Multicentre cross-sectional study for bone-articular lesions associated with dialysis related amyloidosis in Japan.

AIM: Dialysis-related amyloidosis (DRA) exhibits multiple bone-articular lesions, such as carpal tunnel syndrome (CTS), trigger finger (TF), spinal canal stenosis (SCS), destructive spondyloarthropathy (DSA), bone cysts, and joint pains. DRA leads to a decrease in activities of daily living (ADL). We investigated the initiation of CTS and TF, and evaluated the relationship between walking disturbances and bone-articular lesions or joint pains. METHODS: A multicentre cross-sectional study was performed. Eighty-two patients with clinical DRA from 20 hospitals in Japan were evaluated. RESULTS: Of the 82 patients, the first symptom of DRA was CTS in 39 patients (47.6%) and TF in 21 (25.6%). The mean new-onset vintages of 21 earlier cases in the CTS and TF groups were 86.1 ± 36.3 and 133.2 ± 56.4 (mean ± SD) months, respectively (P = 0.0091). The development of SCS and DSA appeared to be later than CTS and TF. Multiple regression analysis revealed that knee joint pain was a significant contributor to walking disturbances. CONCLUSION: Carpal tunnel syndrome appeared significantly earlier than TF since the initiation of dialysis. In the advanced phase, knee joint pain was a major cause of decreased ADL in patients with clinical DRA.

TLR4/MD-2 is a receptor for extracellular nucleophosmin 1.

Nucleophosmin 1 (NPM1) primarily localizes to the nucleus and is passively released into the extracellular milieu by necrotic or damaged cells, or is secreted by monocytes and macrophages. Extracellular NPM1 acts as a potent inflammatory stimulator by promoting cytokine production [e.g., tumor necrosis factor-α (TNF-α)], which suggests that NPM1 acts as a damage-associated molecular pattern. However, the receptor of NPM1 is unknown. Evidence indicates that DAMPs, which include high mobility group box 1 and histones, may bind Toll-like receptors (TLRs). In the present study, it was shown that NPM1 signaling was mediated via the TLR4 pathway, which suggests that TLR4 is an NPM1 receptor. TLR4 binds myeloid differentiation protein-2 (MD-2), which is essential for intracellular signaling. Furthermore, the TLR4 antagonist, LPS-Rhodobacter sphaeroides (an MD-2 antagonist) and TAK-242 (a TLR4 signaling inhibitor) significantly inhibited NPM1-induced TNF-α production by differentiated THP-1 cells as well as reducing ERK1/2 activation. Far-western blot analysis revealed that NPM1 directly bound MD-2. Thus, the results of the present study provide compelling evidence that TLR4 binds NPM1, and it is hypothesized that inhibiting NPM1 activity may serve as a novel strategy for treating TLR4-related diseases.

A circulating beta 2-microglobulin intermediate in hemodialysis patients.

BACKGROUND/AIMS: A misfolded beta(2)-microglobulin (beta(2)m) is a principle component in dialysis-related amyloidosis. However, no such conformational variant of beta(2)m has yet been reported in a clinical setting. Capillary electrophoresis is a tool that can identify the conformational variant of beta(2)m. METHODS: Capillary electrophoresis was used to measure a transitional intermediate from native beta(2)m (N-beta(2)m) to the amyloid beta(2)m. This technique was utilized to assay for intermediate beta(2)m (I-beta(2)m) in serum from 31 hemodialysis (HD) patients before and after HD, 5 patients with non-dialysis chronic renal failure (CRF), and 5 healthy persons. RESULTS: The predialysis values of serum I-beta(2)m and N-beta(2)m were 2.7 +/- 1.4 and 29.4 +/- 6.8 mg/l, respectively, in the HD patients. The presence of serum I-beta(2)m correlated weakly with the total serum beta(2)m concentration in all HD patients. The serum N-beta(2)m concentration decreased significantly during two types of dialysis treatment: by 32.8% on HD using a polymethylmethacrylate (PMMA) membrane and by 71.2% on online hemodiafiltration (HDF) with a polysulfone (PS) membrane. On the other hand, a dialysis-associated change in serum I-beta(2)m varied from -36.4 to +203.5% in HD patients using PMMA and from -70.8 to +62.5% in online HDF patients using PS. Moreover, a rebound beta(2)m profile suggested that I-beta(2)m might be immobilized in the extracellular space. CONCLUSION: This study demonstrated that two or three conformational isomers of beta(2)m were probably ubiquitously recognized in human serum. Though no progressive increase in serum I-beta(2)m concentration could be found along with HD, this study shows a significantly poor removal of I-beta(2)m in comparison to N-beta(2)m in patients receiving ongoing dialysis treatment, even with online HDF.

Immunological evidence for in vivo production of novel advanced glycation end-products from 1,5-anhydro-D-fructose, a glycogen metabolite.

The anhydrofructose pathway is an alternate pathway for glycogen degradation by α-1,4-glucan lyase. The sugar 1,5-anhydro-D-fructose (1,5-AF) acts as the central intermediate of this pathway, but its physiological role of in mammals is unclear. Glycation reactions forming advanced glycation end-products (AGEs) are important in the development of complications of diabetes mellitus. We hypothesized that 1,5-AF may contribute to cellular damage by forming 1,5-AF-derived AGEs (AF-AGEs) with intracellular proteins. To clarify the role of 1,5-AF in protein modification, we created a novel antibody targeting AF-AGEs. Serum albumin modified by AF-AGEs was prepared by incubating rabbit serum albumin (RSA) or bovine serum albumin (BSA) with 1,5-AF. After immunizing rabbits with AF-AGEs-RSA, affinity chromatography of anti-AF-AGE antiserum was performed on a Sepharose 4B column coupled with AF-AGEs-BSA or N-(carboxymethyl)/N-(carboxyethyl)lysine-BSA. A novel immunopurified anti-AF-AGE antibody was obtained and was characterized using a competitive enzyme-linked immunosorbent assay. Then an AF-AGEs assay was established using this immunopurified antibody. This assay was able to detect AF-AGEs in human and animal serum samples. Finally, intracellular accumulation of AF-AGEs was shown to be associated with damage to cultured hepatocytes (HepG2 cells). This is the first report about in vivo detection of AF-AGEs with a novel structural epitope.

Role of conformational change in the C-terminus of beta2-microglobulin in dialysis-related amyloidosis.

BACKGROUND: Beta(2)-microglobulin (beta(2)m) has been identified as the precursor protein of dialysis-related amyloidosis (DRA), which is a serious complication for haemodialysis (HD) patients. However, mechanisms underlying beta(2)m amyloid fibril formation remains to be elucidated. We previously demonstrated, in amyloid deposits from HD patients, a conformational isoform of beta(2)m with an unfolded C-terminus. However, no direct experiments have previously been performed to address whether unfolded beta(2)m in the C-terminus may be prone to form amyloid fibrils. METHODS: To evaluate roles of C-terminal amino acids in beta(2)m-induced amyloid formation, we generated six types of recombinant beta(2)m with amino acid substitutions in the C-terminal region. To investigate their conformational change and amyloidogenicity, we measured circular dichroism spectra, the fluorescence intensity of tryptophan and thioflavin-T (ThT) of the recombinant beta(2)m. To analyse morphological change of beta(2)m, we performed electron microscopy (EM) on the samples with elevated ThT fluorescence intensity. We used ultrasonication to enhance beta(2)m destabilization of the protein. RESULTS: Beta(2)M Trp95Leu and Arg97Ala showed conformational changes and increased their amyloidgenicity compared with beta(2)m wild-type (WT). With ultrasonication, beta(2)m Trp95Leu and Arg97Ala generated more amyloid fibrils than did beta(2)m WT even in physiological solution. EM showed that beta(2)m formed amorphous debris containing typical amyloid fibrils at 24 hours, when ThT fluorescence intensity was three-fold lower than that at six hours. CONCLUSIONS: Conformational changes in the C-terminus of beta(2)m may play an important role in DRA and that ultrasonication is useful for analysis of beta(2)m amyloidogenesis.

Selection of DNA Aptamer That Blocks the Fibrillogenesis of a Proteolytic Amyloidogenic Fragment of ß2 m.

Dialysis-related amyloidosis (DRA) is a severe complication of hemodialysis that results in progressive destruction of bones and joints. Elevated concentrations of the ß2 -microglobulin (ß2 m) level in the serum of subjects on hemodialysis promote the formation of amyloid fibrils in osteoarticular tissues. ß2 m lacking the N-terminal six residues of the mature protein (ΔN6ß2 m) constitutes 25-30% of ß2 m in ex vivo DRA amyloid. Unlike full-length wild-type ß2 m, ΔN6ß2 m forms amyloid fibrils at neutral pH in vitro. However, the role of ΔN6ß2 m in DRA is, at present, poorly understood. In the present study, we screened novel phosphorothioate-modified aptamers directed against ΔN6ß2 m using combinatorial chemistry in vitro. We identified 11 ΔN6ß2 m aptamers; among the identified aptamers, clone #2, #8, and #10 aptamers had higher binding affinity to ΔN6ß2 m than the others. Biolayer interferometry analysis revealed that KD values of clone #2, #8, and #10 aptamers were 56, 23, and 44 nM, respectively. Furthermore, the clone #8 aptamer inhibited fibril formation in a dose-dependent manner, as assessed by Thioflavin T fluorescence assay. Fibrils formed from ΔN6ß2 m bind to Congo red, displaying changes in the absorbance spectrum of the dye characteristic of binding to amyloid fibrils, which was completely blocked by treatment with clone #8 aptamer. These results suggest the potential of ΔN6ß2 m aptamers as tools for elucidating co-assembly mechanisms in amyloid formation.

Decreased Expression of Thrombomodulin in Endothelial Cells by Fibroblast Growth Factor-23/α-Klotho.

Chronic kidney disease (CKD) has been known to be a state of excessive fibroblast growth factor-23 (FGF23) and α-Klotho deficiency. Patients undergoing hemodialysis have an increased mortality risk associated with cardiovascular disease and endothelial dysfunction. The mechanism responsible for the relationship of FGF23 to endothelial damage in these patients has been unclear. On the other hands, increasing evidences have demonstrated that thrombomodulin (TM) plays an important role in the endothelial barrier. Here, we report the suppression of membrane TM, in a dose-dependent manner, in human umbilical vein endothelial cells after FGF23 and FGF23/α-Klotho stimulation. In addition, the levels of soluble TM, which reflect endothelial cell injury, were much higher in cell supernatants after FGF23 and FGF23/α-Klotho stimulation than in the control supernatant. This study indicates a possible mechanism by which excessive levels of FGF23 are involved in endothelial TM disruption, which has been implicated as a potential cardiovascular risk factor in patients with CKD, especially in HD patients.

Lipid droplets are present in amyloid deposits in familial amyloidotic polyneuropathy and dialysis related amyloidosis.

It has been well documented that transthyretin (TTR) shows an affinity for lipoproteins and amyloid is deposited around adipocytes in patients with familial amyloidotic polyneuropathy (FAP). We examined the involvement of lipids in amyloid fibrils in the tissues by histopathologic methods. Sudan black B staining for frozen tissues of autopsied FAP patients and biospied dialysis related amyloidosis (DRA) patients revealed colocalization of lipids in the tissue sections where Congo red staining was positive, while no such positive staining was observed in paraffin embedded tissues. Immunohistochemical study using lipoprotein antibodies revealed that only anti-high density lipoprotein (HDL) antibody showed immunoreactivity in both types of amyloid specimens where Congo red was positively stained. Measurement of the components of lipids in the frozen cardiac samples from an FAP patient and an amyotrophic lateral sclerosis (ALS) patient revealed that concentrations of triglyceride and cholesterol in each lipoprotein, except for HDL-triglyceride, were markedly elevated in the FAP patient's material, compared to that of the ALS patient. These results suggest that interaction of amyloid fibrils with HDL may play an important role in amyloid formation in FAP.

Influence of heparin molecular size on the induction of C- terminal unfolding in ß2-microglobulin.

Dialysis-related amyloidosis (DRA) is characterized by accumulation of amyloid ß2- microglobulin (ß2m) in the interstitial matrix. Matrix substances such as heparin have reportedly been strongly implicated in the pathogenesis of dialysis-related amyloidosis. In clinical setting of hemodialysis, two types of heparin, i.e., high and low molecular heparin (H.M.H. and L.M.H.) have been routinely used. Still commonly used is H.M.H., followed by L.M.H. preparations with distinct advantages. Here, we studied that the interaction of native and two amyloidogenic ß2m variants: ΔN6ß2m and D76N ß2m with H.M.H. and L.M.H. We also investigated whether heparin could induce ß2m to have an amyloidogenic conformation. Biolayer interferometry revealed that ΔN6ß2m had a strong reaction and D76N ß2m had a moderate reaction with H.M.H.. Furthermore,H.M.H. induced the C-terminal unfolding in a native ß2m. By contrast, L.M.H. showed no reaction even with ΔN6ß2m. This study showed firstly a direct binding of ß2m with H.M.H.. H.M.H. would provoked a C-terminal unfolding of ß2m, which indicated production of an amyloidogenic intermediate, i.e., ß2m92-99. In addition, our findings also suggest that L.M.H. may provide beneficial effects against the development of the DRA.

Role of extracellular superoxide dismutase in patients under maintenance hemodialysis.

BACKGROUND: It has been well documented that free radical injury is involved in the progression of chronic renal failure. Extracellular superoxide dismutase (EC-SOD), localized on the endothelial cell surface, plays an important role in reducing oxidative stress especially in the vessels by binding to the endothelial cell surface via the heparin-binding domain. Although EC-SOD Arg213Gly, which cannot bind on endothelial cells, has been considered a polymorphism, the effect of EC-SOD on hemodialysis patients has not been well examined. METHODS: In 178 hemodialysis patients, the following examinations were performed. EC-SOD Arg213Gly was examined by polymerase chain reaction (PCR)-induced mutation restriction analysis (PCR-IMRA). As indexes of atherosclerosis, the annual progression in intima-media thickness (DeltaIMT), plaque score, pulse wave velocity (PWV) and plasma-oxidized low-density lipoprotein (OxLDL) values were examined. RESULTS: PCR-IMRA revealed that 20 of 178 patients possessed the mutation (11.2%), and the incidence was about twice as high as that in a previously reported Japanese population. Although there were no statistical differences in plaque score and PWV with and without EC-SOD Arg213Gly, DeltaIMT and plasma OxLDL values in patients with EC-SOD Arg213Gly were significantly higher than those in patients without the mutation. CONCLUSION: EC-SOD Arg213Gly is an accelerating factor for the progression of renal failure and atherosclerosis.

C-terminal unfolding of an amyloidogenic ß2-microglobulin fragment: ΔN6ß2-microglobulin.

OBJECTIVES: A ß2-microglobulin (ß2m) fragment that lacks the first six amino acids, i.e., ΔN6ß2-microglobulin (ΔN6ß2m), is an endogenous, proteolytically derived, amyloidogenic fragment of ß2m, the precursor protein in Aß2M amyloidosis (dialysis-related amyloidosis). As reports suggest the importance of C-terminal unfolding for the amyloidogenicity of ß2m, in this study we aimed to investigate conformational characteristics of ΔN6ß2m related to amyloidogenicity. We also measured the concentration of an amyloidogenic intermediate of ß2m with C-terminal unfolding (ß2m92-99) in serum samples from 10 patients undergoing hemodialysis (HD). METHODS: We utilized capillary electrophoretic analysis, surface plasmon resonance and enzyme-linked immunosorbent assay. RESULTS AND CONCLUSIONS: We confirmed the normal core structure of ΔN6ß2m with a commercial monoclonal anti-ß2m antibody. In addition, using the specific monoclonal antibody for the C-terminal peptide, i.e. mAb 92-99, we confirmed unfolding in the C-terminal region of ΔN6ß2m. On the basis of these findings, we established an ELISA to measure ß2m92-99 using ΔN6ß2m as a standard molecule in circulation. However, we did not detect ß2m92-99 in serum from 10 HD patients, despite the absence of uremic inhibitors in the serum.

Transplantation of nonvascularized kidney tissue fragments into the rat liver with the aim of preserving renal function.

For research in regenerative medicine, not only the study of cellular pluripotency but also knowledge of the reorganization of tissue structure is crucial. However, the latter will probably be more difficult to acquire. When small fragments of kidney (approx. 1 x 1 mm) were implanted in the liver of syngeneic LEW rats, the tissue survived at least 2 weeks with retention of normal structure including glomeruli and tubules. In contrast, no kidney structure survived when transplanted to subcutaneous sites, omentum, or spleen. Molecules involved in renal tubular function, such as megalin and glut2 transporter protein, were detectable in the implanted tissue by immunohistochemistry, suggesting that the cells were biologically active. Survival of cortex, medulla, and calyx tissues was then compared. All three components were still detectable 8 weeks after transplantation but cortex and medulla were replaced by granuloma at 6 months. Only calyx tissue survived for up to 12 months after transplantation. There was no marked difference in tissue survival, either when the recipient liver was partially resected or when infantile donor kidney was implanted instead of adult kidney. The present method opens new avenues in the development of regenerative medicine (i.e., tissue transplantation) as an intermediate modus between organ transplantation and cell transplantation.

A possible involvement of the carboxymethylation process in carpal tunnel syndrome in hemodialysis patients.

A median motor nerve latency (DML) is generally prolonged in the carpal tunnel syndrome (CTS) of hemodialysis patients. Meanwhile, the advanced glycation process of proteins has been reported to be involved in the pathogenesis of the dialysis related amyloidosis. To investigate the role of carboxymethylation in dialysis related CTS, we measured a circulating carboxymethyllysine-hemoglobin (CML-Hb) level and nerve conduction velocity in 44 hemodialysis patients. The circulating CML-Hb level was 6.56 +/- 3.18 nmol CML/mg Hb, median motor nerve conduction velocity (NCV) was 49.8 +/- 4.64 m/s, median DML was 4.44 +/- 1.06 ms, and difference between median DML and ulnar DML (Delta DML) was 1.68 +/- 1.09 ms. Median and ulnar nerve NCV showed no correlation with circulating CML-Hb level. Both median DML and Delta DML were significantly correlated with CML-Hb (r = 0.429, P = 0.003, r = 0.472, P = 0.001). This study provided additional clinical evidence of an involvement of an advanced glycation process in the pathogenesis in CTS in hemodialysis patients.

[New molecular marker in dialysis setting--misfolded beta 2-microglobulin].

More than 25 clinical settings in amyloidosis have been acknowledged in which a peculiar criminal protein, a precursor protein, has been identified. As of now, however, the mechanism of amyloidogenesis, by which a precursor protein is transformed irreversibly into an amyloid protein, remains to be clarified. We speculated that a study of the molecular conformation of beta 2-microglobulin (beta 2 m), a precursor protein in dialysis-related amyloidosis (DRA), might provide a typic model of amyloidogenesis in other precursor proteins. Therefore, we investigated the misfolding of beta 2 m in DRA using a specific monoclonal antibody against C-terminal peptide 92-99 of beta 2 m. Our study indicated the possibility that the monoclonal antibody specific for C-terminal 92-99 of beta 2 m can detect a pre-amyloid state in amyloidogenesis in vivo, which might take place in the extravascular space.

Effect of heparin on conformation of the ß2-microglobulin molecule.

Heparin, one of the essential molecules called glycosaminoglycans (GAGs), is the anticoagulant that is commonly used in regular hemodialysis, during which dialysis-related amyloidosis (DRA) may develop. The pathogenic protein, i.e. precursor protein, in DRA is ß(2)-microglobulin (ß(2)m). Recent studies defined amyloidosis as a protein misfolding disease of precursor proteins including ß(2)m. Because the analytic technique capillary electrophoresis can identify molecular variants of the folded ß(2)m, i.e. conformational variants, we utilized it to investigate the effect of heparin on ß(2)m conformation and thus determined whether heparin can promote DRA development by inducing a conformational change in the amyloidogenic ß(2)m molecule. Heparin had a slight but significant effect on intermediate ß(2)m conformation but no effect on native ß(2)m conformation and on conversion of native to intermediate ß(2)m. Our findings thus suggest a possible association of ß(2)m with GAGs containing a sulfate moiety, including heparin, in HD patients.

Capillary electrophoretic profile of ß2-microglobulin intermediate associated with hemodialysis.

We previously identified an intermediate ß(2)-microglobulin (I-ß(2) m), which is an amyloidogenic ß(2) m variant, via capillary electrophoresis (CE) and reported hemodialysis (HD)-associated variations in the serum concentrations of each ß(2) m component, including that found in the rebound phase. Recent research has indicated that I-ß(2) m can bind, via the SO(3)(-) moiety, with glycosaminoglycan or proteoglycan, which are major components of interstitial tissue. Because alterations in I-ß(2) m are likely to be important in view of the possible accumulation of amyloidogenic precursor proteins in the interstitial space, we studied the I-ß(2) m profile as related to HD. We used CE to determine the I-ß(2) m profile both at the start and at the end of HD and during the rebound phase in 12 HD patients. We found both an unfolded ß(2) m and a destructured I-ß(2) m. More important, two peaks appeared in the rebound phase, one suggesting a refolding and one suggesting an irreversible destruction. Given that the intercompartmental transfer coefficient for ß(2) m is 1.0, our results indicated concomitant processes occurring after HD: refolding of the ß(2) m conformation and trapping of destructured I-ß(2) m in the extravascular space. Because the trapping of destructured I-ß(2) m supposedly leads to accumulation of ß(2) m in the interstitial space, we have proposed a new concept-a "shuttle" concept-for amyloid formation from ß(2) m in the HD setting.

Efficacy of once-weekly intravenous administration of epoetin-beta as a maintenance treatment for anemia in Japanese hemodialysis patients: a multicenter, open-label clinical study.

Epoetin-beta is extremely useful as a drug for treating anemia in hemodialysis (HD) patients and is widely used for that purpose. The aim of this study was to determine whether once-weekly intravenous administration of epoetin-beta is as effective in maintaining hemoglobin (Hb) concentration as the same weekly dose administered 2 or 3 times per week as maintenance treatment of anemia in HD patients. The subjects were stable HD patients who had been receiving HD for at least 12 months. Using a fixed weekly dose of 3000 or 6000 IU of epoetin-beta, this study evaluated maintenance of improvement of anemia by comparing Hb concentration in the study period (once-weekly) with Hb concentration in the prestudy period (2 or 3 times per week). Of the 112 patients treated with epoetin-beta, 111 patients (full analysis set; 3000 IU, 52 patients; 6000 IU, 59 patients) were evaluated, after excluding one patient whose dose was changed immediately before study initiation. The change in the Hb concentration was maintained within +/-1.5 g/dL in 89.2% of patients (3000 IU, 88.5%; 6000 IU, 89.8%). The mean Hb concentration was 10.42 +/- 0.73 g/dL at study initiation and 10.14 +/- 1.00 g/dL at study completion. Adverse reactions occurred in 9.8% of patients (11 out of 112 patients). The main adverse reactions were malaise and increased blood pressure. Once-weekly intravenous administration of epoetin-beta is useful as maintenance treatment of anemia in HD patients and may be a treatment option.

Pigment epithelium-derived factor inhibits advanced glycation end product-induced retinal vascular hyperpermeability by blocking reactive oxygen species-mediated vascular endothelial growth factor expression.

Pigment epithelium-derived factor (PEDF) is the most potent inhibitor of angiogenesis, suggesting that loss of PEDF contributes to proliferative diabetic retinopathy. However, the role of PEDF against retinal vascular hyperpermeability remains to be elucidated. We investigated here whether and how PEDF could inhibit the advanced glycation end product (AGE) signaling to vascular hyperpermeability. Intravenous administration of AGEs to normal rats not only increased retinal vascular permeability by stimulating vascular endothelial growth factor (VEGF) expression but also decreased retinal PEDF levels. Simultaneous treatments with PEDF inhibited the AGE-elicited VEGF-mediated permeability by down-regulating mRNA levels of p22(phox) and gp91(phox), membrane components of NADPH oxidase, and subsequently decreasing retinal levels of an oxidative stress marker, 8-hydroxydeoxyguanosine. PEDF also inhibited the AGE-induced vascular hyperpermeability evaluated by transendothelial electrical resistance by suppressing VEGF expression. Furthermore, PEDF decreased reactive oxygen species (ROS) generation in AGE-exposed endothelial cells by suppressing NADPH oxidase activity via down-regulation of mRNA levels of p22(PHOX) and gp91(PHOX). This led to blockade of the AGE-elicited Ras activation and NF-kappaB-dependent VEGF gene induction in endothelial cells. These results indicate that the central mechanism for PEDF inhibition of the AGE signaling to vascular permeability is by suppression of NADPH oxidase-mediated ROS generation and subsequent VEGF expression. Substitution of PEDF may offer a promising strategy for halting the development of diabetic retinopathy.

Studies on unfolded beta-microglobulin at C-terminal in dialysis-related amyloidosis.

BACKGROUND: In 1997, Stoppini et al reported that monoclonal antibody specific to the C-terminal 92-99 of beta(2)-microglobulin (beta(2)m) had been capable of inhibiting fibrillogenesis of beta(2)m in vitro. Meanwhile, recent studies have indicated that an acidifying procedure can unfold conformation of the precursor protein, leading to fibril formation of beta(2)m as well as a transthyretin. METHODS: We thus prepared monoclonal antibody specific to the C-terminal 92-99 (mAb 92-99), and investigated its reactivity in plasma ultrafiltrate and amyloid tissues from 18 hemodialysis patients with dialysis-related amyloidosis (DRA). RESULTS: beta(2)m extracted from ultrafiltrate showed no reaction for mAb 92-99, whereas acidified beta(2)m from ultrafiltrate showed a reaction for mAb 92-99. Similarly, a homogenate of carpal amyloid tissues showed a strong reaction for mAb 92-99 on immunoblotting. Immunohistochemical study showed also a distinct staining for mAb 92-99 in 7 Congophilic specimens from DRA patients. More interestingly, staining for mAb 92-99 could be found in most, though not all, non-Congophilic tissues. CONCLUSION: This study demonstrates that the monoclonal antibody specific to the C-terminal 92-99 of beta(2)m can detect the conformational intermediate in amyloidogenesis of beta(2)m ex vivo, and demonstrates that an unfolded beta(2)m at C-terminal could be found not only in Congophilic area but even in non-Congophilic area as well.

An in vitro evaluation of the glycation potential of a natural disaccharide, trehalose.

BACKGROUND: Glucose, an osmotic agent generally used in continuous ambulatory peritoneal dialysis (CAPD) dialysate, has a critical characteristic of forming advanced glycation endproducts (AGEs). We undertook this study to investigate whether a possible osmotic agent, trehalose, formed fewer AGEs than glucose. METHODS: Hemoglobin (Hb), a counter-protein of AGE, was incubated in four kinds of medium; glucose-phosphate buffered saline (PBS), autoclaved glucose-PBS, trehalose-PBS, and autoclaved trehalose-PBS, for 3, 7, 14, and 30 days, respectively. Polymerization of the Hb molecule was detected by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and carboxymethylated Hb was detected by Western blotting, using specific mono-clonal antibody for carboxymethylated N-terminal valine-Hb (CMV-Hb). RESULTS: PBS containing glucose showed bands of polymerized Hb molecule, a phenomenon which was markedly exaggerated by autoclaving. Likely, PBS containing glucose showed the formation of CMV-Hb in the long incubation of 30 days, and PBS containing autoclaved glucose showed accelerated formation of CMV-Hb in an incubation as short as 3 days. By contrast, PBS containing trehalose showed much less increase in a band of 30 k Dalton and in CMV-Hb formation even in autoclaved medium. CONCLUSIONS: Our present in vitro study clearly showed the superior characteristic of trehalose to produce fewer AGEs. Based upon the results of this study, we propose that the application of trehalose should be considered for CAPD solution.