of phagocyte oxidative metabolism detected in vivo and in vitro within microamounts of whole blood

The production of reactive oxygen species by phagocytes from uremic patients undergoing hemodialysis was monitored by chemiluminescence (CL) within microamounts of whole blood or isolated polymorphonuclear (PMN) cells, and compared on the basis of the dialysis membrane, cuprophane (CUP) or polyacrilonitrile (PAN). Compared to control subjects, resting and stimulated CL (with latex, zymosan, phorbol myristate acetate (PMA) but not formyl-methionyl-leucyl-phenylalanine (FMLP) were decreased in 10(-2) diluted blood sampled before dialysis. After 15 min of dialysis (ti), resting whole blood (10(-1) and 10(-2) diluted) CL increased sharply in patients dialyzed with the CUP but not the PAN membrane, while it returned to its predialysis level at the end of the session. This sharp resting CL increase found in whole blood at ti was not observed in isolated PMN cells except when tested with ti plasma from CUP dialyzed patients, suggesting that it was mediated via activated plasma compounds. In vitro treatment of normal blood, plasma, and isolated PMN cells with CUP membrane fragments reproduced this in vivo dialysis-induced activation of phagocyte oxidative metabolism strikingly and demonstrated additionally the requirement of complement for its induction. We propose this model as an effective means of evaluating dialysis membrane biocompatibility.     

Permeability of cellulosic and non-cellulosic membranes to endotoxin subunits and cytokine production during in-vitro haemodialysis.

The possibility of endotoxin transfer across haemodialysis membranes remains a controversial issue. Additional concern has arisen because of the recent introduction in clinical practice of highly permeable, synthetic dialysis membranes and of bacteria-contaminated bicarbonate concentrate with potential short-term and long-term hazards for haemodialysis (HD) patients. Therefore, we performed experiments in an in-vitro dialysis recirculation system using three different types of HD membranes, namely standard regenerated cellulose (Cuprophan, CU), polyacrylonitrile AN-69 (PAN), and polysulphone F-60 (PS). When radiolabelled lipopolysaccharide (125I M-LPS) from E. coli, together with 10 micrograms/ml unlabelled LPS, was added to the recirculating solution in the dialysis compartment, radioactivity could be detected in the blood compartment after 15 min and increased progressively with time up to respectively 6.7% (CU), 10.3% (PAN), and 10.3% (PS) of initial activity on the dialysate side. The addition of albumin to the solution on the blood side led to a decreased permeability of radioactivity (7.3% vs 10.3%), compared to the absence of albumin (tested only for PS membrane). Furthermore, 73% of 125I M-LPS transferred across the PS membrane in the presence of albumin was TCA-precipitable. In contrast, free iodine (Na 125I) incubated in an albumin-containing solution did not precipitate with albumin after the addition of TCA (precipitation of only 0.6%). Moreover, kinetics of transmembranous transfer of Na-125I were strikingly different from that of 125I M-LPS. Analysis by the method of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of the blood side solution, after LPS addition in the dialysis solution and 30 min of back-filtration, revealed the presence of several silver-stainable and autoradiographic bands of low-molecular-weight range, probably LPS fragments. Finally, the presence of LPS in the dialysate compartment led to a moderate increase in interleukin 1 (IL-1) and tumour necrosis factor alpha (TNF) concentrations in plasma as well as in monocyte culture supernatants after isolation from recirculating normal human whole blood exposed to CU, PAN, or PS membrane. In conclusion, our study provides evidence for the permeation of low-molecular-weight LPS subunits across cellulosic and non-cellulosic HD membranes. The clinical significance, if any, of such a transfer has, however, still to be demonstrated.     

Apoptotic markers on lymphocytes and monocytes are unchanged during single hemodialysis sessions using either regenerated cellulose or polysulfone membranes

BACKGROUND: There is an increased rate of apoptosis of peripheral blood mononuclear cells (PBMCs) in patients undergoing hemodialysis (HD), but little is known about how different dialysis membranes may contribute to the process. We, therefore, studied the influence of two different dialysis membranes on apoptotic markers during HD. METHODS: 8 healthy controls and 8 patients on regular HD 3 times per week were enrolled in this cross-controlled study. Patients received HD using polysulfone and then regenerated cellulose dialysis membranes for one week each, sequentially. Serum was collected for C-reactive protein (CRP) detection; flow cytometry with dual antibody staining was used to measure the apoptotic markers Fas (CD95), FasL (CD 178) and TNF-R2 (CD120b) in T cells (CD3+), B cells (CD19+), and monocytes (CD14+) at 0, 15, 120 and 240 min after starting HD. We also measured total leukocyte numbers and differential white cell counts. RESULTS: Hemodialysis patients revealed lymphocytopenia, monocytopenia, higher CRP levels and higher Fas and TNF-R2 expression on lymphocytes and monocytes at baseline when compared with normal controls. Leukocyte numbers, including neutrophils, lymphocytes and monocytes, dropped significantly after 15 min of dialysis. There were no significant differences in Fas levels during hemodialysis on T and B lymphocytes or on monocytes. T lymphocyte FasL (CD 178) levels remained unchanged throughout the process. There was a significantly lower overall level of CD120b at 15 min of HD, whereas this marker was higher on monocytes after dialysis. There were no significant differences in the levels of apoptotic markers between the two membranes. CONCLUSION: Our results suggest that uremia itself contributes to PBMC apoptosis. The two different dialysis membranes used in this study did not influence apoptotic markers on PBMCs significantly, but increased TNF-R2 expression on monocytes during a single dialysis session.     

Evaluation of haemodialysis membrane biocompatibility by parallel assessment in an ex vivo model in health y volunteers

Background. Precise evaluation of the haemocompatibility of prototype membranes, flow configurations and anticoagulant regimens is an essential step in the development of dialysis systems minimizing blood activation. An ex vivo model in humans currently employed in our laboratory has recently been adapted to allow the parallel evaluation of two minimodule dialysers with blood from a single donor, thus eliminating differences due to donor variability in the comparison of test and control dialysis modules. Methods. The ex vivo flow system is designed to reproduce the haemodynamic conditions of clinical dialysis on a 1/50 scale. A blood line from the forearm vein of the volunteer donor is divided at a Y-shaped junction, two roller pumps assure equivalent blood flow (5 ml/min) in the branches leading to two minimodule dialysers and heparin (0.1 IU/ml final concentration) is injected into each branch immediately after the Y junction. Samples for analysis of blood activation markers are collected at the exits of the two minimodules over a test period of 27 min In the present series of tests, a new polyacrylonitirile membrane (PAN) was evaluated relative to standard commercial polysulphone (PS), acrylonitrile copolymer (AN 69) and cuprophan (CUP) membranes. Results. A steady minimal level of anticoagulation corresponding to a slightly less than two-fold prolongation of APTT (activated partial thromboplastin time) was maintained throughout testing in both branches of the ex vivo flow system. Time curves for the accumulation of activation markers (thrombin-antithrombin II complexes, prothrombin fragment 1+2, platelet {beta}-thromboglobulin, and complement fragment C3a) showed all four types of minimodule dialyser to induce comparable low levels of activation of coagulation parameters and platelets, together with similar mild activation of complement for AN 69, PAN, and PS dialysers as compared to stronger activation for CUP modules. Overall results thus confirmed the acceptable haemocompatibility of the prototype polyacrylonitrile (PAN) membrane. Conclusions. Among current methods for evaluation of the biocompatibility of haemodialysis systems, ex vivo flow models in humans avoid problems arising from species differences and may be designed to closely reproduce the conditions of clinical dialysis. A parallel configuration eliminates artefacts due to individual variations in donor response. This not only facilitates the direct comparison of test and control membranes under close to identical experimental conditions, but also provides a model particularly well adapted to studies of the effects of different anticoagulation regimens, flow configurations and dialysates, or alternative methods of sterilization, rinsing and priming of the dialysers.     

In Vivo Evaluation of Platelet Activation by Different Cellulosic Membranes

Abstract: This study was undertaken to evaluate platelet activation in vivo induced by different cellulosic membranes by measuring the expression of P-selectin on the platelet surface during hemodialysis in 9 uremic patients. Hollow fiber dialyzers of similar surface with different cellulosic membranes (Cuprophan, cellulose acetate, cellulose triacetate, and Hemophan) were evaluated and compared to a synthetic membrane (polysulfone). Blood samples were obtained before hemodialysis and from the efferent and afferent limbs 5 min after the beginning of dialysis. P-select in exposure was evaluated by flow cytometry (FACScan) using a monoclonal antibody (RUU 2.17). The percentage of platelets expressing P-select in before hemodialysis and the percentage from the arterial line during hemodialysis were similar. All membranes evaluated induced platelet activation (estimated as the increase in percentage of platelets expressing P-selectin in samples obtained from the venous line with respect to the arterial line). Cuprophan induced more platelet activation than any other membrane (p < 0.05). The activation induced by cellulose acetate and cellulose triacetate membranes was also higher than that observed with Hemophan (p < 0.05). Hemophan-induced platelet activation was similar to that of polysulfone. These results indicate that all cellulosic membranes induce platelet activation during hemodialysis although there are quantitative differences among them. While Cuprophan induced the highest degree of platelet activation, Hemophan was the only cellulosic membrane that showed a degree of platelet activation similar to the biocompatible membrane polysulfone.     

Dialysis filter type determines the acute effect of haemodialysis on endothelial function and oxidative stress.

BACKGROUND: Endothelial function of large arteries is impaired in chronic haemodialysis patients and oxidative stress due to the dialysis procedure has been suggested as a causal factor. However, it is not clear whether different types of dialysis membranes affect endothelial function differently. Therefore we determined endothelium-dependent, flow-mediated dilatation (FMD) of the brachial artery as well as markers of oxidative stress immediately before and after haemodialysis (HD) with either a cellulosic cuprophane or a synthetic polysulphone dialyser in a blinded, randomized, cross-over study. METHODS: Twelve haemodialysis patients (age 55+/-3 years, time on dialysis 20+/-2 months, mean fluid change -1782+/-21 ml, systolic/diastolic blood pressure 139/75 mmHg) were included. Using a multi-gate-pulsed Doppler system (echo-tracking device) brachial artery FMD and nitroglycerine-induced, endothelium-independent vasodilatation (NMD) were measured. Patients were randomized to HD with either a polysulphone or a cuprophane membrane and were crossed over to the other filter. Investigators were blinded to the type of membrane used. Serum concentrations of oxidized LDL (oxLDL) and alpha-tocopherol as markers of oxidative stress were measured before and after each dialysis session. RESULTS: Data are given as mean+/-SEM. Treatment with polysulphone filter HD did not significantly affect FMD (baseline 9.3+/-2.0% vs after HD 9.6+/-1.8%). After dialysis with a cuprophane membrane FMD decreased from 9.4+/-2.1 to 7.4+/-1.8% (P<0.05). NMD was not significantly affected by HD irrespective of the membrane material used. Serum levels of oxLDL were not changed by either treatment; however, alpha-tocopherol concentrations fell significantly after dialysis with the cuprophane filter (baseline 18.0+/-2.3 after HD 16.6+/-1.3 micro g/ml, P<0.05), while alpha-tocopherol levels remained unchanged when the polysulphone membrane was used. CONCLUSIONS: The type of dialysis filter membrane determines the acute effect of haemodialysis on arterial endothelial function. Differences in biocompatibility and oxidative stress may account for the observed differential effects, since the decrease of FMD after dialysis with a cellulosic cuprophane membrane-but not with a synthetic polysulphone membrane-was associated with a reduction in serum vitamin E.     

Comparison of cellulose diacetate and polysulfone membranes in the outcome of acute renal failure. A prospective randomized study.

BACKGROUND: Whether the nature of haemodialysis (HD) membranes can influence the outcome of acute renal failure (ARF) remains debatable. Recent studies have suggested that dialysis with bioincompatible unsubstituted cellulosic membranes is associated with a less favourable patient outcome than dialysis with biocompatible synthetic membranes. Since we generally use a modified cellulosic membrane with substantially lower complement- and leukocyte-activating potential than cuprophane, for dialysis of patients with ARF, and because there are no data in the literature regarding the influence of modified cellulosic membranes on the outcome of patients with ARF, we compared the outcome of ARF patients dialysed either with cellulose diacetate or with a synthetic polysulfone membrane. We also investigated the potential role of permeability by comparing membranes with high-flux versus low-flux characteristics. METHODS: This prospective, randomized, single centre study included 159 patients with ARF requiring HD. Patients were stratified according to age, gender, and APACHE II score and then randomized in chronological order to one of three dialysis membranes: low-flux polysulfone, high-flux polysulfone and meltspun cellulose diacetate. RESULTS: Aetiologies of ARF and the prevalence of oliguria were similarly distributed among the three groups. There was no significant difference between the three groups for survival (multivariate Cox's proportional hazards model, P=0.57), time necessary to recover renal function (P=0.82), and number of dialysis sessions required before recovery (P=0.86). Multivariate analysis showed that survival was significantly influenced only by the severity of the disease state (APACHE III score, P<0.0001), but not by the nature of the dialysis membrane (P=0.57) or the presence of oliguria (P=0.24). CONCLUSIONS: Among patients with ARF requiring HD survival and recovery time are not significantly influenced by the use of either meltspun cellulose diacetate or the more biocompatible high-flux or low-flux polysulfone. Dialysis using modified cellulose membranes is just as effective as dialysis using synthetic polysulfone membranes, but at a lower cost. In addition, the flux of the membrane did not influence patient outcome.     

Myeloperoxidase serves as a marker of oxidative stress during single haemodialysis session using two different biocompatible dialysis membranes.

BACKGROUND: There is increased oxidative stress in patients undergoing haemodialysis (HD); however, little is known of how different dialysis membranes contribute to the oxidative stress induced by the dialysis procedure per se. We therefore studied the influence of two different dialysis membranes on oxidative stress during HD. METHODS: Eight patients undergoing HD three times per week were enrolled in this cross-controlled study. Patients sequentially received HD using polysulphone (PS) and regenerated cellulose (RC) dialysis membranes for 1 week each. Blood samples were collected in the last section of each hollow fibre 0, 15, 120 and 240 min after starting HD. We determined superoxide anion production derived from neutrophils, superoxide dismutase (SOD) and glutathione peroxidase (GPx) derived from washed red cells, plasma myeloperoxidase (MPO), plasma thiobarbituric acid-reactive substances (TBARS), plasma advanced oxidation protein products (AOPP) and serum 8-hydroxy-2'-deoxyguanosine (8-OHdG). RESULTS: Leukocyte numbers, including neutrophils, lymphocytes and monocytes, decreased significantly after 15 min of dialysis, more so with RC than with PS membrane. For both membranes, superoxide anion production transiently increased during the first 15 min whereas the post-dialysis production was decreased. Plasma MPO levels persistently increased during dialysis with the two membranes. Moreover, the increase was more marked with RC than with PS membrane. AOPP and 8-OHdG levels increased progressively when using RC membranes. There were no significant differences in SOD, GPx, TBARS, AOPP and 8-OHdG levels between the two membranes. CONCLUSIONS: The biocompatibility of the dialyser affects oxidative stress production during a single dialysis session. The measurement of MPO may serve as a reliable marker of the degree of oxidative stress induced using dialysis membranes of different biocompatibilities.     

Biocompatibility of cellulosic and synthetic membranes assessed by leukocyte activation

BACKGROUND/AIMS: The contact of blood with artificial surfaces may activate blood leukocytes and platelets and initiate the leukocyte inflammatory response. We have investigated the effect of a hemodialysis (HD) with a cellulosic- and a synthetic-based membrane on circulating leukocyte activation. METHODS: Samples were obtained from patients with ESRD at baseline, and at 15 and 120 min of a hemodialysis session from both the arterial and venous lines. Leukocyte respiratory burst was analyzed by luminol chemiluminescence. Actin polymerization, expression of CD11b, and heterotypic aggregation were studied by flow cytometry, leukocyte labeling with NBD phallacidin and monoclonal antibodies, respectively. RESULTS: HD with a cellulosic membrane induced a transient fall in neutrophil (1.2 +/- 0.5 x 10(9) vs. 3.6 +/- 0.6 x 10(9) cells/l; p < 0.05) and monocyte counts (0.2 +/- 0.1 x 10(9) vs. 0.7 +/- 0.1 x 10(9) cells/l; p < 0.05). There was also an increase in respiratory burst in the venous line during a HD with a cellulosic membrane, at 15 and 120 min (100 +/- 41 and 143.2 +/- 45.3 vs. 23.8 +/- 15.7; p < 0.05). Polymerized actin, expressed as fluorescence arbitrary units, was increased in baseline samples from uremic patients versus control subjects (327.8 +/- 60.8 for a cellulosic membrane, p < 0.005, and 205 +/- 26.5 for a synthetic one, p < 0.05 vs. 97.8 +/- 27.6 in controls). The percentage of CD11b+ cells increased in samples during a HD with a cellulosic membrane at the venous line at 15 and 120 min (9.6 +/- 4.5 and 18.4 +/- 7.1% vs. 3.3 +/- 1.9%; p < 0.05%). Changes in heterotypic aggregation during HD did not reach statistical significance, but levels were higher in patients treated with a cellulosic membrane at all points than in patients dialyzed with a synthetic one. CONCLUSION: There is evidence of a priming state of leukocytes from uremic patients, which is more evident in patients dialyzed with a cellulosic membrane. Cellulosic membranes also induce greater leukocyte activation than synthetic membranes during hemodialysis.     

Activation of platelets, coagulation and fibrinolysis in patients on long-term haemodialysis: influence of cuprophan and polyacrylonitrile membranes

This study was designed to evaluate platelet activation, enhancement of coagulation and fibrinolysis in patients with chronic renal failure on long-term haemodialysis. Beta thromboglobulin (BTG), platelet factor 4 (PF4), fibrinopeptide A, tissue plasminogen activator (t-PA) activity and antigen, tissue plasminogen activator inhibitor (PAI), fibrin, and fibrinogen degradation products were studied during dialysis. The influence of two types of membrane on these parameters was also evaluated. The patients comprised 24 individuals on long-term haemodialysis on either cuprophan membrane (CUP) (12 patients) or polyacrylonitrile membrane (AN 69) (12 patients). Blood samples were collected before, at 15 min, and at the end of dialysis. The results demonstrated that platelet activation was permanent and increased during haemodialysis. However, no difference could be demonstrated between patients treated on CUP and patients treated on AN 69. Coagulation was also enhanced permanently but did not show modification during haemodialysis. Fibrinolysis was activated at the end of haemodialysis in half the patients, but again no difference could be demonstrated between patients treated on AN 69 and CUP membranes. It was concluded that the process of haemodialysis itself enhanced platelet activation, coagulation, and fibrinolysis but that both membranes were of equal effect.     

Dialysis-induced hypoxemia: membrane dependent and membrane independent causes

Hypoxemia during hemodialysis may result from several differing processes. We initially studied patients undergoing standard acetate hemodialysis. At 15 minutes of dialysis, leukopenia (primarily neutropenia), a decline of platelet count, and hypoxemia occurred, but without a significant change in mean minute ventilation. Complement activation (V/A ratios of C5a greater than 1.0) persisted throughout dialysis. Leukocyte count returned to baseline by one hour. To separate the effects of solute and/or gas fluxes from those of blood-membrane interaction we studied changes in Po2, WBC, C5a, TxB2, and PGI2 during a period of blood membrane interaction without dialysis, and during subsequent acetate dialysis. Patients were studied with both polyacrylonitrile (PAN) and cuprophan membranes containing different priming solutions during membrane contact alone. Despite leukopenia and complement activation, hypoxemia failed to occur during membrane contact alone. At 15 minutes of subsequent acetate dialysis, significant hypoxemia occurred with both membranes. However, the degree of hypoxemia was twice as great with a cuprophan membrane primed with acetate (18.6 +/- 3.3 mm Hg) compared with air or bicarbonate (9.1 +/- 1.4 and 7.0 +/- 2.0 mm Hg, respectively), or compared with PAN (8 +/- 2.8 mm Hg). Changes in thromboxane B2, PGI2, and C5a did not correlate with changes in Po2. We conclude that there are two major components to dialysis related hypoxemia. One is membrane independent, and may relate to the metabolic effects of acetate or to dialyzer CO2 loss. The remaining portion is membrane dependent, occurring with cuprophan, but not with PAN, and is conditioned by an acetate dependent interaction between blood and membrane.     

Dialyzed polymorphonuclear neutrophil oxidative metabolism during dialysis: a comparative study with 5 new and reused membranes

Dialyzed neutrophils were isolated at time 0, 5, 15 and 60 min after the onset of hemodialysis in patients successively treated on 5 new and reused membranes, that is cuprophan (CU), cellulose acetate (CA), polysulfone (PS), polycarbonate (PC) and polyacrilonitrile (PAN). Production of oxygen radicals was monitored by luminol and lucigenin-enhanced chemiluminescence (CL). During dialysis with CU and PC, cells remaining in circulation at the maximum neutropenia showed a significant decrease of luminol-enhanced CL, whether stimulated with opsonized zymosan or phorbol myristate acetate. This defect was transient and the responses normalized at 60 min or upon reuse of the membranes. Among the other membranes tested, only cells collected during the first use of PS showed an impaired CL response to phorbol myristate acetate, but not to opsonized zymosan. CL again normalized upon reuse. At 5 min of dialysis with each membrane, a plasma factor appeared that was able to stimulate oxygen radical production by autologous dialyzed and control cells. A dissociation between the oxidative responses of dialyzed neutrophils and neutropenia was observed depending on the nature of the membranes, suggesting that neutropenia is a multifactorial process in which oxygen radical production appears as an early disturbance.     

Ex vivo flow cytometry determination of intracytoplasmic expression of IL-2, IL-6, IFN-gamma, and TNF-alpha in monocytes and T lymphocytes, in chronic hemodialysis patients

AIMS: To determine the intracytoplasmic expression of TNF-alpha, IL-2, IL-6 and IFN-gamma, ex vivo and in vitro, in both monocytes and T lymphocytes by flow cytometry after appropriate stimulation using phorbol myristate acetate (PMA)/ionomycin or lipopolysaccharides (LPS) in the presence of monensin, in order to assess the bio(in)compatibility of different dialysis membranes. METHODS: We examined monocytes and T lymphocytes taken from chronic hemodialysis patients (using either cuprophane (CUP), n = 6; polyacrylonitrile (AN 69), n = 6; or polysulfone (PS), n = 6 membranes), before and after a dialysis session. We compared the results with those obtained from end-stage chronic renal failure patients (n = 3) and healthy volunteers (n = 11). RESULTS: Before any stimulation there was a statistically significant difference in the percentages of TNF-alpha, IL-6, and IFN-gamma- expressing monocytes with respect to the dialysis membrane used. The highest percentages were observed for CUP and AN69 patients with figures of around 30% for each cytokine; the lowest percentages were found in PS patients and healthy volunteers. One hour after LPS stimulation the patterns remained unchanged for TNF-alpha and IFN-gamma, whereas the percentages of IL-6-expressing cells in PS patients and in healthy volunteers reached the figures obtained in the other groups. When we examined the percentage of IFN-gamma-, TNF-alpha- and IL-6-expressing monocytes in patients before and after a dialysis session, before any stimulation, we found that the results were significantly different for the three membranes (p = 0.01). Thus, a dialysis session with polysulfone membranes had no significant effect on the precentages of IFN-gamma-, TNF-alpha-, and IL-6-expressing monocytes, whereas percentages were significantly lower after the dialysis session when using cuprophane or AN69 membranes, suggesting a release of these cytokines by the monocytes during dialysis. A significant number of IFN-gamma- and IL-2-expressing T lymphocytes were only detected after 18 hours of PMA/ionomycin stimulation. The percentages of IFN-gamma-expressing T cells recorded for the different membranes were not statistically different from those recorded for healthy subjects or pre-dialysis patients, i.e., they were between 11.5 and 20%. However, the percentages of IL-2-expressing T lymphocytes were significantly different between the 5 groups, i.e., 31.3, 30.5, 18.6, 13.9 and 7. 6%, respectively, for CUP patients, pre-dialysis patients, healthy volunteers, PS and AN69 patients. This suggests that pre-dialysis and CUP patients have, at baseline, a stimulation of their T lymphocytes. Finally, a 4-hour dialysis session had no impact on the percentages of IL-2-expressing T lymphocytes, whereas it was associated with a significant decrease in the percentage of IFN-gamma-expressing cells, but only when cuprophane membranes were used. CONCLUSION: Cytokine flow cytometry enables one to study, ex vivo, i.e., without any stimulation of the cells, and in vitro after appropriate stimulation, the bio(in)compatibility of dialysis membranes assessed by the intracytoplasmic cytokine profiles of TNF-alpha, IFN-gamma, IL-6 and IL-2, evaluated at the single cell level.     

The effect of elastase on aminonucleoside nephrosis--on renal vascular system]

This study was performed to investigated the relationship between cross sectional areas of glomerular arterioles and glomerular volume in rats with puromycin aminonucleoside (PAN)-induced nephrosis, which is an experimental model of glomerular sclerosis. We also evaluated the effect of an anti-arteriosclerotic agent, elastase on these parameters. Six-week-old male Sprague-Dawley rats were divided into 3 groups; control, PAN-treated (70 mg/kg i.p.), or PAN+elastase-treated (5 mg/kg/day i. m.) group. After 12 weeks of experimental period, microvascular cast of the kidney was prepared, and the cross sectional areas of afferent and efferent arterioles as well as glomerular volume were measured using scanning electron microscopy. In the juxtamedullary glomeruli with minor abnormalities, the efferent arteriole was narrower in PAN-treated rats than in the control rats and the elastase-treatment partially corrected it (control 55.7, PAN 35.9, PAN+elastase 43.7 x 10 microns 2). On the other hand, the cross sectional area of afferent arteriole was not different among the 3 groups. In glomeruli showing sclerosis, the cross sectional area of efferent arteriole correlated positively with the glomerular volume. There results suggest that constriction of the efferent arterioles contributes to the development of glomerular sclerosis in PAN-induced nephrosis. In addition, elastase possibly inhibits this process and thereby protect glomeruli from sclerosis.     

Complement activation retards resolution of acute ischemic renal failure in the rat.

We investigated the role of complement activation on the resolution of acute ischemic renal failure in the rat. Acute renal failure was induced by clamping of the renal arteries of Sprague-Dawley rats for 45 minutes (Day 0). On subsequent days, groups of rats with acute renal failure were exposed to daily zymosan infusion (an activator of the complement system), or to blood incubated with cuprophane (CUP) or polyacrylonitrile (PAN) dialysis membranes. We serially measured the change in BUN daily, glomerular filtration rate and 24-hour proteinuria on Day 3 and Day 5 following ischemia. On Day 6, the animals were sacrificed and their kidneys examined histologically. Zymosan and cuprophane exposed rats had a significant delay in the recovery of renal failure, reduced glomerular filtration rate, and histologically had more neutrophil infiltration than control or PAN exposed animals. To investigate the potential pathophysiology of these observations, we assessed the response of zymosan-exposed rats to infusion of deferoxamine (DFO), a potent inhibitor of hydroxyl radical formation (OH.). Infusion of DFO prior to zymosan significantly improved recovery of renal function. We also measured urinary thromboxane B2 levels in these groups of rats. While the groups of rats exposed to zymosan had the highest levels of thromboxane B2, these levels were not different between the groups exposed to zymosan alone, or to zymosan and DFO. These observations suggest a role for hydroxyl radicals in the prolongation of renal failure in this model. Taken together, these findings may have implications for the dialytic intervention in patients with acute renal failure.     

Effect of different membranes on amino-acid losses during haemodialysis

Background: We analysed amino-acid losses during haemodialysis, their influence on plasma amino-acid concentration, and their possible effects on nutritional state. Methods: Five patients were dialysed with three membranes: cuprophan (CUP), polysulphone (PS), and polyacrylonitrile AN69 (PAN). We compared anthropometric and biochemical parameters after 6 months in patients dialysed with CUP respect to patients with PAN. Results: Total losses of amino acids were higher with PAN than with PS and CUP (6.1±2.3 vs 3.8±1.3, P <0.05, and 3.7±1.3 g/session, P <0.01 respectively). Losses of essential amino acids (EAA) and non-essential amino acids (NEAA) were also higher with PAN respect to PS and CUP (1.8±0.8 vs 1±0.3 and 0.8±0.3, and 4.3±1.6 vs 2.8±1 and 2.9±1.1 g/session, P <0.05). The percentage reduction for plasma EAA and NEAA were lower with CUP respect to PS and PAN (11±5% and 20±14% vs 25±10% and 33±11%, and 30±11% and 25±17% respectively, P <0.05). There was no difference in the nutritional state between patients with CUP and PAN. However, plasma valine in patients with PAN was lower than in those with CUP (1.88±0.12 vs 2.13±0.32 mg/dl) and almost reached statistical significance. Conclusions: New synthetic membranes are advantageous with respect to conventional ones, but a disadvantage is the higher amino-acid losses, especially with polyacrylonitrile. Long-term studies are necessary to evaluate the impact of amino-acid losses on nutritional state in patients dialysed with these membranes.     

Improved Biocompatibility by Modified Cellulosic Membranes: The Case of Hemophan

The rising problem of biocompatibility is encouraging the development of new dialysis membranes, but the high cost of synthetic ones precludes their wide use. The authors compared the biocompatibility of cuprophan (CU), cellulose acetate (CA), and hemophan (HE), evaluating both in vitro and in vivo polymorphonuclear leukocyte (PMN) oxidative metabolism activation by resting chemiluminescence and complement activation by C3a; in vivo PMN counts during dialysis were also performed. The lowest increase in in vitro PMN resting chemiluminescence using HE was + 71.3% with CA, +49.3% with CU, and + 21.4% with HE (p less than 0.001 versus CA and CU); furthermore, HE did not significantly stimulate PMN resting chemiluminescence during in vivo hemodialysis: + 56.6% with CA, + 38.8% with CU, and + 3.7% with HE (p less than 0.01 versus CU and p less than 0.001 versus CA). C3a concentration increased with all membranes both in vitro and in vivo, but HE (in both experimental conditions) showed the lowest increase at any time (p less than 0.001 versus CA and CU). After 15 min of dialysis, PMN count dropped to 20.3% of basal values with CU, to 49.8% with CA, and to 76.5% with HE (p less than 0.001 versus CU and CA). Among cellulosic membranes, HE is the most biocompatible and appears to be an important step in preventing blood-membrane interactions and related complications.     

Effect of vitamin E-bonded membrane on the 8-hydroxy 2'-deoxyguanosine level in leukocyte DNA of hemodialysis patients

BACKGROUND: 8-Hydroxy 2'-deoxyguanosine (8-OHdG) of leukocyte DNA has been identified as a surrogate marker of oxidative stress in chronic hemodialysis (HD) patients. In this study, we focused on the determinants of the 8-OHdG level in leukocyte DNA of HD patients. We further investigated the influence of vitamin E-modified, regenerated cellulose (CL-E) membrane on the oxidative DNA damage, intracellular reactive oxygen species (ROS) production of granulocytes, and plasma alpha-tocopherol concentration. METHODS: 8-OHdG content in cellular DNA of leukocytes was measured by a high-performance liquid chromatography-electrochemical detection (HPLC-ECD) method. Intracellular production of ROS, H2O2 and O2-. were analyzed by flow cytometry in leukocytes with and without phorbol-12-myristate-13-acetate (PMA) stimulation before dialysis, as well as at 15 and 30 minutes of dialysis. Plasma alpha-tocopherol concentration was measured by a HPLC method, and the value of alpha-tocopherol was corrected by total blood lipid concentration. RESULTS: In the prospective cross sectional study, the mean 8-OHdG level in leukocyte DNA was equally lower in the patients of the CL-E, polymethylmethacrylate (PMMA), and polysulfone (PS) groups as compared with the cellulosic group (ANOVA, P < 0.001). The leukocyte 8-OHdG level correlated negatively with plasma alpha-tocopherol and blood lipid-adjusted plasma alpha-tocopherol, but correlated positively with serum iron and percentage of transferrin saturation. Forward stepwise multiple regression showed that dialysis membrane type, serum iron, and blood lipid-adjusted plasma alpha-tocopherol were the independent determinants of the leukocyte 8-OHdG level in HD patients. Like synthetic membranes, granulocyte ROS production was less augmented during dialysis with the CL-E membrane as compared with the cellulose membrane. Exposure to cellulose membrane impaired intracellular ROS production of granulocytes in response to PMA challenge, whereas the CL-E and synthetic membranes improved the granulocyte responsiveness to PMA. In the longitudinal cross-over study, the 8-OHdG level significantly decreased, and blood lipid-adjusted plasma alpha-tocopherol increased after switching the cellulose membrane to CL-E or synthetic membrane for eight weeks. In contrast, the 8-OHdG level dramatically rose, and blood lipid-adjusted plasma alpha-tocopherol declined after shift of CL-E or synthetic membrane to the cellulose membrane. CONCLUSIONS: CL-E membrane exhibited biocompatible and bioactive characteristics. Like synthetic membranes, treatment with a CL-E dialyzer effectively reduced the 8-OHdG content in leukocyte DNA, suppressed intracellular ROS production of granulocytes, and preserved the plasma level of vitamin E. It could further improve granulocyte responsiveness to a PMA challenge. Reduced DNA damage and improved immune function of leukocytes may reduce the cancer and infection risks in chronic HD patients.     

PDGF-AB release during and after haemodialysis procedure.

BACKGROUND: During haemodialysis blood membrane contact causes the release of the content of platelet alpha-granules, which contain platelet-derived growth factor (PDGF). In view of its possible role in accelerated atherosclerotic processes, we evaluated the intra- and post-dialytic changes in PDGF-AB serum levels during haemodialysis sessions performed using a cellulosic membrane. METHODS: Using the ELISA method, PDGF-AB, platelet factor-4 (PF4) and beta-thromboglobulin (beta-TG) levels were determined in peripheral blood, as well as in arterial and venous haemodialyser lines, in 10 patients each of whom underwent five consecutive dialysis sessions with a CU membrane. Blood samples were taken at 0, 15, 30, 60, 120, 180 and 240 min during dialysis and at 1, 4 and 20 h after the end of the session. In the same group of patients the levels of the same molecules were also determined after a heparin bolus injection of 4500 IU, blood samples were taken at 0, 15 and 30 min after injection of the bolus. RESULTS: PDGF-AB serum levels increased, remained consistently high during the haemodialysis session (in particular +134+/-20% after 30 min, P<0.001, and +140+/-5% after 240 min, P<0.001) and returned to basal values only after 20 h following the end of the session. PF4 and beta-TG showed a similar trend to PDGF. The heparin bolus injection caused only a small increase (+15+/-5% at 30 min) in PDGF-AB serum levels. CONCLUSIONS: PDGF-AB is released during dialysis mainly as consequence of the blood-membrane contact and it returns only slowly to basal values.     

Early increase in blood nitric oxide, detected by electron paramagnetic resonance as nitrosylhaemoglobin, in haemodialysis

The objective of this study was to determine intradialytic blood levels of nitrosylhaemoglobin, as a surrogate marker of nitric oxide (NO) generation, in patients undergoing chronic haemodialysis. This was done by detection of nitrosylhaemoglobin by a sensitive technique of spin trap electron paramagnetic resonance at 0, 5, 15, 60, 180 and 240 min of a 4-h standard bicarbonate dialysis, using the same dose (6000 U) of heparin and different dialysis membranes. The study group included 12 patients treated with cellulose-derived dialysis membranes (nine with cuprophan and three with cellulose triacetate) and 10 patients treated with synthetic membranes (five with polysulfone and five with polymethylmethacrylate). Control groups included 11 normal subjects and six patients with endstage renal failure who were receiving intermittent peritoneal dialysis. Basal blood levels of nitrosylhaemoglobin in haemodialysis patients were significantly higher than normals, but similar to peritoneal dialysis patients. A significant increase (P <0.01) in nitrosylhaemoglobin level was detected at 15 min of haemodialysis irrespective of the membrane used. A decrease to basal levels at 180 min was observed in all but two cuprophan-treated patients who, in contrast to the others, had a symptomatic hypotension at the end of the session and a further increase in blood nitric oxide. Patients undergoing peritoneal dialysis did not show any change in blood levels of nitrosylhaemoglobin during the first 180 min of the procedure. Thus, a constant increase in nitrosylhaemoglobin levels was observed early in haemodialysis, but not in peritoneal dialysis patients. Very preliminary evidence was obtained for a role of nitric oxide in the vascular instability at the end of haemodialysis in a few patients who had hypotensive episodes.