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Dialysis-associated β2microglobulin (β2M) amyloidosis is a disabling complication affecting patients maintained on long-term hemodialysis.1, 2, 3 Early studies showed that accumulation and deposition of β2microglobulin amyloid proteins (Aβ2M) occurs predominantly in the bones, tendons and joints.4, 5, 6, 7, 8 The major clinical manifestations of this disease include carpal tunnel syndrome, chronic synovytis, progressive bone destruction and pathological bone fractures. Marked increase in β2M plasma concentration is probably necessary, but it is not sufficient to induce β2M amyloidosis. Attempts were made to determine the precise chemical structure of Aβ2M proteins in order to reveal subtle structural changes responsible for the amyloid fibril formation and deposition. Although some authors stated that proteolysis of β2M is necessary for fibrilogenesis,9 other studies showed that amyloid deposits are composed of intact 12 kDa molecules.4, 8 A possibility was raised that in contrast to the normal circulating β2M, the deposited Aβ2M proteins contain some acidic isoforms.10, 11, 12 The appearance of these isoforms was explained by deamination of Asn1713, 14 or modification by advanced glycation end products (AGE),10, 12 but these findings have not been confirmed by other authors.15 In addition, presence of acidic β2M isoforms has been demonstrated in normal urine, thus bringing up the question whether these isoforms are specific to dialysis-related amyloidosis.15 The importance of the β2M structure-unrelated factors in the pathogenesis of dialysis-related amyloidosis has been proposed by implying the role of other proteins and cells under chronic inflammatory stress conditions and by raising questions of bioincompatibility of membranes or insufficient purity of water used for dialysis.16, 17 Thus, the existence of the disease-specific modifications of β2M structure and their role in the pathogenesis of amyloid disease remain questionable.

Although dialysis-associated amyloidosis was initially believed to be limited to osteoarticular tissue, the involvement of visceral organs (gastrointestinal tract, liver, heart, prostate, kidneys, endocrine glands and subcutaneous tissue) is now well recognized.2, 3, 4 However, in contrast to the extensive studies of Aβ2M in the osteoarticular tissues, amyloid deposition in the extra-articular tissues was examined only by routine histological and immunohistochemical techniques, and not biochemically. As a result, little is known about the precise chemical structure of Aβ2M deposited in the extra-articular tissues of patients with dialysis-related amyloidosis.

We present here a first report on the biochemical analysis of intestinal amyloid deposits found in two cases of hemodialysis-related amyloidosis. To overcome the difficulties of biochemical analysis of amyloid proteins related to small amounts of available tissue material and its fixation with formalin, we employed our new microtechnique allowing the extraction and immunochemical/chemical characterization of amyloid proteins in tiny amounts of fixed tissue.18 In both cases studied, the molecular mass of the identified amyloid β2M was close to that of normal intact β2M, with no evidence of the products of proteolytic fragmentation of these molecules. The isoelectrofocusing (IEF) of the purified Aβ2M demonstrated a shift to more acidic pI values as compared to the normal β2M. The obtained data suggest that the intestinal amyloid deposits in dialysis-related amyloidosis contain disease-specific β2M isoforms, which may play a role in the pathogenesis of amyloid disease.

Patients and methods

Patients

In case I, the 64-year-old female was started on hemodialysis in 1980. The cause of kidney failure was unknown. In 1998, the patient was admitted to the hospital because of the obstructive ileus due to volvulus. Subtotal colectomy was performed and a specimen was examined histopathologically and biochemically as described below. After 4 years, she succumbed due to septicemia complicating femoral fracture. In case II, the 78-year-old female with end-stage renal failure of an unknown etiology was maintained on hemodialysis for 13 years. She was admitted to the hospital for acute abdomen secondary to colonic perforation. Hemicolectomy was performed, and a segment of the resected colon was subjected to histopatological and biochemical examination as described below.

Pathological Methods

Tissues were fixed in 4% buffered formalin. Formalin-fixed paraffin-embedded colon tissue specimens were subjected to histological and immunohistochemical examination. Sections were stained with hematoxylin and eosin (H&E), Crystal Violet and Congo-red according to the standard methods. Microwave antigen retrieval technique was employed for the immunostaining of tissue sections with rabbit anti-human antibodies to β2M, 1:100 (Zymed, San-Francisco, CA, USA). Bound antibodies were detected by the avidin–biotin–peroxidase method according to the established protocols.

Extraction and Purification of Amyloid Proteins

Amyloid proteins were recovered from the formalin-fixed paraffin-embedded biopsy tissue by the formic acid extraction procedure described previously.18, 19 Briefly, tissue specimen was deparaffinized at 60°C, washed in xylene and rehydrated in decreasing concentrations of alcohol. The obtained tissue material was homogenized, washed with phosphate-buffered saline and centrifuged. The resulting pellet was suspended in concentrated formic acid overnight, centrifuged, and the obtained supernatant was dried in a Speed Vac apparatus.

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

SDS-PAGE was performed using the precast 20–25% polyacrylamide LongLife microgels (Gradipore, Frenchs Forest, Australia) as described.18, 19 The material extracted from 2–5 mg fixed tissue sample was applied per well and run with Tris HEPES buffer (Gradipore). For the visualization of the proteins, the electrophoresed proteins were blotted onto Sequi-Blot PVDF (polyvinylidene difluoride) membranes (Bio Rad, Hercules, CA, USA) using Gradipore LongLife Transfer buffer, and afterwards stained with Coomassie blue R-250.

Western Blot Analysis

The electrophoresed proteins were transferred onto nitrocellulose (Schleicher and Schuell, Dassel, Germany) with a Gradipore LongLife Transfer buffer. The blots were immunostained with polyclonal rabbit antibodies to human immunoglobulin kappa (κ) and lambda (λ) light chains, and to β2M (Dako, Carpinteria, CA, USA) as described earlier.18, 19, 20 Proteins were visualized using Super-Signal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL, USA).

Amino-Acid Sequence Analysis

For N-terminal amino-acid sequence analysis, proteins run by SDS-PAGE were blotted onto Sequi-Blot PVDF (polyvinylidene difluoride) membranes (Bio Rad). The blotted protein bands (visualized with Coomassie blue R-250) were excised and subjected to amino-acid sequence analysis as described.18, 19

Micropreparative Purification of the Extracted Amyloid Proteins

The SDS-PAGE procedure described above was employed for concentration and micropreparative purification of amyloid proteins. The material extracted from about 15 mg fixed biopsy tissue was loaded onto 3–4 wells, run and electrotransferred onto Sequi-Blot PVDF membranes. Commercial urinary β2M (Sigma-Aldrich Inc., St Louis, MO, USA) was referred as a normal circulating β2M.21 It was applied to SDS-PAGE (2–2.5 μg/well) and run together with the extracted sample under the same experimental conditions. The 12 kDa protein bands were excised, pooled and eluted with TFA/acetonitrile/water (3:4:3) as described.22 The eluted material was dried in a Speed Vac apparatus.

Isoelectrofocusing

IEF was performed in a Model 111 Mini IEF Cell (Bio-Rad) on 0.4 mm thick gels consisting of 5.4% acrylamide, 0.17% bis-acrylamide, 2.3% pH 3–10 ampholyte (Bio-Rad), 6.5% glycerol, and 6 M urea. Samples of the purified amyloid Aβ2M and the normal β2M were solubilized in an IEF sample buffer consisting of 2% NP-40 (Sigma Chemical Co., St Louis, MO, USA), 9 M urea and 0.002% β-mercaptoethanol for 0.5 h at 37°C. Samples of the purified Aβ2M recovered from about 3 mg starting tissue material and those of the normal β2M (2 μg) were loaded per well. Gels were run first for 15 min at 100 V, then 15 min at 250 V and finally for 1 h at 450 V.

For detection of proteins, gels were stained with Fast Silver stain (Geno Technology, St Louis, MO, USA). For Western blotting, the IEF run samples were transferred onto nitrocellulose membrane and immunostained with polyclonal rabbit antibodies to human β2M (Dako) as described above.

Results

Pathological Findings

In case I (Figure 1a), the subtotal colectomy specimen included part of the terminal ileum (19 cm length) and right colon up to the sigmoid level (total length 135 cm). The main findings were marked dilatation, thinning of the colonic wall and flattening of the mucosal folds. In case II (Figure 1b), the specimen consisted of a colon segment (29 cm length) showing a perforation and fibrinopurulent deposition over the serosal surface. The mucosa was flattened, markedly congested and covered with white exudates at the perforation site.

Figure 1
figure 1

Macroscopic (a and b) and microscopic (cf) examination of colon tissue specimens in two cases of dialysis-related amyloidosis. (a) Macroscopic examination of case I shows marked dilatation of a large bowel with flattening of the mucosal surface. (b) Mucosal elevation of the perforated area into the gut lumen in case II; (c) H&E staining (original magnification, × 200) shows massive deposits of amorphous pink material of amyloid and multinucleated giant cells in the muscularis propria; (d) Congo-red-positive staining of amyloid deposits in the blood vessel walls (× 200); (e) amyloid deposits in the blood vessel walls exhibit apple green birefringence under polarized light (× 100); and (f) amyloid deposits demonstrate strongly positive immunostaining for β2M (× 100).

The histological changes were similar in both cases. H&E-stained sections presented a dense pink amorphous material deposition seen in the submucosal blood vessels wall and in the muscularis propria (Figure 1c). Several giant cells of foreign body-type reaction were seen surrounding these deposits. Additional histological findings in case II were ischemic colitis and acute inflammation. Staining with Crystal Violet showed metachromatic reaction. Tissue sections were Congo-red-positive (Figure 1d) and showed an apple green birefringence under polarized light (Figure 1e), that proved the amyloid nature of the amorphous material seen with H&E staining. The immunohistochemical analysis (Figure 1f) revealed strong immunoreactivity with anti β2M antibodies.

Biochemical Findings

The formalin-fixed paraffin-embedded biopsy specimens (cases I and II) were deparaffinized, rehydrated and subjected to the extraction procedure as described above. In both cases, the SDS-PAGE (Figure 2) and Western blot (Figure 3) analyses of the extracted proteins showed the presence of 12 kDa proteins immunoreactive with anti-β2M antibodies. The immunoreactivity of the 24 kDa band was also observed, probably indicating the dimeric form of this protein. The 5 kDa bands present in crude tissue extracts showed no immunoreactivity with anti-β2M antibodies. None of the extracted proteins reacted with either anti-κ light-chain or anti-λ light-chain antibodies. Amino-terminal amino-acid sequence analysis of the 12 kDa band (case I) revealed a sequence typical of β2M: IQRTPKIQVY (positions 1–10).

Figure 2
figure 2

SDS-PAGE analysis of the amyloid-containing intestinal tissue extracts obtained from formalin-fixed paraffin-embedded diagnostic biopsy specimens in two cases of dialysis-related amyloidosis. Proteins were visualized by Commassie blue staining. (a) Track 1—molecular weight markers, tracks 2 and 3—tissue extract in case I; (b) track 1—molecular weight markers, tracks 2 and 3—tissue extract in case II.

Figure 3
figure 3

SDS-PAGE based Western blot analysis of the amyloid-containing intestinal tissue extracts obtained from formalin-fixed paraffin-embedded diagnostic biopsy specimens in two cases of dialysis-related amyloidosis. Proteins were immunostained with anti-β2M (a. β2M), anti-kappa (a. κ), and anti-lambda (a. λ) light-chain antibodies. Tracks 1, 3 and 5—tissue extract in case I; tracks 2, 4 and 6—tissue extract in case II.

The comparative SDS-PAGE and IEF analysis of the tissue Aβ2M and normal β2M was carried out according to the Scheme in Figure 4. For the purification of these proteins, the samples run on SDS gels were blotted onto PVDF, and the12 kDa proteins were eluted from the membrane (Figures 5 and 6). The electrophoretic mobility of the Aβ2M was close to that of the normal β2M. The purified Aβ2M proteins were subjected to IEF, where the closely spaced bands were revealed with Fast Silver stain (Figure 7). These bands were reactive with anti-β2M antibodies (Figure 8). Although the IEF pattern in case I was different from that in case II, in both cases the Aβ2M protein bands migrated to a more acidic position as compared to the normal β2M.

Figure 4
figure 4

Flowchart of a comparative analysis of the amyloid-containing tissue Aβ2M and normal β2M proteins.

Figure 5
figure 5

SDS-PAGE of the normal β2M (tracks 1–4) and Aβ2M containing tissue extract (tracks 5–9). The electrophoretically run proteins were blotted to PVDF membranes, and the 12 kDa protein bands (in brackets) were excised, pooled and eluted. The purified normal β2M and the tissue Aβ2M were further examined by IEF (Figures 7 and 8).

Figure 6
figure 6

SDS-PAGE of the tissue Aβ2M purified following SDS electrophoresis, blotting to and elution from PVDF membranes. Tracks 1 and 2—purified Aβ2M; tracks 3 and 4—normal β2M.

Figure 7
figure 7

IEF of the tissue Aβ2M (track 1—case II, track 2—case I) and normalβ2M (track 3). Proteins were detected with Fast Silver stain.

Figure 8
figure 8

IEF based western blot analysis of tissue Aβ2M (track 1 – case II, track 2 – case I) and the normal β2M (track 3). Proteins were immunostained using anti-β2M antibodies.

Discussion

Systemic amyloid deposition and visceral organ involvement in dialysis-related amyloidosis are now being reported with increasing frequency;23, 24, 25, 26, 27, 28, 29, 30, 31 however, the data on biochemical features of these amyloid deposits are still lacking. We present here a first report on the biochemical analysis of intestinal amyloid deposits found in two cases of hemodialysis-related amyloidosis. The results of our study showed that the extensive amyloid deposition observed in these cases was of β2M origin, whereas the presence of other amyloid types, such as amyloid A (AA) or immunocyte-derived amyloid proteins (AL), was excluded. In both cases, the molecular mass of the deposited protein was very close to that of normal β2M (12 kDa). None of the lower molecular mass β2M degradation products were found, thus supporting the view that β2M proteolysis is not necessary for fibril formation.4, 8 The extracted amyloid proteins were purified by our recently developed micropreparative SDS-PAGE technique22 and afterwards subjected to IEF analysis, which demonstrated a shift to the more acidic pI values as compared to the normal β2M. These findings support the hypothesis that amyloid deposits contain the acidic β2M isoforms specific to dialysis-related amyloidosis. Further studies are needed to reveal the precise chemical nature of structural alterations responsible for the appearance of the more acidic β2M isoforms in the gastrointestinal amyloid deposits.

It has been shown that the onset, development and severity of the gastrointestinal amyloidosis in hemodialysis patients correlated with the time of dialysis.27 Life-threatening complications such as gastrointestinal hemorrhage and/or intestinal obstruction have been documented in some cases26, 27, 28, 29, 30 and were also observed in the present study. These findings support the view that gastrointestinal amyloidosis should be considered in any patient maintained on hemodialysis for 10 years or more.26, 27 As clinical symptoms may be unreliable indicators of gastrointestinal amyloidosis, examination of biopsy specimens with Congo-red and immunostains becomes critical.27 It should also be noted that besides β2M amyloidosis, deposition of other amyloid types, such as AA and AL, has been documented in several cases of dialysis-related amyloidosis.31, 32, 33, 34 Since the treatment of amyloidosis depends on the chemical type of this disease, the reliable confirmation of the β2M amyloid origin excluding the presence of other amyloid types is imperative in dialysis-related amyloidosis.33 The immunohistochemical amyloid typing methods are used routinely to provide this information, but in some instances, the obtained results can be inconclusive, especially in relation to AL proteins.35 In this respect, our biochemical micromethods15, 16 applied in this study were helpful in identifying unequivocally the chemical nature of the deposited amyloid.

It should be emphasized, however, that biochemical examination of amyloid proteins in the diagnostic biopsy specimens faces two major difficulties: small quantities of the available tissue material and decreased solubility of proteins due to tissue fixation with formalin.36, 37 In fact, previous biochemical studies on Aβ2M proteins were performed using larger amounts of fresh (unfixed) tissues obtained at autopsy5, 8, 12 or at carpal tunnel release operations.4, 10 In these studies, the classical amyloid isolation methods, which include the water extraction method of Pras10, 12, 38 and the guanidine hydrochloride solubilization technique,4, 5, 8 were successfully used, but these methods were found unsuitable for biochemical examination of small fixed biopsy specimens. Recently, we reported a new simple formic acid microextraction technique allowing the recovery and chemical typing of amyloid in small formalin-fixed biopsy specimens.15, 16 Application of this microtechnique in the present study, together with our small-scale amyloid purification method,22 made possible the first biochemical characterization of the intestinal amyloid deposits in dialysis-related amyloidosis. Our study was based on previous findings39, 40 showing that amyloid, in contrast to many other proteins, is not affected by formalin fixation, probably because beta-pleated sheet conformation of amyloid prevents formalin infiltration. In fact, formalin fixation is even thought to be advantageous for the purification of amyloid proteins.40 Thus, it is most likely that the observed shift of Aβ2M proteins to more acidic pI values represents a disease-related condition rather than an artifact caused by tissue fixation with formalin.

Finally, application of our microtechniques was useful in obtaining precise diagnosis of amyloidosis, which is necessary for effective therapy. Usage of these techniques in the analysis of different visceral tissues affected by dialysis-related amyloidosis could provide new important information on the chemical structure of amyloid proteins and contribute to our understanding of the pathogenesis of this devastating disease.