Dialysis-related Amyloidosis Research Articles

Mouse model to study human A β2M amyloidosis: generation of a transgenic mouse with excessive expression of human β2-microglobulin

Patients on long-term hemodialysis can develop dialysis-related amyloidosis (DRA) due to deposition of β2-microglobulin (β2m) into amyloid fibrils (Aβ2M). Despite intensive biochemical studies, the pathogenesis of amyloid deposition in DRA patients remains poorly understood. To elucidate the mechanisms that underlie Aβ2M fibril formation in DRA, we generated transgenic mice that overexpress human β2m protein in a mouse β2m gene knockout background (hB2MTg+/+ mB2m+/+). The hB2MTg+/+mB2m−/− mice express a high level of human β2m protein in many tissues as well as a high plasma β2m concentration (192.8 mg/L). This concentration is >100 times higher than that observed in healthy humans and >4 times higher than that detected in patients on dialysis. We examined spontaneous and amyloid fibril-induced amyloid deposition in these mice. Amyloid deposition of β2m protein was not observed in aged or amyloid fibril injected animals. However, mouse senile apolipoprotein A-II amyloidosis (AApoAII) was detected, particularly in the joints of mice that were injected with AApoAII amyloid fibrils.This study demonstrates that this mouse model could be valuable in studying the components and conditions that promote DRA, and indicates that high plasma concentrations of hβ2m as well as seeding with pre-existing amyloid fibrils may not be sufficient to induce Aβ2M.

Stacked Sets of Parallel, In-register β-Strands of β2-Microglobulin in Amyloid Fibrils Revealed by Site-directed Spin Labeling and Chemical Labeling

β2-microglobulin (β2m) is a 99-residue protein with an immunoglobulin fold that forms β-sheet-rich amyloid fibrils in dialysis-related amyloidosis. Here the environment and accessibility of side chains within amyloid fibrils formed in vitro from β2m with a long straight morphology are probed by site-directed spin labeling and accessibility to modification with N-ethyl maleimide using 19 site-specific cysteine variants. Continuous wave electron paramagnetic resonance spectroscopy of these fibrils reveals a core predominantly organized in a parallel, in-register arrangement, by contrast with other β2m aggregates. A continuous array of parallel, in-register β-strands involving most of the polypeptide sequence is inconsistent with the cryoelectron microscopy structure, which reveals an architecture based on subunit repeats. To reconcile these data, the number of spins in close proximity required to give rise to spin exchange was determined. Systematic studies of a model protein system indicated that juxtaposition of four spin labels is sufficient to generate exchange narrowing. Combined with information about side-chain mobility and accessibility, we propose that the amyloid fibrils of β2m consist of about six β2m monomers organized in stacks with a parallel, in-register array. The results suggest an organization more complex than the accordion-like β-sandwich structure commonly proposed for amyloid fibrils.

Fibrillar vs Crystalline Full-Length β-2-Microglobulin Studied by High-Resolution Solid-State NMR Spectroscopy

Elucidating the fine structure of amyloid fibrils as well as understanding their processes of nucleation and growth remains a difficult yet essential challenge, directly linked to our current poor insight into protein misfolding and aggregation diseases. Here we consider beta-2-microglobulin (beta2m), the MHC-1 light chain component responsible for dialysis-related amyloidosis, which can give rise to amyloid fibrils in vitro under various experimental conditions, including low and neutral pH. We have used solid-state NMR to probe the structural features of fibrils formed by full-length beta2m (99 residues) at pH 2.5 and pH 7.4. A close comparison of 2D (13)C-(13)C and (15)N-(13)C correlation experiments performed on beta2m, in both the crystalline and fibrillar states, suggests that, in spite of structural changes affecting the protein loops linking the protein beta-strands, the protein chain retains a substantial share of its native secondary structure in the fibril assembly. Moreover, variations in the chemical shifts of the key Pro32 residue suggest the involvement of a cis-trans isomerization in the process of beta2m fibril formation. Lastly, the analogy of the spectra recorded on beta2m fibrils grown at different pH values hints at a conserved architecture of the amyloid species thus obtained.

The study of the resin adsorption on patients of dialysis-related amyloidosis in clinical efficacy and quality of life

目的 探讨树脂吸附对维持性血液透析(MHD)患者透析相关性淀粉样变(DRA)的防治作用及生活质量的影响.方法 将40例合并DRA患者随机分为树脂吸附+血液透析组(HP+HD)和常规血液透析组(HD).HP+HD组在每周3次常规血液透析的同时,接受每2周1次的HP治疗;HD组行每周3次常规血液透析.分别于治疗前、治疗3个月时应用SF-36量表进行生活质量评分,检测血清β2-M、Hs-CRP的变化;并用磁共振(MRI)检查患者双侧肩关节,检测肩关节MRI阳性征象,观察临床症状改变情况.结果 HP+HD组治疗后患者生活质量评分由(86.39±26.46)升到(104.30±20.69)(P<0.05) β2-M从(13.48±4.69)mg/ml降至(5.91±1.73)mg/ml(P<0.01);Hs-CRP从(15.86±5.12)pg/ml降至(8.86±3.36)pg/ml(P<0.01),MRI影像学征像和临床症状治疗后均明显改善(P<0.05),治疗前后比较差异均有显著性.结论 DRA患者在常规HD治疗的同时接受HP治疗能有效的改善或缓解其DRA,并提高其生活质量。

Native-unlike Long-lived Intermediates along the Folding Pathway of the Amyloidogenic Protein β2-Microglobulin Revealed by Real-time Two-dimensional NMR

Beta2-microglobulin (beta2m), the light chain of class I major histocompatibility complex, is responsible for the dialysis-related amyloidosis and, in patients undergoing long term dialysis, the full-length and chemically unmodified beta2m converts into amyloid fibrils. The protein, belonging to the immunoglobulin superfamily, in common to other members of this family, experiences during its folding a long-lived intermediate associated to the trans-to-cis isomerization of Pro-32 that has been addressed as the precursor of the amyloid fibril formation. In this respect, previous studies on the W60G beta2m mutant, showing that the lack of Trp-60 prevents fibril formation in mild aggregating condition, prompted us to reinvestigate the refolding kinetics of wild type and W60G beta2m at atomic resolution by real-time NMR. The analysis, conducted at ambient temperature by the band selective flip angle short transient real-time two-dimensional NMR techniques and probing the beta2m states every 15 s, revealed a more complex folding energy landscape than previously reported for wild type beta2m, involving more than a single intermediate species, and shedding new light into the fibrillogenic pathway. Moreover, a significant difference in the kinetic scheme previously characterized by optical spectroscopic methods was discovered for the W60G beta2m mutant.

Molecular Insights into the Role of Serum Amyloid-P Component in the Stabilzation of Fibrillar Beta-2 Microglobulin

Serum amyloid-P component is found ubiquitously in amyloid deposits and has been shown to stabilize fibrillar structures and prevent clearance by the host's defences. Here we report on solid-state NMR studies on fibrillar deposits formed from β2-microglobulin, typically found in patients with dialysis related amyloidosis, and their interactions with serum amyloid-P component.We have successfully undertaken the expression, purification and refolding of the 99 residue β2-microglobulin and established conditions for optimal binding of serum amyloid-P component. High resolution solid-state magic-angle spinning (MAS)-NMR spectra obtained from the fibrils indicate that within the fibrils the β2-microglobulin adopts a homogeneous structure. Using two-dimension homo- and hetero-nuclear correlation spectroscopy we have been able to assign several of the sites within the protein. Currently we are using a range of labelling schemes and acquiring three-dimensional data-sets to complete this assignment. Comparison of the assignment with that obtained from monomeric β2-microglobulin in solution is beginning to provide valuable insights into the structural changes occurring upon fibrilization. Similar comparisons with fibres decorated with serum amyloid-P component should provide valuable insights into how this molecule interacts and stabilizes amyloid fibrils at a molecular level.During the course of these experiments we have also obtained 2D correlation data on inclusion bodies of β2-microglobulin. The resolution attained is not as high as that observed in the fibrillar spectra, however they permit the assignment of resonances to amino acids with the β2-microglobulin. This suggests that within the inclusion bodies the β2-microglobulin adopts a well defined conformation with the lower spectral resolution arising from reduction in dynamics in these highly packed structures. Detailed comparisons of the data with that obtained from the soluble and fibrillar β2-microglobulin should provide insights into the nature of this structure.

Seed-Conjugated Polymer Bead for β2-Microglobulin Removal at Neutral pH

beta2-Microglobulin (beta2m) is known to be a major factor for dialysis-related amyloidosis. We have studied beta2m removal through an aggregation process, which was initiated by a ligand that could catch the beta2m monomer and promote its aggregation into fibril. As a ligand, we have prepared beta2m fibril fragments and used them as a seed. The seed was coupled to PEGylated-PS beads to remove the monomeric beta2m from solution. The beta2m seed-conjugated resin effectively adsorbed the beta2m monomers with a capacity of 3.6 mg/ml via promoting their aggregation into fibrils on the resin at pH 7.4.

K3 Fragment of Amyloidogenic β2-Microglobulin Forms Ion Channels: Implication for Dialysis Related Amyloidosis

Beta(2)-microglobulin (beta(2)m) amyloid deposits are linked to dialysis-related amyloidosis (DRA) in hemodialysis patients. The mechanism by which beta(2)m causes DRA is not understood. It is also unclear whether only the full-length beta(2)m induces pathophysiology or if proteolytic fragments are sufficient for inducing this effect. Ser20-Lys41 (K3) is a digestion fragment of full-length beta(2)m. Solid state NMR (ssNMR) combined with X-ray diffraction and atomic force microscopy (AFM) revealed the characteristic oligomeric amyloid conformation of the U-turn beta-strand-turn-beta-strand motif stacked in parallel and stabilized by intermolecular interactions also shown by Abeta(9-40)/Abeta(17-42) and the CA150 WW domain. Here we use the K3 U-turn atomic coordinates and molecular dynamic (MD) simulations to model K3 channels in the membrane. Consistent with previous AFM imaging of other amyloids that show channel-like structures in the membrane, in the simulations K3 also forms ion channels with 3-6 loosely attached mobile subunits. We carry out AFM, single channel electrical recording, and fluorescence imaging experiments. AFM images display 3D ion channel topography with shapes, morphologies, and dimensions consistent with the theoretical model. Electrical conductance measurements indicate multiple single channel conductances, suggesting that various K3 oligomer sizes can constitute the channel structure. Fluorescence measurements in kidney cells show channel-mediated cell calcium uptake. These results suggest that the beta(2)m-induced DRA can be mediated by ion channels formed by its K3 fragment. Because the beta-strand-turn-beta-strand motif appears to be a universal amyloid feature, its ability to form ion channels further suggests that the motif may play a generic role in toxicity.

Utilisation de l’AN69 en hémodiafiltration « online »

Removal of the middle molecules (MM) weight toxins, and particularly of beta-2-microglobuline (b2M) is made first by convection technique with membranes habitually used in hemodiafiltration online (HDFOL). AN69 was in standard hemodialysis (HD) the reference membrane for the removal of b2M mostly (60%) by adsorption. Its use with convective methods is generally restricted to low efficiency modalities with low substitution rates (Qs), either with continuous HF or HDF for acute renal failure, or with acetate free biofiltration (AFB) for chronic renal failure. So-called adsorptive membranes do not have, at the present time, a well-defined indication for high efficiency HDFOL, which necessitates substitution rates (Qs) higher than 15 litres per session. The objective of this study is to demonstrate the feasibility of such an association between polyethyleneimine (PEI) surface treated AN69 and HDFOL both in qualitative and quantitative terms. Five patients were dialysed, respectively, with Nephral 500ST (AN69, PEI, surface 2.15m(2), Hospal France) in HD (HDNEP), postdilution HDF (PostNEP), predilution HDF (PreNEP) and as a reference, with FX80 (Helixone, surface 1,8m(2), Fresenius) in PostD (PostFX). For the small molecules (SM), equilibrated Kt/V (Kt/VEq) and the phosphorus (Ph) reduction ratio (RR)Ph have been measured. Considering the predominating adsorption of b2M by AN69, the removal of MM - b2M and myoglobine (Myo) - was also assessed by their RR (RRb2M and RRMyo). Results are convincing in terms of behaviour in relation to convective techniques, the 2.15m(2) membrane used during 4 hour sessions accepting Qs up to 25 litres with PostD and up to 59 litres with PreD, without any alarm of transmembrane pressure (TMP) or any fiber coagulation incident, proving without any ambiguity the absence of deleterious membrane plugging and its compatibility with high efficiency HDFOL. The behaviour of AN69 in HDFOL in relation to SM does not differ from HDNEP or FXPost. For MM, results for b2M are significantly lower (P=0.01) for PostNEP (RR=71.4%) in comparison with PostFX (RR=79.3%), but the behaviour of AN69 is above all particular for higher molecular weight substances since results are significantly reversed (P=0.03) for Myo (PM=17,800Da) in favour of PostNEP (RR=73.6%) compared to PostFX (RR=65.7%). These results open up new horizons for HDFOL and encourage us to focus future studies on the consequences of an optimized removal, mainly by absorption, of high molecular weight toxins, such as factor D, C3a, C3b and cytokines (IL-1 and TNF). The expected beneficial consequences concern complications linked to inflammation and oxidative stress, which could account notably, beyond the mere quantitative removal of b2M, for the quasi disappearance of any clinical expression of dialysis-related amyloidosis.

Atypical rapid progression of osteoarticular amyloidosis involving the hip in a patient on hemodialysis using polyacrylonitrile membranes

Amyloidosis related to dialysis is a well-known complication affecting many organ systems, in particular the musculoskeletal system. In 1985 Shirahama et al. (Biochem Biophys Res Commun 53:705-709, 1985) identified beta-2 microglobulin (MG) as the offending constituent by using protein purification techniques. Amyloidosis has been increasing in prevalence because of longer life spans and increased chronic medical conditions such as end-stage renal disease. When dialysis-related amyloidosis involves the musculoskeletal system, it affects the shoulder girdle, the so called shoulder pad sign, the wrist, hip, knee, and spine (Resnick, Diagnosis of bone and joint disorders, 4th edn., pp. 2054-2058 and 2176-2183, 2002). Other osteoarticular manifestations of amyloidosis include osteoporosis, lytic lesions, and pathologic fractures. It has been well documented that the prevalence of amyloid is dependent on duration of dialysis-over 90% in patients on dialysis for over 7 years (Jadoul, Nephrol Dial Transplant 13:61-64, 1998). However, a recent changeover to high-flux membranes used in hemofiltration has been reported to delay its onset (Campistol et al., Contrib Nephrol 125:76-85, 1999). We report on the radiographic, nuclear medicine, and computed tomography (CT) findings of osteoarticular amyloidosis involving the hip, and sequence its atypical rapid onset. The imaging, histopathological findings, and differential diagnosis are discussed.

Molecular dynamics simulations to investigate the relationship between the structural stability and amyloidogenesis of the wild-type and N-terminal hexapeptide deletion ΔN6 β2-microglobulin

β2-Microglobulin (β2m) forms amyloid fibrils in patients undergoing long-term haemodialysis, leading to dialysis-related amyloidosis. Proteolysis of the N-terminal region of β2m results in a truncation of the six N-terminal residues (ΔN6 β2m) in ∼30% of the β2m molecules extracted from ex vivo fibrils. The ΔN6 β2m has been shown to exhibit a higher tendency for self-association comparing to the wild-type (wt) β2m, particularly at neutral pH. In order to gain atomic insights into the early stages of amyloid formation of the wt and ΔN6 β2m, various molecular dynamics simulations were conducted to investigate the stability and dynamics of these two molecules at various temperatures and neutral pH in this study. Our results, in agreement with previous experimental results, indicate that the structural stability of the ΔN6 β2m is lower than that of the wt β2m. It can be attributed to fact that the removal of the N-terminal six residues results in the loss of the salt–bridge interaction between residues R3 and D59, leading to the increased solvent exposure of the K3 peptide. It further allows water molecules to destabilise the interior region of the K3 peptide, leading to the elongation between the B- and E-strands. It may further accelerate the conformational changes of the ΔN6 β2m, leading to the formation of amyloid fibrils more readily at neutral pH. Our results also suggest that the K3 peptide may be a potential initiation site of amyloid formation for the ΔN6 β2m due to its increased solvent exposure. We further suggest that fibril morphology of the ΔN6 β2m formed at neutral pH is similar to that of the wt β2m formed at low pH (1.5–3) since they adopt the similar conformation with the elongation between B- and E-strands for their partially unfolded amyloidogenic intermediates.

NMR‐based characterization of a refolding intermediate of β2‐microglobulin labeled using a wheat germ cell‐free system

In patients with dialysis-related amyloidosis, beta2-microglobulin (beta2-m) is a major structural component of amyloid fibrils. It has been suggested that the partial unfolding of beta2-m is a prerequisite to the formation of amyloid fibrils, and that the folding intermediate trapped by the non-native trans-Pro32 isomer leads to the formation of amyloid fibrils. Although clarifying the structure of this refolding intermediate by high resolution NMR spectroscopy is important, this has been made difficult by the limited lifetime of the intermediate. Here, we studied the structure of the refolding intermediate using a combination of amino acid selective labeling with wheat germ cell-free protein synthesis and NMR techniques. The HSQC spectra of beta2-ms labeled selectively at either phenylalanine, leucine, or valine enabled us to monitor the structures of the refolding intermediate. The results suggested that the refolding intermediate has an overall fold and cores similar to the native structure, but contains disordered structures around Pro32. The fluctuation of the beta-sheet regions especially the last half of the betaB strand and the first half of the betaE strand, both suggested to be important for amyloidogenicity, may transform beta2-m into an amyloidogenic structure.

Ultrasonication-dependent production and breakdown lead to minimum-sized amyloid fibrils

Because of the insolubility and polymeric properties of amyloid fibrils, techniques used conventionally to analyze protein structure and dynamics have often been hampered. Ultrasonication can induce the monomeric solution of amyloidogenic proteins to form amyloid fibrils. However, ultrasonication can break down preformed fibrils into shorter fibrils. Here, combining these 2 opposing effects on beta(2)-microglobulin (beta2-m), a protein responsible for dialysis-related amyloidosis, we present that ultrasonication pulses are useful for preparing monodispersed amyloid fibrils of minimal size with an average molecular weight of approximately 1,660,000 (140-mer). The production of minimal and monodispersed fibrils is achieved by the free energy minimum under competition between fibril production and breakdown. The small homogeneous fibrils will be of use for characterizing the structure and dynamics of amyloid fibrils, advancing molecular understanding of amyloidosis.

Structural modeling and biochemical studies reveal insights into the molecular basis of the recognition of β‐2‐microglobulin by antibody BBM.1

Human beta-2-microglobulin (beta2m) is the light chain of human leucocyte antigen-I (HLA-I). It can disassociate from HLA-I and accumulate to cause serious dialysis-related amyloidosis (DRA) in long-term hemodialysis patients. Monoclonal antibody (mAb) BBM.1 can recognize both free-form and HLA-I associated beta2m. It can be used for specific elimination of beta2m from serum and can induce apoptosis of several types of tumor cells, and thus has great therapeutic potential. In this study, we constructed structural models of the BBM.1 Fv (fragment of the variable domain) and the BBM.1 Fv-beta2m complex, followed by biochemical evaluation. Analysis of the optimal complex model reveals that the previously identified immunodominant residues Glu(44) and Arg(45) of beta2m have direct interactions with BBM.1, while Asp(38) exerts its function mainly via stabilization of Arg(45). In addition, Arg(81) of beta2m is a newly identified immunodominant residue to have direct interaction with BBM.1. Further modeling study shows no steric conflict between the antibody and the HLA-I heavy chain. These results provide insights into the molecular basis of the recognition of beta2m by BBM.1 and explain why BBM.1 can bind both free-form and HLA-1 associated beta2m. This information could be exploited in the engineering and improvement of BBM.1 and the development of other beta2m-targeting mAbs for therapeutic purposes.

Mechanism of Lysophosphatidic Acid-Induced Amyloid Fibril Formation of β2-Microglobulin in Vitro under Physiological Conditions

Beta(2)-microglobulin- (beta2m-) based fibril deposition is the key symptom in dialysis-related amyloidosis. beta2m readily forms amyloid fibrils in vitro at pH 2.5. However, it is not well understood which factors promote this process in vivo, because beta2m cannot polymerize at neutral pH without additives even at elevated concentration. Here we show that lysophosphatidic acid (LPA), an in vivo occurring lysophospholipid mediator, promotes amyloid formation under physiological conditions through a complex mechanism. In the presence of LPA, at and above its critical micelle concentration, native beta2m became sensitive to limited proteolytic digestion, indicating increased conformational flexibility. Isothermal titration calorimetry indicates that beta2m exhibits high affinity for LPA. Fluorescence and CD spectroscopy, as well as calorimetry, showed that LPA destabilizes the structure of monomeric beta2m inducing a partially unfolded form. This intermediate is capable of fibril extension in a nucleation-dependent manner. Our findings also indicate that the molecular organization of fibrils formed under physiological conditions differs from that of fibrils formed at pH 2.5. Fibrils grown in the presence of LPA depolymerize very slowly in the absence of LPA; moreover, LPA stabilizes the fibrils even below its critical micelle concentration. Neither the amyloidogenic nor the fibril-stabilizing effects of LPA were mimicked by its structural and functional lysophospholipid analogues, showing its selectivity. On the basis of our findings and the observed increase in blood LPA levels in dialysis patients, we suggest that the interaction of LPA with beta2m might contribute to the pathomechanism of dialysis-related amyloidosis.

Glimpses of the molecular mechanisms of β2-microglobulin fibril formation in vitro: Aggregation on a complex energy landscape

β2-microglobulin (β2m) is a 99-residue protein that aggregates to form amyloid fibrils in dialysis-related amyloidosis. The protein provides a powerful model for exploration of the structural molecular mechanisms of fibril formation from a full-length protein in vitro. Fibrils have been assembled from β2m under both low pH conditions, where the precursor is disordered, and at neutral pH where the protein is initially natively folded. Here we discuss the roles of sequence and structure in amyloid formation, the current understanding of the structural mechanisms of the early stages of aggregation of β2m at both low and neutral pH, and the common and distinct features of these assembly pathways.

New Therapeutic Strategy for Amyloidosis

Evaluation of: Ozawa D, Yagi H, Ban T et al.: Destruction of amyloid fibrils of a β2-microglobulin fragment by laser beam irradiation. J. Biol. Chem. 284(2), 1009–1017 (2009). In this study, the authors discovered that laser beam irradiation with thioflavin T could stop the extension of β2-microglobulin fibrils and even degrade preformed amyloid fibrils. These reactions require both thioflavin T and a laser beam at 442 nm; active oxygen plays an important role. This approach may be a new therapeutic strategy for destroying fibrils in dialysis-related amyloidosis as well as other types of amyloidosis or related disorders, including Alzheimer’s disease, familial amyloid polyneuropathy, Parkinson’s disease and Huntington’s disease.

Thermal Response with Exothermic Effects of β2-Microglobulin Amyloid Fibrils and Fibrillation

Calorimetric measurements were carried out using a differential scanning calorimeter to characterize the thermal response of β2-microglobulin amyloid fibrils, the deposition of which results in dialysis-related amyloidosis. The fibril solution showed a large decrease in heat capacity (exothermic effect) before the temperature-induced depolymerization of the fibrils, which was characterized by a definite dependence on heating rate. To understand the factors that determine the heating-rate-dependent thermal response, the concentration dependence of polyethylene glycol, which inhibits the association of amyloid fibrils with heating, on exothermic effect was examined in detail and showed a causal link between the exothermic effect and fibril association. The results suggest that the transient association driven by a spatial approach and the concomitant dehydration of hydrophobic areas of amyloid fibrils may be significant factors determining the thermal response with exothermic effect, which has not been observed in calorimetric studies of monomolecular globular proteins. The heating-rate-dependent thermal response with the exothermic effect was observed not only for other amyloid fibrils formed from amyloid β-peptides but also during the processes of the temperature-induced conversion of β2-microglobulin protofibrils and hen egg-white lysozyme into amyloid fibrils. These results highlight the physics related to the heating-rate-dependent behaviors of heat capacity in terms of interactions between the specific structures of amyloid fibrils and water molecules.

Recent progress in understanding dialysis-related amyloidosis

Dialysis-Related Amyloidosis (DRA) is a general amyloidosis, which is specifically found in CKD stage 5 patients. DRA causes various osteoarticular lesions in dialysis patients, and therefore it is not practical to regard this condition separately from chronic kidney disease–mineral and bone disorder (CKD–MBD), at least from the viewpoint of daily clinical practice. However, it is still controversial whether this disease condition should be included in CKD–MBD. Recently, a better understanding of the pathogenesis of DRA has been obtained by examination of β 2-microglobulin-related amyloid fibril formation, extension, and depolymerization in vitro. Apoliprotein E, proteoglycans, and glycosaminoglycans stabilize the amyloid fibrils. In addition, some lysophospholipids and non-esterified fatty acids accelerate amyloid fibril formation and extension under physiological conditions in vitro. Those molecules may enhance the amyloid deposition in vivo. The frequency and severity of osteoarticular disorders that may be associated with DRA accelerate with the duration of dialysis therapy. We have shown that patients undergoing dialysis therapy for 30 years or more survive with serious complications from osteoarticular disorders. DRA is one of the most harmful osteoarticular complications with regard to the maintenance of daily activities and quality of life in patients undergoing long-term dialysis therapy, in addition to the classical complications of CKD–MBD.

A β 2-microglobulin cleavage variant fibrillates at near-physiological pH

β 2-microglobulin (β 2m) deposits as amyloid in dialysis-related amyloidosis (DRA), predominantly in joints. The molecular mechanisms underlying the amyloidogenicity of β 2m are still largely unknown. In vitro, acidic conditions, pH < 4.5, induce amyloid fibrillation of native β 2m within several days. Here, we show that amyloid fibrils are generated in less than an hour when a cleavage variant of β 2m—found in the circulation of many dialysis patients—is exposed to pH levels (pH 6.6) occurring in joints during inflammation. Aggregation and fibrillation, including seeding effects with intact, native β 2m were studied by Thioflavin T fluorescence spectroscopy, turbidimetry, capillary electrophoresis, and electron microscopy. We conclude that a biologically relevant variant of β 2m is amyloidogenic at slightly acidic pH. Also, only a very small amount of preformed fibrils of this variant is required to induce fibrillation of native β 2m. This may explain the apparent lack of detectable amounts of the variant β 2m in extracts of amyloid from DRA patients.