β2-Microglobulin and Low-Flux SyntheticDialyzers

Abstract: The aim of this study was to compare the effect on β₂-microglobulin (β₂-M) plasma levels of dialyzers with 3 low-flux synthetic membranes and regenerated cellulose (Cuprophan) in 12 chronic dialysis patients. The synthetic membrane materials chosen were low-flux polymethylmethacrylate (PMMA), low-flux polysulfone (PS 400), and polycarbonate-polyether (Gambrane). Adequate and comparable removal of small solutes was provided by dialyzers with all 4 membrane materials used under similar conditions. A significant reduction of β₂-M plasma levels was seen only with Gambrane while the other 2 synthetic membrane materials gave rise to increases similar to those known to occur with Cuprophan. After correction for the hemoconcentration caused by ultrafiltration, dialysis with Gambrane showed a 24% lower plasma β₂-M level while the β₂-M concentrations with the other 3 membrane materials were practically unchanged. In addition, the efficiency of Gambrane dialyzers in β₂-M removal was able to significantly lower the predialysis plasma β₂-M levels after only 5 dialysis sessions. The hemocompatibility of the 3 synthetic low-flux membranes as judged by the white blood cell (WBC) count and complement activation was similar and therefore cannot be used to explain the different β₂-M plasma levels. In anticipation of gaining further insight into the mechanisms of accumulation and deposition of β₂-M in dialysis patients, a worthwhile approach may be to use a low-flux membrane such as Gambrane which combines removal with protection against potential activating factors in the dialysis fluid.     

Clinical Experience and Model Analysis on Beta-2-Microglobulin Kinetics in High-Flux Hemodialysis

Abstract: Beta-2-microglobulin (β₂M) is associated with amyloidosis. The study of β₂M kinetics can provide information on the elimination of this uremic toxin. A β₂M kinetic model modified from Gotch, considering the volume changes between intracellular, interstitial, and intravascular compartments and the generation stimulation and inhibition during hemodialysis is proposed. The clinical experiments on 8 stable hemodialysis patients treated with polysulfone (F80) and polymethyl methacrylate (PMMA, BK2.1p) 3 times a week were conducted. There was an 18% decrease of β₂M clearance in the period from 30 to 180 min with a time-averaged β₂M clearance of 48 ml/min using polysulfone dialyzers (F80). In PMMA dialyzers, there was a 64% decrease of β₂M clearance from 30 to 180 min with a time-averaged clearance of 56.3 ml/min. During hemodialysis, the generation rate was 0.379 mg/min in polysulfone and 0.828 mg/min in PMMA dialyzers. There was a stimulation generation of 0.309 mg/min in polysulfone and 0.749 mg/min in PMMA during hemodialysis. In conclusion, we provide a β₂M kinetic model including volume changes, polymerization, generation stimulation, and inhibition that is similar to the human physiological condition. This model can be used for further clinical study.     

Influence of Blood Flow on Adsorption of β2-Microglobulin onto AN69 Membrane

Abstract: Adsorption onto the dialyzer membrane is a contributing factor to the elimination of β₂-microglobulin (β₂M) from the sera of uremic patients. The purpose of this prospective study was to ascertain the influence of the blood flow rate on adsorption of β₂M onto the polyacrylonitrile (AN69) hollow-fiber dialyzer membrane in 8 pa tients during regular hemodialysis (HD). Blood first passed through a low-flux polysulfone dialyzer and then through an AN69 dialyzer, which was not in contact with the dialysis fluid. During the investigation period (first hour of the HD session), the blood flow rate was 100 ml/min (first part of the study), 200 mumin (second part of the study), and 300 ml/min (third part of the study). Ultrafiltration was not performed during the investigation period. At the start of the HD sessions, the serum concentration of β₂M in the afferent blood line did not differ significantly among the 3 parts of the study. Serum β₂M was measured in samples taken from the afferent and efferent blood lines of the AN69 dialyzer at 5,10, 15, 30, 45, and 60 min. The serum β₂M concentration decreased significantly in blood that had passed through the AN69 dialyzer. This decrease, indicating membrane adsorption, was maximal during the first part and minimal during the third part of study. The decrease in the contact time between the blood and the AN69 could be the underlying cause. The calculated quantities of β₂M adsorbed onto the AN69 membrane (44.2 ± 10.2, 43.2 ± 12.1, and 42.6 ± 17.3 mg) did not differ significantly among the 3 parts of the study. These results suggest that an increase in blood flow rate from 100 to 300 ml/min did not significantly affect the quantity of β₂M adsorbed onto the AN69 membrane.     

Lixelle Adsorbent to Remove Inflammatory Cytokines

It is the goal of this section to publish material that provides information regarding specific issues, aspects of artificial organ application, approach, philosophy, suggestions, and/or thoughts for the future.
A β₂-microglobulin (β₂M) selective adsorbent (Lixelle) for direct hemoperfusion has been used for the treatment of hemodialysis patients with the long-term complication of dialysis related amyloidosis (DRA), but there is no significant correlation between the serum level of β₂M and the occurrence of DRA. Inflammatory cytokines such as interleukin (IL)-1, IL-6, and tumor necrosis factor alpha (TNFα) are related to the development of DRA. We studied the adsorptive rates of cytokines in vitro using the Lixelle adsorbent. The adsorptive rates were 98. 5% for IL-1β, 98.0% for interleukin-1 receptor antagonist (IL-1RA), 82.9% for IL-6, 99.9% for IL-8, 31.2% for TNF α, and 46.1% for soluble TNF receptor (sTNFr), respectively. As the molecular weights of cytokines increase, the adsorptive rates decrease. The Lixelle column adsorbed β₂M and various inflammatory cytokines as well. Therefore, the removal of both β₂M and inflammatory cytokines may play an important role in the treatment of DRA.     

A New Therapeutic Approach to Dialysis Amyloidosis: Intensive Removal of β2-Microglobulin with Adsorbent Column

Abstract: Amyloidosis, in which amyloid protein consists of β₂-microglobulin (β₂-M), is both a common and a serious complication of long-term hemodialysis. The mechanism of its development is not completely understood. Since (β₂-M is an amyloid protein, it is essential to try to remove as much of it as possible. A specific adsorbent of β₂-M has been developed for use in direct hemoperfusion. The adsorbent is a porous cellulose bead to which hydrophobic organic compound is bound covalently. A combination of a high-flux membrane dialyzer and an adsorption column (BM-01) would make it possible to efficiently eliminate β₂-M. Dialysis with a combination of direct hemoperfusion (DHP) and an adsorption column led to the elimination of more than 200–300 mg of β₂-M. We observed 5 patients who received treatment with this column (BM-01) in combination with high-flux dialysis 3 times a week for periods of 1 week (3 patients), 6 months (I patient), or 14 months (1 patient). It is demonstrated that the adsorbent column (BM-01) provides an intensive method to eliminate β₂-M from the blood with no serious adverse effect. It thus has the potential to suppress the progression of dialysis amyloidosis. The use of this adsorbent column (BM-01) in combination with a high-flux dialyzer may present an improved approach to removing β₂-M from the body.     

Dialysis-related amyloidosis: Pathogenetic aspects and therapeutic considerations

Summary: Beyond renal transplantation and the provision of symptomatic relief, approaches to treat dialysis-related amyloidosis (DRA), an important long-term complication in patients on regular dialysis, must be based on the knowledge of the underlying pathogenetic process. Retention of beta₂-microglobulin (β₂m) is the prerequisite; biochemical alterations of β₃₂m increasing its amyloidogenicity, and local predisposing tissue factors together with age appear to be relevant. A growing body of evidence points toward the importance of pro-inflammatory effects of dialysis (blood-membrane interactions, pyrogen-related priming of cytokine producing mononuclear cells) in the development of DRA. Advanced glycation endproduct formation (AGE-β₂m) may represent a central element in the pathogenesis of DRA. For non-transplant therapy of DRA, the main goals must be the optimization of β₂m removal (high-flux haemodialysis, haemofiltration, especially pre-dilution haemofiltration) and reduction of pro-inflammatory effects of dialysis (use of non-complement activating biocompatible membranes, pyrogen free dialysate). At least patients at high risk for DRA should be treated according to these guidelines.     

Protein Adsorption by Artificial Membrane Materials Under Filtration Conditions

Abstract: Elevated plasma levels of numerous low molecular weight proteins (LMWP) in renal insufficiency are likely to contribute to the uremic syndrome. Dialysis-related amyloidosis, caused by the accumulation of β₂-microglobulin (β₂M), has highlighted the need for a renal replacement therapy that allows the elimination of LMWP in addition to small solutes. Synthetic membrane materials employed under hemofiltration conditions proved to be most effective in lowering elevated β₂M plasma levels. In addition to convection, protein adsorption to artificial membrane materials is an important mechanism for β₂M removal. Using an in vitro setup, 12 commercially available hemofilters representing 11 different membrane materials were perfused with human blood containing ¹²⁵I-labeled plasma proteins. Under filtration conditions, total protein adsorption ranged from 338–2,098 mg/m² of membrane surface, and the fraction of adsorbed LMWP varied between 14–70% of total protein adsorption and was negatively correlated to total protein adsorption. β₂M adsorption showed up to an 8-fold difference between membranes, and was negatively correlated with total protein adsorption and positively correlated with the adsorption of LMWPs.     

Long-Term Clinical Evaluation of an Adsorbent Column (BM–01) of Direct Hemoperfusion Type for (β2–Microglobulin on the Treatment of Dialysis-Related Amyloidosis

Abstract: The clinical efficacy and safety of a β₂–microglobulin (β₂M) adsorbent column, BM–01, on the treatment of dialysis-related amyloidosis were investigated in 7 hemodialysis patients for more than 6 months. The percent reduction of serum β₂M was more than 60–70%, and the level at the end of each session was less than 10 mg/L in almost all patients. The amount of β₂M removed was calculated as more than 200–300 mg/session. The results demonstrated that BM–01 performed very well for removing β₂M, was capable of maintaining less than 25 mg/L of time average concentration (TAC) for β₂M, and improved the clinical symptoms. Clinically severe side effects were not observed. We recommend that BM–01 should undergo further evaluation for its usefulness in the long-term treatment of dialysis-related amyloidosis, though treatment with the column may not be successful in preventing the onset of the disease.     

Clinical Evaluation of a New Dialyzer, FLX-12 GW, with a Polyester-Polymer Alloy Membrane

Abstract: The performance of a membrane in renal failure therapy is determined by its structure, its overall mass transfer properties, and its blood compatibility. In this regard. removal of β₂-microglobulin (β2M) has become a major objective of dialysis therapy. In the present study, a newly developed high-flux membrane composed of a polyester-polymer alloy (PEPA) with the components of polyarylate and polyethersulfone (dialyzer FLX-12 GW; Nikkiso Co., Japan) has been evaluated with regard to both hiocompatibility and elimination capacity for β2M during hemodialysis of 8 stable chronic uremic patients. The clearance values of low molecular weight solutes were in the same range as those reported for high-flux dialyzers of comparable surface area. There was no drop in leukocyte counts and only a minimal fall in platelet counts nearly in the same range as has been observed by other investigators using polyamide membrane. C3a Des Arg generation was low, and C5a Des Arg formation was not significantly influenced. There was a sharp drop in the serum β2M level (-35%) during dialysis with a clearance between 59.7 ± 5.6 ml/min ( Q_B 200 ml/min) and 70.1 ± 9.7 ml/min ( Q_B 300 ml/min), respectively. Accordingly, the sieving coefficient was calculated to be 0.2 at 30 min after start of dialysis and 0.6 1 h later. The membrane was able to remove 184.0 ± 22.3 mg/4 h due to an apparent rate of adsorption during the first hour of treatment in combination with high transmembrane transfer in the following time.     

Localization of integrin β1, α1, α5 and α9 subunits in the rat testis

Very late activation ( VLA, β₁; α₁; α₅, α₉) integrins were studied by immunoblotting and immunohistochemistry in the testes of sexually mature rats. All integrin subunits were present in membrane fractions of homogenized testes. Immunohistochemistry revealed that the anti β₁ antibody recognized peritubular cells and the basement membrane of blood vessels. Immunoreactivity was also demonstrated in the lamina propria, basement membrane, and the basal cytoplasm of Sertoli cells. In elongating spermatids, β₁ integrin was localized to the acrosome. The α₁ subunit was expressed in peritubular cells and in the lamina propria. In the adluminal compartment, round spermatids were stained diffusely for the α₁ subunit. Immunoreactivity for α₁ integrin was found additionally in the acrosomes of elongating spermatids shortly before their release into the seminiferous tubule lumen. The α₅ subunit was expressed in the acrosomes of elongating spermatids as well as in their distal cytoplasm during stages III–VI; the cytoplasmic lobes of elongate spermatids and/or residual bodies also appeared to be immunostained in seminiferous tubules at stages VII–VIII. The α9 subunit was immunolocalized only in the basement membrane and in peritubular cells. These data suggest that integrins are involved in spermatogenesis, in particular in the process of spermatid maturation.     

Urinary Excretion of Thromboxane and Markers for Renal Injury in Patients Undergoing Cardiopulmonary Bypass

Abstract: Urinary excretion of selected markers for renal injury, as well as urinary excretion rates of the thromboxane metabolite, 11–keto–thromboxane B₂ (llk–TXB₂), was studied in 36 male patients undergoing coronary bypass surgery using cardiopulmonary bypass (CPB). In all patients, excretion of both tubular (AT–acetyl–(β–D–glucosaminidase [βNAG]; α₁–microglobulin [α, –MG]) and glomerular markers (albumin [Alb]; transferrin [Trf]; immunoglobulin G [IgG]) sharply increased on Day 1 after CPB, and they remained elevated throughout the observation period of 5 days. Urinary excretion rates of 11k–TXB₂ markedly increased on Day 1 after surgery, and they rapidly decreased thereafter. In 12 of the 36 patients, a temporary increase of serum creatinine levels (>1. 30 mg/dl) was noted following surgery. A positive correla tion was found between serum creatinine levels and excretion of the tubular enzyme βNAG (r = 0. 36; p < 0. 05), but not between creatinine levels and α₁–MG or the glomerular markers. Furthermore, no correlation between urinary excretion of llk–TXB₂ and any of the urinary markers for renal injury could be detected. Our data do not strengthen the hypothesis that acute renal injury observed during CPB is related to exaggerated thromboxane biosynthesis in these patients. Monitoring of urinary markers for incipient renal damage, particularly excretion of βNAG, might be of additional diagnostic value for detection of otherwise subclinical renal injury in patients undergoing CPB.     

Serum levels of alpha-1 microglobulin and beta-2 microglobulin in bone marrow transplant recipients treated with cyclosporin A

Abstract. The levels of alpha-1 microglobulin (α₁m) and beta-2 microglobulin (β₂m) in serum were estimated in 77 bone marrow transplant recipients. In comparison to pretransplant levels, the highest levels of α₁m and β₂m were found during impairment of renal function, i. e., during cyclosporin-induced nephrotoxicity and during treatment with other nephrotoxic drugs ( P < 0.001). The α₁m levels were less elevated during infections and acute graft-versus-host disease ( P < 0.01), while β₂m levels were markedly elevated during the same conditions ( P < 0.001). The linear correlations between serum creatinine and α₁m and creatinine and β₂m were r = 0.7 and 0.8, respectively ( P < 0.001). The overall correlation between α₁m and β₂m was 0.4 ( P < 0.001). It is concluded that α₁m might be a complement to serum creatinine levels in monitoring renal function after bone marrow transplantation.     

Hemodynamics and Survival of Patients with Acute Renal Failure Treated by Continuous Dialysis with Two Synthetic Membranes

Synthetic membranes are not identical and have specific interactions that may be harmful or beneficial. We have investigated the incidence of hypotension and the outcome of acute renal failure (ARF) in ventilated patients treated by continuous venovenous dialysis with 2 different synthetic membranes. In Study 1, the mean arterial pressure (MAP) and systemic vascular resistance (SVR) were monitored during the first 12 min of dialysis with polyacrylonitrile (PAN). In Study 2, the MAP and survival rates were compared in patients randomly assigned to either PAN or polysulfone. No subjects were receiving angiotensin converting enzyme inhibitors. In Study 1, the MAP decreased due to a reduction in the SVR during the first 6 min of dialysis but returned to the baseline value by 12 min in 22 patients during 27 dialysis treatments. In Study 2, the MAP was lower than the baseline value at 6 min during 233 dialysis treatments in 133 patients randomly assigned to PAN or polysulfone membranes (PAN group, 81.5 ± 15 to 78.7 ± 15.6 mm Hg, p =0.001; and polysulfone group, 81.3 ± 15.4 to 80.0 ± 15.7 mm Hg, p =0.06). Severe reductions in the MAP were seen during 13.2% of the PAN and 7.2% of the polysulfone treatments (χ², p =NS). The age, APACHE II score, MAP, inotrope requirement, and primary diagnosis did not differ according to membrane material in a total of 197 consecutive patients (PAN, n =97; polysulfone, n =100). Patient survival was 29% (PAN) and 27% (polysulfone). In multivariate analysis, APACHE II score, inotrope requirement, and liver failure were significant determinants of survival. In conclusion, PAN and polysulfone membranes were not different with respect to hypotensive reactions or survival in critically ill patients undergoing continuous venovenous hemodialysis.     

Adsorption of Complement, Cytokines, and Proteins by Different Dialysis Membrane Materials: Evaluation by Confocal Laser Scanning Fluorescence Microscopy

The membranes tested in the present study were cellulose triacetate (CTA), polymethylmethacrylate (PMMA), and polyacrylonitrile (PAN). The adsorption by each membrane of albumin, IgG, C3a, interleukin-1β (IL-1β), interleukin-6 (IL-6), human neutrophil elastase (HNE), and tumor necrosis factor α (TNFα) was examined and semiquantitatively graded by confocal laser scanning fluorescence microscopy (CLSFM). After clinical use the dialyzers were treated with antibodies for these proteins and cytokines. Then the samples were incubated with fluorescein isothiocyanate-labeled anti-IgG antibody and observed by CLSFM. The changes in the blood levels of C3a and cytokines were also studied. In the CTA membrane, the adsorption of these substances, except for albumin and HNE, was less than in the synthetic membranes. The PAN membrane revealed the most abundant adsorption, especially for IL-1β, IL-6, and TNFα. Although a marked elevation of C3a in the blood was observed in the CTA membrane, considerable adsorption was evident in the PMMA and the PAN membranes. Because the changes in the blood levels could be affected by membrane adsorption, both the blood levels and the adsorption of the biocompatibility parameters should be evaluated when membrane biocompatibility is discussed.     

Principles and Practice of Hemofiltration and Hemodiafiltration

There is growing interest in the convective dialysis therapies, hemofiltration (HF) and hemodiafiltration (HDF). Both require dialysis membranes which are highly permeable to solutes as well as fluid, and in both cases large volumes of ultrafiltration are the condition for convective transport. In HDF the convection is combined with diffusion, and as a consequence, maximum clearance over the entire molecular weight spectrum is achieved. Optimal forms of HDF provide urea clearance 10–15% higher than the corresponding diffusive mode. The larger the solute, the greater is the impact of convection, and β₂-microglobulin (β₂m) levels may be up to 70% reduced. Traditional postdilution HF provides high clearance of medium sized and large molecules. Satisfactory clearance of small solutes requires blood flows in excess of 500 ml/min. With access to practically unlimited volumes of substitution solution through on-line ultrafiltration, predilution HF can now be used. This increases the clearance of small solutes to an acceptable range. For HDF as well as HF, large patient populations consistently treated for longer periods of time are needed to make valid outcome comparisons with other therapies.     

End-stage renal failure in New Zealand Maori: an analysis of circulating transforming growth factor-β1

SUMMARY: There is a high incidence of end-stage renal disease in New Zealand Maori. Reasons for this have not been established. Transforming growth factor-β, (TGF-β₁) is a profibrogenic cytokine, which stimulates the secretion of extracellular matrix components, and has been implicated in the pathogenesis of kidney failure. the aim of this study was to examine TGF-β₁ in the serum of haemodialysis patients at our institution, in order to determine whether there was an upregulation of TGF-β₁ in Maori. A TGF-Prspecific sandwich enzyme-linked immunosorbant assay was used to measure active TGF-β from the sera of 74 haemodialysis patients, and 19 healthy Maori without renal disease, diabetes or hypertension. In addition, clinical and laboratory markers were examined in the haemodialysis patients studied. There was no association between TGF-β₁ and ethnicity in the groups studied. Transforming growth factor-β₁ protein appeared to be inversely related to age. but was not associated with parameters of survival on dialysis such as serum albumin or measures of dialysis adequacy. Although there was a significantly higher incidence of type II diabetes mellitus in the Maori ( P < 0.001) in comparison to European patients, the glycaemic control was comparable between the groups, as were all other laboratory and clinical parameters studied. This is the first study to examine the fibrogenic growth factor TGF-β₁ in New Zealand Maori. Thus, an endogenous increase in TGF-β₁ in Maori does not appear to be implicated in the increased incidence of end-stage renal disease in this population.     

Characterization of a murine monoclonal antibodv specific for swine β1 integrin

Abstract: Murine monoclonal antibodies were raised against porcine platelets in order to provide tools for investigating interactions of human blood cells and natural antibodies with porcine tissues. Hybridomas were screened by cellular ELISA on porcine platelets and endothelial cells. Positive clones were tested by flow cytometry for reactivity with isolated endothelial cells. One clone, NaM160–1A3, produced an antibody that stained porcine but not human endothelial cells and lymphocytes. The antibody bound to a 116 kDa glycoprotein on Western blot of both platelets and endothelial cells. The antigen was purified from a platelet lysate by affinity chromatography, first on a ConA column and then on a column presenting the immobilized NaM 160–1 A3 antibody. Two glycoproteins were obtained: one (116 kDa) was recognized by the antibody and one (150 kDa) was not. The 116 kDa protein had an internal decapeptide identical with human β₁ integrin, and the 150 kDa protein had an internal amino acid sequence belonging to porcine α₂ integrin. Therefore, the NaM 160–1 A3 antibody was directed against porcine β₁ integrin and allowed the purification of the complex α₂β₁ also termed Very Late Antigen 2 (VLA-2). It did not recognize human β₁ integrin.     

109Near Infrared Spectroscopy of Partial Thickness Burns

Impaired wound healing is a problem for immobilized patients, diabetics, and the elderly. The 43 amino acid angiogenic peptide thymosin β₄ has previously been found to promote accelerated dermal wound repair in rats, aged mice and db/db diabetic mice, and corneal repair in normal rats. It has been found in great abundance in wound fluid. Here, we hypothesized that thymosin β₄ may regulate matrix metalloproteinase (MMP) expression in cells that are involved in wound repair. Western blot analysis of keratinocytes, endothelial cells, and fibroblasts that were treated with increasing concentrations of thymosin β₄ showed changes in the expression of the MMP‐1, −2, and −9. Zymographic analysis of whole excised mouse wounds taken after homogenization also showed changes in MMP‐2 and‐9 expression over a 3‐day period. These results were confirmed in 2‐day‐old wounds by RT‐PCR. We conclude that part of the wound healing activity of thymosin β₄ resides in its ability to increase protease activity. Since thymosin β₄‐induced protease activity can be further controlled by inflammatory cytokines, a regulatory role for thymosin β₄ is proposed in wound healing. These studies suggest that thymosin β₄ may play a pivotal role in extracellular matrix remodeling during wound repair and may be effective in the treatment of chronic wounds in humans.     

Hemodialysis with Cellulose Membranes Primes the Neutrophil Oxidative Burst

Abstract: Hemodialysis with cellulose membranes causes a complement-mediated neutropenia. Changes in neutrophil function have also been reported; however, it is unclear if these changes indicate a direct effect of the membrane on neutrophils or if they are a consequence of the neutropenia. We tested the hypothesis that neutrophil oxidative burst activity is enhanced during dialysis with cellulose membranes. Resting and Staphylococcus aureus -stimulated H₂O₂ production were determined predialysis and in blood entering and leaving the dialyzer during the first 30 min of dialysis and in blood leaving the membrane module in a single-pass on-line model of hemodialysis. Resting H₂O₂ production increased slightly but significantly during the first 30 min of dialysis. Transit of neutrophils through the dialyzer caused a marked increase in stimulated H₂O₂ production, indicating priming of the oxidative burst. However, priming was limited to the first 5 min of dialysis before the onset of neutropenia. In contrast, stimulation and priming of H₂O₂ production persisted throughout 30 min of single-pass on-line perfusion. These results indicate that cellulose membranes both stimulate and prime neutrophil oxidative burst activity but that these effects are partially obscured by neutropenia.