Glycoxidative modification of AA amyloid deposits in renal tissue

Noriko Uesugi1, Noriyuki Sakata1, Ryoji Nagai2, Tadashi Jono2, Seikoh Horiuchi2 and Shigeo Takebayashi1

1 Second Department of Pathology, School of Medicine, Fukuoka University, 7–45–1 Nanakuma, Jonan-ku, Fukuoka 814–0133, Japan and 2 Department of Biochemistry, Kumamoto University School of Medicine, Honjo, 2–2-1, Kumamoto, 860–0166, Japan

Correspondence and offprint requests to: Professor S. Takebayashi, MD, Second Department of Pathology, School of Medicine, Fukuoka University, 7–45–1 Nanakuma, Jonan-ku, Fukuoka, 814–0133, Japan.



   Abstract
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Background. N{varepsilon}-carboxymethyllysine (CML) is a product of the oxidative modification of glycated proteins, which damages proteins with ageing, diabetes, uraemia and Alzheimer's disease. In contrast, pyrraline is one of the advanced glycation end products, which is independent of oxidative processes. CML has been identified in ß-amyloid of Alzheimer's disease and ß2-microglobulin-associated amyloid. We investigated whether CML and pyrraline are formed in AA and AL amyloid of the kidney.

Method. Renal specimens from 19 cases of AA amyloidosis and 14 cases of AL amyloidosis were investigated for immunolocalization of CML, pyrraline, collagen type IV and laminin in amyloid deposits. Renal biopsies of 10 age-matched cases with thin basement membrane disease and normal renal function were used as controls. The fractional areas of amyloid, CML, laminin and collagen IV in glomeruli and interstitium (%amyloid, %CML, %laminin and %collagen, respectively) were calculated using the point counting method. The correlation between these parameters was evaluated using Spearman's rank correlation test.

Results. CML colocalized with AA amyloid, but not AL amyloid, except in two cases of the latter with a long history of nephropathy exceeding 14 years. In contrast, pyrraline was not observed in either type of amyloid. Mean %CML in AA amyloid was significantly higher than %collagen and %laminin in glomeruli and interstitium, indicating that AA amyloid is modified by CML independent of colocalized extracellular matrix. %CML significantly correlated with %amyloid both in glomeruli and interstitium in AA amyloidosis. AL amyloid cases with a long history of nephropathy showed positive staining for CML in glomeruli and interstitium but no staining for collagen IV and laminin in amyloid deposits.

Conclusion. CML modification may occur in amyloid deposits of AA amyloidosis, independent of extracellular matrix components. Glycoxidative modification may have a functional link to AA amyloid deposition in renal tissues.

Keywords: AA amyloid; CML; extracellular matrix; glycoxidative modification; kidney



   Introduction
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
During the normal ageing process, body proteins are progressively and irreversibly modified with advanced glycation end products (AGEs) [1]. AGEs may produce adverse effects on tissues and cells via a variety of mechanisms involving the alteration of tissue protein structure and function [2,3]. Furthermore, the progression of several diseases, including diabetes and atherosclerosis, is influenced by AGE formation [1,4]. AGEs have been identified immunohistochemically in amyloid proteins of senile plaques in patients with Alzheimer's disease and in elderly individuals [5] and also in amyloid fibrils of damaged synovial tissues from patients with haemodialysis-related amyloidosis [6]. Although AGEs might form in AA and AL amyloid proteins, their formation has not been fully investigated [7].

AGE formation commences with a chemical reaction between reducing sugars and amino acids, and after subsequent reactions over time, a heterogeneous group of products, AGEs, are formed. The formation of a subgroup of AGEs, including N{varepsilon}-carboxymethyllysine (CML) and pentosidine, requires both glycation and oxidation, and accordingly these are called `glycoxidation products'. However, the formation of other AGEs, such as pyrraline and imidazolone, is independent of oxidation. Highly reactive carbonyl compounds and free oxygen radicals are produced during the formation of glycoxidation products. These agents induce several biological effects [3] and have been suggested to play an important role in the tissue damage caused by AGE modification [8,9]. However, no studies have examined the oxidative stress caused by AGE modification in AA and AL amyloidosis. To clarify the modification of AGEs in AA and AL amyloid proteins in relation to oxidative stress, we investigated the formation of AGEs, including glycoxidation products, CML, and nonoxidative AGEs, pyrraline, in amyloid deposits using renal tissues from amyloid patients.

Extracellular matrix proteins, including collagen type IV and laminin, are present in amyloid deposits [10,11]. AGE modification of collagen and other extracellular matrix proteins has been reported previously [1,4,8]. Here we also investigated a possible colocalization of collagen type IV and laminin in amyloid deposits to answer the question whether AGE formation in amyloid is related to colocalized extracellular matrix proteins.

We applied morphometric analysis to immunohistochemically stained serial sections of the kidney and quantified the extent of modification by CML and the area of colocalization of extracellular matrix in affected amyloid proteins. With this quantification, we were able to compare the degree of involvement of CML with that of extracellular matrix proteins in amyloid deposits.



   Patients and methods
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Patients
Renal biopsy specimens were obtained from 33 cases of systemic amyloidosis, including 19 patients with AA amyloidosis and 13 patients and one autopsy case of AL amyloidosis. The clinical data from all subjects are summarized in Tables 1 and 2GoGo. In addition, we examined 10 age-matched cases (age range 30–70 years, mean 59±10) with no renal disease and with normal renal function as the control group.


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Table 1. Clinical peofile of AA amyloid subjects
 

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Table 2. Clinical peofile of AL amyloid subjects
 
Routine staining methods
Samples were fixed in 10% buffered formalin and embedded in paraffin. For light-microscopy, sections were stained with haematoxylin–eosin, periodic acid-Schiff, periodic acid-methenamine-silver stain and Congo red with or without pretreatment with potassium permanganate (KMnO4). Frozen samples of AL amyloidosis cases were subjected to a direct immunostaining method using antibody for {lambda}- and {kappa}-light chain (Dako, Glostrup, Denmark). Amyloid deposits were determined by the positive green birefringence under polarized light in Congo red-stained sections. AA amyloid protein was identified by positive staining with both Congo red and anti-AA amyloid antibody. However, AL amyloid protein was determined by positive staining with Congo red, which was resistant to KMnO4 reaction and negative staining with anti-AA amyloid antibody. Ten cases were subclassified to A {lambda}-type and A {kappa}-type (n=8, 2, respectively) according to the positive staining of {lambda}-and {kappa}-light chain, respectively.

Preparation of anti-CML antibody and anti-pyrraline antibody
Preparation of anti-CML antibody (6D12) was as described previously [12]. A recent study from our group showed that monoclonal antibody, 6D12 recognized CML as an epitope among AGE-structures [13]. To prepare monoclonal mouse anti-pyrraline antibody, 6 mg of caproyl pyrraline, synthesized as described previously [12], was conjugated with 6 mg of human serum albumin (HSA). The conjugate was immunized in Balb/c mice and splenic lymphocytes were fused with myeloma P3U1 cells. The hybrid cells were screened and two cell lines positive for both caproyl pyrraline-HSA and caproyl pyrraline-conjugated with keyhole limpet haemocyanin (KLH), but negative for HSA, were selected through successive subcloning. On-line, termed H12, was produced from ascitic fluid of Balb/c mice and futher purified by protein G-affinity chromatography to IgG1. Immunochemical studies showed that the reactivity of H12 to caproyl pyrraline-conjugated HAS was selectively inhibited by caproyl pyrraline, but not by caproic acid, indicating that the epitope of H12 was the pyrraline moiety.

Immunohistochemistry
Paraffin-embedded samples were cut into 3-µm sections and subjected to indirect immunohistochemical staining. The alkaline phosphatase and anti-alkaline phosphatase method (APAAP) was applied for anti-CML, pyrraline, amyloid A and ß2-microglobulin (ß2-M) and labelled avidine-streptavidine (LSAB) method for collagen type IV, laminin and heparan sulfate proteoglycan core protein (HSPG). The following primary antibodies were used, monoclonal mouse anti-CML (6D12, provided by Kumamoto University, Japan) [12], mouse anti-pyrraline (H12, provided by Kumamoto University), mouse anti-amyloid AA protein (Dako), mouse anti-HSPG (Chemicon International Inc., Temecula, CA, USA) antibodies and polyclonal rabbit anti-ß2-M (Dako), mouse anti-collagen type IV (Shiseido Co. Japan) and rabbit anti-laminin (LSL, USA) antibodies. Sections were subjected to the following protease pretreatment: 5 mg/dl of pronase (Dako) for 30 min at 37°C for CML, 0.5 mg/dl of pronase for 20 min at room temperature for amyloid AA, 0.5 mg/dl of pronase for 30 min at 37°C for pyrraline, 2.5 mg/dl of pronase for 20 min at 37°C for ß2-M, and 0.05 mg/dl of pronase for 10 min at room temperature, followed by 1% pepsin for 90 min at 37°C for laminin, collagen type IV and HSPG. After treatment with 4% skim milk for 20 min at room temperature, sections were incubated overnight with various primary antibodies (other than anti-CML and anti-pyrraline antibodies) at 4°C. CML and pyrraline were examined by incubation with anti-CML and anti-pyrraline antibodies overnight at room temperature, respectively. Each primary antibody was used at the following dilution: CML, 1:750; pyrraline, 1:500; AA amyloid, 1:100; ß2-M, 1:500; laminin, 1:2000; collagen type IV, 1:1000; and HSPG, 1:25. After washing with phosphate-buffered saline (PBS), sections were incubated with the secondary antibody for 60 min at room temperature. The sections were then incubated with alkaline phosphatase-conjugated streptavidine and monoclonal alkaline phosphatase–anti-alkaline phosphatase complex (Dako) for 60 min at room temperature for LSAB and APAAP methods, respectively. All immunostained sections were visualized by reaction with a solution of new fuchsin (naphthol AS-BI phosphate). Couterstaining was performed using Mayer's haematoxylin.

Morphometry
To evaluate the degree of staining for various antigens in each section, morphometric analysis was performed using the point counting method of Mackensen et al. [14]. The examiner was blinded to the clinical background. The section was examined using a 100-point grid placed in the 10x ocular of a light microscope. We selected several glomeruli observed in suitable aspect, such as those wihout obvious peripheral cut planes, global sclerosis and severe shrinkage, using 40x objective and randomly chose several fields with amyloid protein-positive interstitium, excluding large vessels, using a 10x objective. Points falling on stained area (Pp) and points falling on non-stained area (Pn) were counted for each glomerulus and interstitium. The area, which was immunohistochemically positive for AA amyloid protein, was classified as the AA-amyloid positive area. In contrast, the Congo red-positive and AA-amyloid negative area was classified as the AL-amyloid positive area for AL amyloidosis. The volume fraction of amyloid-protein positive area (%amyloid) was calculated as (Pp/Pp+Pn)x100. Pp for CML, laminin and collagen type IV were counted in amyloid-positive sites in the glomeruli and interstitia. The volume fractions of areas positive for CML, laminin and collagen type IV (%CML, %laminin, %collagen) were calculated separately using the above formula. The relative %CML, %laminin and %collagen were defined as a percentage of Pp of CML, laminin, collagen type IV to Pp of amyloid, respectively. Approximately, 43 and 65 points were counted per each glomerulus and interstitium, respectively. Moreover, the average of these parameters per subject was calculated.

Statistical analysis
The correlation between %amyloid and %CML, %laminin and %collagen was examined in each glomerulus and interstitium or in each subject using Spearman's rank correlation test. The correlation between %amyloid and various clinical parameters, including the duration of underlying disease, that of nephropathy, 1/serum creatinine and the degree of proteinuria, was also determined by Spearman's rank correlation test. Mann–Whitney's U-test was applied to evaluate differences in %amyloid, %CML, %laminin and %collagen between AA and AL amyloidosis. A P-value of less than 0.05 was taken as the level of significance.



   Results
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Clinical profile
The clinical features of patients at renal biopsy are shown in Tables 1 and 2GoGo. The average age and sex distributions were not different between patients with AA and AL amyloidosis. The average duration of the underlying disease in AA amyloidosis was longer than in AL amyloidosis (P<0.001). The duration of nephropathy, defined by urinary abnormalities or nephrotic syndrome, was not different between AA and AL amyloidosis. Nephrotic syndrome was observed less frequently in AA (47%) than in AL amyloidosis (85%). AA amyloidosis showed a higher incidence of renal dysfunction (49%) than AL amyloidosis (19%), which was defined as blood urea nitrogen (BUN) >30 mg/dl, serum creatinine >1.5 mg/dl or creatinine clearance <50 ml/min.

Two cases of primary AL amyloidosis (cases 13 and 14) had a longer duration of disease and nephropathy than other AL amyloid cases. Case 13 had had proteinuria for 18 years, but showed no other complications. Case 14, the autopsy case, was diagnosed as primary AL amyloidosis with plasma cell dysclasia and nephrotic syndrome at age 63 years. She had undergone haemodialysis for 14 years.

Amyloid deposition
The morphological distribution of glomerular amyloid deposition was classified into four types, including mesangial–nodular, mesangial–capillary, perimembranous and hilar patterns, according to Shiiki et al. [15]. In AA amyloidosis, glomerular amyloid distribution was more frequently of the mesangial nodular pattern (13 cases, 72%), than mesangial-capillary (three cases, 17%) and hilar patterns (two cases, 10%). One case of AA amyloidois had no glomeruli in the specimen. However, in AL amyloidosis, mesangial–capillary distribution was more frequently observed (11 cases, 79%) than mesangial nodular (two cases, 14%) and hilar patterns (one case, 7%). Two cases of AL amyloidosis with a long history of nephropathy showed a mesangial nodular amyloid deposition pattern.

As shown in Table 3Go, %amyloid in glomeruli was not different between AA and AL amyloidosis. However, AA amyloidosis showed a significantly higher %amyloid in the interstitium than AL amyloidosis. The two cases of AL amyloidosis with long history of nephropathy showed a significantly higher amyloid deposition per glomeruli and per interstitium compared with other cases of AL amyloidosis (P=0.001, Table 3Go, right).


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Table 3. Volume fractional area of amyloid, CML, laminin and collagen type IV per glomerulus and interstitium and per subject
 
AGE deposition
CML colocalized with AA amyloid proteins in glomeruli (Figure 1A and BGo) and interstitium (Figure 2A and BGo), while it was scarcely present in AL amyloid positive areas (Figure 3AGo) except for the two cases with a long history of nephropathy (Figure 3DGo). As shown in Table 3Go, the level of relative %CML was high in glomeruli and interstitium positive for AA amyloid protein, suggesting that a significant amount of AA amyloid protein may be modified by CML. Although the mean relative %CML of AL amyloidosis in cases with a long history of nephropathy was similar to that of AA amyloid patients, it was significantly lower in cases with nephropathy of <4 years than in AA amyloid patients. Pyrraline was not detected in any site of amyloid deposits in AA and AL amyloidosis. Apart from areas with amyloid deposits, positive staining for CML was noted in the proximal tubular epithelium and sclerotic arteries in both AA and AL amyloidosis. In comparison, pyrraline was found in the interstitium and fibrous intima of arteries but not in the proximal tubular epithelium.



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Fig. 1. Immunohistochemical localization of AA amyloid protein (A), CML (B) and collagen type IV (C) in the glomerulus. Sections were obtained from the same glomerulus of AA amyloid case (case 5, Table 1Go). The glomerulus showed mesangial-nodular distribution of AA amyloid protein. Note the abundant and intense staining for CML in the AA-amyloid-positive area. CML was also found in the proximal tubular epithelium. In contrast, collagen type IV is faintly stained in AA amyloid positive area (arrow). A–C x400.

 


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Fig. 2. Immunohistochemical localization of AA amyloid protein (A), CML (B), pyrraline (C) and laminin (D) in the interstitium. Sections were obtained from the same glomerulus of AA amyloid case (case 19, Table 1Go). Note the marked colocalization of CML with AA amyloid protein. In contrast, pyrraline was almost negligible in AA amyloid protein. Laminin was partially observed within interstitial AA amyloid deposits (arrow). A–D x150.

 


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Fig. 3. Comparative analysis of localization of AGEs and laminin in AL amyloid cases with a short or long history of nephropathy. (A–C) were obtained from an AL amyloid patient with nephropathy of 1-year duration (case 11, Table 2Go). (D–F) were from a patient with primary AL amyloidosis associated with nephropathy for more than 14 years (case 13). The glomerulus of case 11 showed mesangial–capillary amyloid distribution and no staining for CML (A), pyrraline (B) or collagen type IV (C). In contrast, the glomerulus of case 13 showed mesangial–nodular amyloid distribution. CML (D) was intensely distributed in Congo red positive areas, but pyrraline (E) and collagen type IV (F) were negative in amyloid protein. A–F x400.

 
For control cases, CML was positive in the epithelium of proximal tubules and intima of artery in all cases. In contast, pyralline was negative in tubular epithelium but positive in interstitium and in fibrous intima. Neither CML nor pyrraline were detected in glomeruli.

Extracellular matrix components in amyloid deposits
Laminin and collagen type IV were detected in some AA-amyloid positive glomerular areas (Figure 1CGo, arrow) and interstitium (Figure 2DGo, arrow). As shown in Table 3Go, %laminin and %collagen of AA amyloidosis were less than %CML in the glomeruli and interstitium. The relative %laminin and %collagen were significantly greater in the interstitium than in glomeruli, suggesting that laminin and collagen type IV are probably more abundant in interstitial amyloid deposits than in glomeruli in AA amyloidosis. HSPG showed focal deposits within amyloid stained areas of the interstitium in some cases of AA amyloidosis. In contrast, laminin, collagen type IV and HSPG were scarcely seen in amyloid positive areas of glomeruli and interstitium in all cases of AL amyloidosis (Figure 3C and FGo, Tables 3 and 4GoGo).


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Table 4. Relative volume fractional area of amyloid, CML, laminin and collagen type IV within amyloid deposition per glomerulus and interstitium and per subject
 
Correlation between %amyloid, %CML, %laminin and %collagen
In both glomeruli and interstitia of AA amyloidosis, %amyloid correlated significantly with %CML, %laminin and %collagen, as shown in Figures 4–6GoGoGo. However, the correlation coefficient between %amyloid and %CML was significantly higher in both glomeruli and interstitium than that between %amyloid and %laminin or %collagen. Furthermore, %amyloid correlated significantly with %CML in the glomeruli and interstitia of the two cases of AL amyloidosis with a long history of nephropathy (Figure 7Go).



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Fig. 4. Correlation between %amyloid and %CML in glomeruli (A) and interstitium (B) of AA amyloidosis. %Amyloid correlated significantly with %CML in both glomeruli and interstitium.

 


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Fig. 5. Correlation between %amyloid and %laminin in glomeruli (A) and interstitium (B) of AA amyloidosis. %Amyloid correlated significantly with %laminin in both glomeruli and interstitium.

 


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Fig. 6. Correlation between %amyloid and %collagen in glomeruli (A) and interstitium (B) of AA amyloidosis. Positive correlation was observed between %amyloid and %collagen both in glomeruli and interstitium.

 


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Fig. 7. Correlation between %amyloid and %CML in glomeruli (A) and interstitium (B) in patients with AL amyloidosis associated with nephropathy for more than 14 years. Positive correlation was observed between %amyloid and %CML both in glomeruli and interstitium.

 
Correlation between clinical parameters, %amyloid and %CML
In both glomeruli and interstitium, there was no correlation between %amyloid or %CML and various clinical parameters, including duration of nephropathy, that of underlying disease and 1/serum creatinine. %Amyloid and %CML in glomeruli tended to correlate with the degree of proteinuria, but the correlation was not statistically significant (Table 5Go, rs=0.395, P=0.13 and rs=0.407, P=0.118).


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Table 5. Correlation between clinical parameters and %amyloid and %CML in AA amyloidosis
 


   Discussion
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Our study provided strong evidence for the presence of CML-modified amyloid protein in the renal tissues of patients with AA amyloidosis. CML has been identified immunohistochemically in ß-amyloid protein of senile plaques in patients with Alzheimer's disease and in elderly individuals [5]. CML-modified ß2-M has been detected in amyloid fibril from damaged synovial tissue of patients with haemodialysis-related amyloidosis [6]. However, the formation of CML in AA and AL amyloid proteins has not been investigated fully. Previous studies using immunohistochemical analysis showed no staining of CML in renal tissues in either experimental murine AA amyloidosis [7] or human autopsy material of AA and AL amyloidosis [16]. The different results of CML staining in AA amyloid deposits seem to be due to different methods used for the pretreatment of paraffin-embedded sections. In a series of preliminary studies, we obtained partial positive staining for CML in AA amyloid deposits in only a limited number of cases, using sections pretreated with 100% formic acid. However, the technique used in the present study showed that pretreatment with high concentrations of pronase (5 µg/ml) for 30 min in 37°C was optimal for immunohistochemical detection of CML in AA amyloid deposits in renal tissue. Furthermore, pretreatment of renal tissue with this enzyme allowed the detection of CML in the proximal tubular epithelium and fibrous intima of arteries, a finding consistent with the results reported by Horie et al. [17]. Negative controls, using nonimmunized serum and PBS instead of the primary antibody showed negative staining in the AA-amyloid positive area. These results suggest that the immunohistochemical staining using this pretreatment with pronase is specific for CML. Tissue-deposited amyloid proteins are shown to be resistant to several proteases [18]. AGE formation is known to cause protease-resistant cross-linking of peptides [8,9]. Thus, the use of high concentrations of pronase may be necessary to express CML epitope in deposited AA amyloid protein.

Previous studies have shown immunohistochemical staining of CML in ß-amyloid [5] and ß2-M-associated amyloid proteins [6]. However, none of these studies provided a quantitative analysis of the degree of CML formation in tissue-deposited amyloid proteins. Our study demonstrated that amyloid deposits positive for CML were present both in glomeruli and interstitium in all cases of AA amyloidosis. In addition, morphometric analysis indicated that ~80% of AA-amyloid positive areas exhibited colocalization of CML in the glomeruli and interstitium. A significant correlation was also observed between %CML and %amyloid in glomeruli and interstitium in AA amyloidosis. These findings suggest that the formation of CML in AA amyloid protein may have a functional link to deposition of AA amyloidosis.

AGE formation starts with the reaction of amino acids, particularly the side chain of lysine, arginine and histidine, with reducing sugars. This process leads via reversible Schiff's base adducts to protein-bound Amadori products. Through subsequent rearrangement, dehydration and oxidation, a heterogeneous group of products, AGEs, are formed over time. AGEs are considered to be formed by a chemical reaction dependent or independent of oxidation [9,10]. CML is produced by an oxidation-dependent pathway and is thus termed a glycoxidation product. In contrast, the formation of pyrraline and imidazolone is independent on oxidation (nonoxidative AGEs). Both CML and imidazolone [19] have been detected in ß2-M-associated amyloid deposits. However, it is unclear whether glucoxidative products and nonoxidative AGEs are formed in AA amyloid deposits. In our study, immunohistochemical analysis showed that CML, a glycoxidative product, was formed in amyloid deposits in all cases of AA amyloidosis. However, pyrraline, one of the nonoxidative AGEs, was negative in these cases. In contrast, in most cases of AL amyloidosis, AL amyloid deposits did not react with the antibody against CML or with that against pyrraline. These results suggest that accumulation of AGEs in AA amyloid is closely related to accelerated oxidation of glycated proteins (glycoxidation). Recent studies have focused on the formation of highly reactive carbonyl compounds and oxygen free radicals during the oxidation pathway of AGE formation [8,9]. These products may contribute to the destruction of bone and synovium in patients with in ß2-M-associated amyloidosis [6]. Since CML modification was found in AA amyloid deposits in renal tissue, agents such as carbonyl compounds and free oxygen radicals might be involved in the irreversible deposition of amyloid protein and tissue damage in patients with AA amyloidosis.

In this study, CML was not detected in AL amyloid except in two cases with a history of nephropathy exceeding 14 years. Foss et al. [20] recently identified the amino acid sequence of the {kappa}-light chain extracted from Bence Jones proteins of urine and tissue-deposited light chain, and found that Asn20 was glycosylated in both proteins. This suggests that AGE modification may occur in AL amyloid proteins. The formation of AGE requires long-term incubation. In fact, only a negligible amount of CML has been identified in ß2-M-associated amyloid of the synovial tissue in patients who underwent haemodialysis for less than 6 years [16]. In the present study, the clinical course of patients with AA amyloidosis, who showed CML formation in amyloid deposits, was significantly longer than that of AL amyloidosis with no formation of CML (17±10 vs 0.9±1.0 years). In contrast, two cases of AL amyloidosis with nephropathy of >14 years showed CML formation in deposited AL amyloid. Thus, CML may require long incubation to form in amyloid protein and can occur in AL amyloid protein after long-term incubation.

Extracellular matrix, including collagen type IV and laminin, has been shown in AA-amyloid positive areas [10,11]. Our study demonstrated colocalization of collagen type IV and laminin in AA-amyloid positive-areas of glomeruli and interstitium. Moreover, there was a positive correlation between %collagen, %laminin and %amyloid in AA amyloidosis. These results suggest a close link between extracellular matrix formation and AA amyloid deposition, a finding consistent with the results of two separate studies [10,11]. Because these extracellular matrix proteins are known to be susceptible to nonenzymatic glycation [8], CML-positive areas of AA amyloid deposits may result from CML modification of matrix proteins. However, the mean values of %laminin and %collagen were lower than that of %CML in AA amyloidosis. Immunohistochemistry showed the presence of CML in amyloid deposits negative for collagen type IV and laminin in AA amyloidosis. Moreover, in two cases of AL amyloidosis with nephropathy of more than 14 years, AL amyloid deposits were positive for CML but negative for collagen type IV and laminin. Combined together, these results suggest that CML modification may occur in amyloid proteins per se, rather than in extracellular matrix components.

In conclusion, our study demonstrates for the first time the formation of CML, a glycoxidation product, in AA amyloid deposits. By contrast, pyrraline, a non-oxidative AGE, could not be detected in either AA or AL amyloidosis. Whether glycoxidative modification is involved in the deposition of AA amyloid in renal tissue or is only a secondary phenomenon remains to be seen.



   References
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

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Received for publication: 29. 4.99
Accepted in revised form: 19.10.99