Uremic toxin removal based on diffusion and/or convection allows eliminating solutes with negative metabolic impact. Uremic solutes can be classified as small and water-soluble compounds, larger “middle molecules,” or protein-bound solutes. The question arises whether additional removal of each of these solute classes affects the patients' survival. Removal of uremic toxin during the dialytic process is mainly influenced by four factors: dialysis membrane performance, dialysis modality, dialysate, and blood flow.
Chauveau et al.1 reported that a better survival rate was observed in the patients treated with high water flux dialysis membrane. Furthermore, the German Diabetes and Dialysis (4-D) Study2 showed that patients with Type II diabetes mellitus on maintenance hemodialysis (mHD) therapy may benefit from greater permeability and better biocompatibility of the dialysis membrane in terms of all-cause mortality. Whether removal of larger uremic toxins and a decreased degree of direct and indirect blood-membrane interactions have a causative effect on patients' survival remains speculative.
β2-Microglobulin (β2M) is an 11.7-kDa nonglycosylated polypeptide composed of 99 amino acids. It is one of the major histocompatibility complex class I (MHC-I) molecules on the cell surface of all nucleated cells. It interacts with and stabilizes the tertiary structure of the MHC-I α-chain.3 Because it is noncovalently associated with the α-chain and has no direct attachment to the cell membrane, free β2M circulates in the blood as a result of shedding from cell surfaces or intracellular release. Once released, β2M is almost exclusively eliminated by glomerular filtration rate.4
In mHD patients, the increase in the serum β2M levels has been only recognized as the source for amyloid deposition and as a surrogate for middle molecule uremic toxins. Predialysis serum levels of β2M may provide dual information both on dialysis efficacy and on internal milieu bioactivity. The European Best Practice Guideline5 has recommended the use of β2M as a marker for middle molecules and to maximize the removal of middle molecules, although previous studies have largely related β2M to amyloidosis. Recently, it has been reported that β2M is an independent predictor of outcome for HD patients as well as the representative substance of middle molecules.6 A substantial number of bioactive middle molecules may be linked to inflammation, malnutrition, and vascular disease, the major areas of current clinical concern in the uremic and dialyzed populations.7
In HD patients, increased biomarkers of inflammation and oxidative stress are highly prevalent and also associated with cardiovascular diseases (CVD), which might be relevant to high morbidity and mortality. In this study, we evaluated the influence of β2M clearance on CVD risk factors and the nutritional state in HD patients.
Outpatients (n = 132, 63% were men, 28.7% were diabetic, mean age was 60.3 ± 1.6 years, mean duration of HD was 7.6 ± 1.3 years) in eight dialysis facilities in Hyogo and Osaka, Japan, were enrolled in this study. All patients had been on standard bicarbonate HD for at least 1 year and were dialyzed three times a week for 3–4 hours with ultrapure dialysate. Patients with malignant disease, chronic inflammatory disease, and severe liver and lung disease were excluded from this study. Almost all patients enrolled in this study had no residual renal function. All patients enrolled in this study gave informed consent in accordance with the requirement of the Institutional Committee on Human Research, and our investigational protocol was approved by the committee (Hyogo College of Medicine No. 293).
Dialysis Membrane Classes
The choice of dialysis membrane was exclusively at the discretion of nephrologists at the dialysis centers. During the study period, all patients had been dialyzed with the same dialysis membrane for at least 6 months.
Based on the property to activate complement and leukocytes, patients were divided into two groups using different classes of membrane biocompatibility: cellulose and synthetic. Furthermore, they were divided into three groups according to β2M clearance: three classes of membrane were 1) low filtration group (LF) (β2M clearance <30 ml/min), 2) middle filtration group (MF) (β2M clearance >30 and <50 ml/min), 3) high filtration group (HF) (β2M clearance >50 ml/min).
Sample Collection and Measurement of Parameters
Blood samples were drawn from all of the patients at the start of their first HD session of the week. Blood levels of hemoglobin (Hb), urea nitrogen (UN), creatinine (Cr), β2M, calcium (Ca), phosphorus (P), total cholesterol (T-CHO), and triglyceride (TG) were measured by standard laboratory techniques using an autoanalyzer. Intact-parathyroid hormone (int-PTH) was determined by immunoradiometric assay. Kt/V was calculated in these patients using formal urea kinetic modeling. We also measured serum levels of hCRP by latex photometric immunoassay, IL-6, and TNF-α by an ESIA kit (Human IL-6 immunoassay kit, Human TNF-α immunoassay kit, Biosource International, Inc., Camarillo, CA), troponin-T (TnT) by immunoassay electrochemiluminescence (ECLIA), myeloperoxidase (MPO) by an EIA kit (BIOXYTECH MPO-EIA kit, Oxis International Inc., Foster City, CA), and prealbumin (PA) by nephlometory. N-Terminal pro-B-type natriuretic peptide was measured using the Elecsys proBNP immunoassay (Roche Diagnostics, Indianapolis, IN).
Ankle-Brachial Index and Brachial-Ankle Pulse Wave Velocity Measurement
Ankle-brachial index (ABI) and brachial-ankle pulse wave velocity (baPWV) were measured using a volume-plethysmographic apparatus (form PWV/ABI, Colin Co., Komaki, Japan). This device records the phonocardiogram, electrocardiogram, and volume pulse form and arterial blood pressure at both the left and right brachia and ankles. Ankle-brachial index was the ratio of ankle systolic blood pressure (SBP) to brachial SBP, and left ABIs were measured simultaneously. In all the patients, baPWV and ABI were obtained after a minimum 5 minutes of rest.
All values are presented as the mean ± SD. Differences between the two groups were analyzed by an unpaired Student's t test. Values of p < 0.05 were taken as statistically significant. Potential associations between β2M and CVD risk factors were assessed by liner regression analysis. Statistical analyses were performed with StatView software version 5.0 for Macintosh computer.
Dialysis Membrane Properties
There was no significant difference in age; % of males; % with diabetes; duration of HD; blood levels of Hb, TP, alb, Ca, P, int-PTH, or T-CHO between the cellulose and synthetic groups. Serum PA level of the synthetic group was significantly higher than that of the cellulose group. On the other hand, there was no significant difference in dialysis efficiency (Kt/V and serum β2M level), indexes of inflammation (IL-6, TNF-α, hCRP, and MPO), or risk factors of CVD (TnT, ABI, and PWV) between the cellulose and synthetic groups (Table 1).
Dialysis Membrane Performance
There was no significant difference in % of males; % with DM; blood levels of Hb, Ca, P, int-PTH, or T-CHO among the LF, MF, and HF groups. In the MF and HF groups, duration of hemodialysis, int-PTH, and TG were significantly (p < 0.05) higher than those of the LF group (Table 2).
There was no significant difference in Kt/V among the three groups. On the other hand, serum β2M, hCRP, TnT, and MPO levels of LF group were significantly (p < 0.05) higher and PA levels were significantly (p < 0.05) lower than those of the MF and HF groups (Figures 1 and 2). There was no significant difference in ABI and baPWV among LF, MF, and HF groups.
Correlation Between β2M and CVD Risk Factors
Serum levels of β2M were positively correlated with markers of inflammations such as hCRP (p = 0.008, R = 0.21), IL-6 (p < 0.0019, R = 0.27), and TNF-α (p < 0.00001, R = 0.53) and also correlated with the marker of oxidative stress, MPO (p < 0.046, R = 0.16) (Figure 3). Furthermore, β2M was positively correlated with serum levels of TnT (p < 0.0001, R = 0.34) and NT-pro BNP (p = 0.0015, R = 0.26), whereas it was inversely correlated with PA (p < 0.0143, R = −0.19) and ABI (p = 0.048, R = −0.21) (Figure 4).
This study is consistent with recent reports regarding two clinical studies in which high-flux HD and hemodiafiltration (HDF) were used for the removal of middle molecules including β2M. The Dialysis Outcomes and Practice Patterns Study (DOPPS)8 showed that total mortality risk was found to be 35% lower for patients who received high-efficiency HDF compared with low-flux HD even after adjustment for all variables, including Kt/V. Furthermore, the hemodialysis (HEMO) study9 showed that the relative risk for all-cause mortality increased by 11% per 10-mg/L increase in β2M over a reference value of 27 mg/L after adjustment for residual renal function and dialysis vintage. Both high-flux HD and HDF have the ability to remove middle molecules (such as β2M) to some degree. Whether removal of middle-molecular-weight compounds and biocompatibility of the dialysis membrane have a causative effect on patient survival remains speculative. In this study, we evaluated the influence of membrane properties and β2M clearance on CVD risk factors.
β2M and Inflammatory Cytokines
Serum level of IL-610,11 and hCRP12,13 are well recognized as markers of the chronic inflammatory process could be associated with the high morbidity and mortality seen in HD patients.
Recently, β2M itself has emerged as a particularly significant biomarker of morbidity and mortality in uremic patients.14 Moreover, Xie and Yi15 showed in an in vitro study that β2M stimulates monocytes to secrete high levels of proinflammatory cytokines (TNF-α, IL-1, IL-6, IL-8, and IL-10). It has also been reported that β2M inhibited cell growth and induced apoptosis or necrosis in tumor cells such as leukemia and myeloma cells.16 Mori et al.17 reported that β2M at high concentrations induces apoptosis or necrosis in normal cells, including endothelial cells and fibroblasts, because apoptotic or necrotic bodies release enzymes and cytokines that could act as chemoattractants for mononuclear cells, and they speculated that β2M may be a potential initiator of the inflammatory response.
In this study, β2M had a significant correlation with indexes of inflammation (IL-6, TNF-α, and hCRP) in MHD patients. Moreover, in the patients using a low β2M clearance dialyzer, serum hCRP levels were significantly higher than those of middle or high β2M clearance dialyzers. It has been already established that cellulose dialysis membranes lead to intense activation of the complement system and blood cells and the formation of reactive oxygen species.18 This might be a trigger of systemic inflammation in patients on hemodialysis, which is well documented to be associated with CVD risk and mortality. However, in our study, there was no significant difference between the cellulose and synthetic membrane group in indexes of inflammation (TNF-α, IL-6, and hCRP) or oxidative stress (MPO).
The intensity of microinflammation is correlated with the use of bioincompatible cellulosic membranes, as well as endotoxins derived from contaminated dialysate, especially using high-flux dialyzers.19 In this study, almost all patients (both using low-flux and high-flux dialyzers) were treated with ultrapure dialysate. So, we presumed that the effect of dialyzate purity on inflammatory and oxidative stress indexes such as hCRP, IL-6, TNF-α, and MPO were minimal in this study. From these results, we suspected that accelerated inflammation and oxidative stress may be induced by β2M levels but not affected by dialysis membrane properties.
β2M and Atherosclerosis
In this study, we found significant correlations between β2M and markers of inflammation, oxidative stress, and malnutrition (serum levels of PA). Furthermore, we found an inverse correlation between serum level of β2M and ABI. Saijyo et al.20 reported that serum levels of β2M as well as high CRP were significantly correlated with PWV. Another study21 showed that in patients with peripheral arterial disease (nonchronic kidney disease patients), the severity of peripheral arterial disease (ABI) was correlated with a high circulating β2M level but not with other risk factors (diabetes, hyperlipidemia, body mass index, and log hCRP).
Many CVD risk factors (hCRP, IL-6, TNF-α, MPO, TnT, NT-proBNP, and PA) correlated with serum β2MG levels in this study. However, we could not find any significant difference in ABI and PWV among the three groups (dialysis performance). We presumed that 6 months may be a too short time for any changes in atherosclerosis indexes, such as ABI and PWV. From these results, we could not rule out of the possibility that increased levels of β2M may accelerate atherosclerosis via the inflammation and oxidative stress in the patients on MHD.
β2M and LV Function
There is the possibility that blood levels of cTNT may be affected by dialyzer performance because of its molecular weight (30 kDa). High flux membranes remove molecules of up to 30 kDa, whereas the threshold for low-flux dialyzers is 9 kDa.22 Therefore, high-flux dialysis membranes may extract cTNT. However, the molecular weight of NT-proBNP (7 kDa) is smaller than cTNT; therefore, both low- and high-flux membrane can remove NT-proBNP efficiently. So, we presumed that the performance of dialysis membranes has little effect on serum levels of NT-proBNP.
Recently, several studies have demonstrated that elevated serum cTnT is a powerful predictor of mortality in HD patients.23,24 Another study found that elevation in serum cTnT in asymptomatic HD patients was a reflection of silent ischemic heart disease and nonischemic cardiomyopathy, and TnT level has been related to LV mass.25 Satyan et al.26 reported that plasma levels of NT-proBNP strongly correlated with left ventricular (LV) systolic dysfunction and was associated more strongly with all-cause and CVD mortality in HD patients.
In this study, serum levels of β2M had a significant correlation with TnT and NT-proBNP. Moreover, in the patients with high clearance of β2M, serum levels of TnT were significantly higher than those of the low group. These results suggest that serum levels of β2M may reflect LV dysfunction and silent ischemic heart disease of HD patients.
A better understanding of uremic solutes and their toxic effect would place dialysis on a more rational basis and lead to more effective therapy. To lower these toxic substances, either their generation should be reduced or strategies for effective removal should be developed. The main conclusions of this study were as follows. 1) An intense relationship between β2M and indexes of inflammation, oxidative stress, malnutrition, and cardiovascular damage was found in HD patients. Thus, high serum β2M level is a possible predictor of CVD in HD patients. 2) Removal of middle-molecular-weight compounds by dialysis therapy may reduce the morbidity and mortality rate, which is associated with CVD in patients with HD.
Supported by a Grant-in-Aid for Research from Hyogo College of Medicine, 2006.
1. Chauveau P, Nguyen H, Combe C, et al
: Dialyzer membrane permeability and survival in hemodialysis patients. Am J Kidney Dis
45: 565–571, 2005.
2. Krane V, Krieter DH, Olschewski M, et al
: Dialyzer membrane characteristics and outcome of patients with type 2 diabetes on maintenance hemodialysis. Am J Kidney Dis
49: 267–275, 2007.
3. Braciale TJ: Antigen processing for presentation by MHC class I molecules. Curr Opin Immunol
4: 59–62, 1994.
4. Acchiardo S, Kraus AP Jr, Jennings BR: Beta 2 microglobulin levels in patients with renal insufficiency. Am J Kidney Dis
13: 70–74, 1989.
5. European Best Practice Guideline Expert Group on Hemodialysis, European Renal Association: Section II. Haemodialysis adequacy. Nephrol Dial Transplant
17: 16–31, 2002.
6. Canaud B, Morena M, Cristol JP, Krieter D: β2-microglobulin, a uremic toxin with a double meaning. Kidney Int
69: 1297–1299, 2006.
7. Bouré T, Vanholder R: Biochemical and clinical evidence for uremic toxicity. Artif Organs
28: 248–253, 2007.
8. Canaud B, Bragg-Gresham JL, Marshall MR, et al
: Mortality risk for patients receiving hemodiafiltration versus hemodialysis: European result from the DOPPS. Kidney Int
69: 2087–2093, 2006.
9. Cheung AK, Rocco MV, Yan G, et al
: Serum β-2 microglobulin levels predict mortality in dialysis patients: Result of the HEMO study. J Am Soc Nephrol
17: 546–555, 2006.
10. Rao M, Guop D, Perianayagam MC, et al
: Plasma interlerukin-6 predicts cardiovascular mortality in hemodialysis patients. Am J Kidney Dis
45: 324–333, 2005.
11. Honda H, Qureshi AR, Heimbüger O, et al
: Serum albumin, C-reactive protein, interleukin 6, and fetuin A as predictors of malnutrition, cardiovascular disease, and mortality in patients with ESRD. Am J Kidney Dis
47: 139–148, 2006.
12. Wang AY, Woo J, Lam CW, et al
: Is a single time point C-reactive protein predictive of outcome in peritoneal dialysis patients? J Am Soc Nephrol
14: 1871–1879, 2003.
13. Zimmermann J, Herrlinger S, Pury A, et al
: Inflammation enhances cardiovascular risk and mortality in hemodialysis patients. Kidney Int
55: 648–658, 1999.
14. Vanholder R, Smet R, Glorieux G, Dhondt A: Survival of hemodialysis patients and uremic toxin removal. Artif Organs
27: 218–223, 2007.
15. Xie J, Yi Q: β2-microglobulin as a potential initiator of inflammatory response. Trends Immunol
24: 228–229, 2003.
16. Min R, Li Z, Epstein J, et al
: β2-microglobulin as a negative growth regulator of myeloma cells. Br J Haematol
118: 495–505, 2002.
17. Mori M, Terui Y, Ikeda M, et al
: Beta(2)-microglobulin identified as an apoptosis-inducing factor and its characterization. Blood
94: 2744–2753, 1999.
18. Hörl WH: Hemodialysis membrane: Interleukins, biocompatibility, and middle molecules. J Am Soc Nephrol
13: S62–S71, 2002.
19. Panich V, Tetta C, Rindi P, et al
: Plasma C-reactive protein is linked to backfiltration associated interleukin-6 production. ASAIO J
44: M415–M417, 1998.
20. Saijyo Y, Utsugui M, Yoshioka E, et al
: Relationship of β2- microglobulin to arterial stiffness in Japanese subjects. Hypertens Res
28: 505–511, 2005.
21. Wilson AM, Kimura E, Harada RK, et al
: β2-Microglobulin as a biomarker in peripheral arterial disease. Protemic profiling and clinical studies. Circulation
116: 1396–1403, 2007.
22. Sommerer C, Heckle S, Schwenger V, et al
: Cardiac biomarkers are influenced by dialysis characteristics. Clin Nephrol
68: 392–400, 2007.
23. Stolear J, Georges B, Shita A, Verbeelen D: The predictive value of cardiac troponin T measurements in subjects on regular hemodialysis. Nephrol Dial Transplant
14: 1961–1967, 1999.
24. Kanderian AS, Francis GS: Cardiac troponins and chronic kidney disease. Kidney Int
69: 1112–1114, 2006.
25. Rosner MH: Measuring risk in end-stage renal disease: Is N-terminal pro brain natriuretic peptide a useful marker? Kidney Int
71: 481–483, 2007.
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26. Satyan S, Light RP, Agarwal R: Relationship of N-terminal pro-B-natriuretic peptide and cardiac troponin T to left ventricular mass and function and mortality in asymptomatic hemodialysis patients. Am J Kidney Dis
50: 1009–1019, 2007.