1 Kidney Center, Saitama Social Insurance Hospital and 2 Department of Internal Medicine, School of Medicine, Keio University, Japan
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Abstract |
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Methods. Peritoneal tissue was obtained by biopsy or at autopsy from 18 CAPD patients (median duration, 122 months), 5 control haemodialysis (HD) patients, and 3 pre-CAPD patients. The distribution of calcium deposits and OPN protein was determined by von Kossa staining and immunohistochemistry, respectively. Smooth muscle cells and macrophages were identified with anti- smooth muscle actin (
-SMA) and anti-CD68 antibodies.
Results. Calcium deposits with various configurations were observed in specimens from 12 of the 18 CAPD patients. They included massive calcification facing the peritoneal cavity, scattered granular or crystalloid deposits in the submesothelial stroma, and oval-shaped deposits formed within hyalinized vasa. Most were present in highly sclerosed areas and accompanied by extracellular OPN precipitation. Cytoplasmic OPN was detected in infiltrating leukocytes, granulation tissue cells, fibroblast-like cells and mast cells. Computerized tomography examination also detected peritoneal calcification in seven of the CAPD patients. No calcium deposits or OPN staining was detected in control specimens.
Conclusions. The results of our study suggest that microscopic peritoneal calcification is frequent in patients on CAPD for more than 10 years. Myofibroblast infiltration, OPN expression, calcium deposition, and associated OPN precipitation seem to be components of the peritoneal changes in such patients.
Keywords: calcification; continuous ambulatory peritoneal dialysis; fibrosis; myofibroblasts; osteopontin; peritoneum
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Introduction |
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While the mechanism of dystrophic calcification in general remains unknown, it is now clear that dystrophic calcification has features in common with normal bone formation. Calcified areas contain abundant type I collagen and various non-collagenous bone matrix proteins, such as bone morphogenic protein type 2, osteocalcin, matrix Gla protein and osteopontin (OPN). Osteopontin is a secreted calcium-binding acidic phosphoprotein and it has recently been shown to have diverse biological functions. Several studies on atherosclerotic lesions have revealed a close association between calcium deposition and local OPN gene expression, and have suggested that it plays an important role in the pathogenesis of atherosclerosis [3].
In this study, we examined the morphology and prevalence of calcium deposits in peritoneal tissue obtained from patients on long-term CAPD therapy. To explore the possible contribution of OPN to peritoneal calcification, we investigated concurrently the distribution of the OPN deposition and of OPN-producing cells.
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Subjects and methods |
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Histological methods
Specimens were fixed in 10% formaldehyde and embedded in paraffin, and 4-µm sections were stained with haematoxylin and eosin and MalloryAzan stain. To demonstrate calcium deposits and OPN protein, adjacent sections were used for von Kossa staining and immunohistochemistry.
For immunodetection of OPN, rehydrated sections were pre-treated with 0.25 M EDTA in PBS, pH 7.4, for 10 min [4], and then incubated with 5% BSA containing TBS for 30 min at room temperature. Monoclonal antibody 10A16 (4 µg/ml, IBL, Japan) or MPIII101 (2.8 µg/ml, Developmental Studies Hybridoma Bank, Iowa University) was used as the primary antibody. After overnight incubation at 4°C, the antibody was localized as a red colour by using an alkaline-phosphatase-labelled secondary antibody (Envision labelled polymer/AP, Dako, USA), levamisole, and fuchsin substrate-chromogen (Dako, USA) according to manufacturer's instructions. In a preliminary experiment, both monoclonal antibodies yielded similar staining patterns on EDTA-treated sections from a heavily calcified peritoneal specimen. All specimens were immunostained with antibody 10A16 and selected specimens were also stained with antibody MPIII101 to confirm the results.
The OPN-stained sections or adjacent sections of some specimens were immunostained with other antibodies to discriminate the cell types expressing OPN. Smooth muscle cells (SMCs) were identified with a peroxidase-labelled monoclonal antibody to human -SMA (EPOS anti-human
-smooth muscle actin/HRP, Dako, Denmark), and macrophages were identified with a monoclonal antibody to CD68 (EPOS anti-human CD68/HRP, Dako, Denmark). Addition of the enzyme substrate 3,3'-diaminobenzidine yielded a dark-brown reaction product. Finally, the sections were counterstained with nuclear fast red for von Kossa staining or with Meyer's haematoxylin for immunostaining.
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Results |
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Calcium deposits
While massive calcifications were easily distinguished by regular haematoxylin and eosin staining, most calcium deposits were small and sporadic and could be identified only by careful high power examination of sections stained by von Kossa's method. Among the 19 biopsy/autopsy specimens from 18 CAPD patients, calcium deposits having various configurations were observed in those from 11 patients (Table 3). The most commonly observed calcium deposits were located on the surface of the peritoneal tissues (Figures 1A
and 1G
) and in the submesothelial stroma (Figures 1B
, 1C
and 1H
). As the mesothelial cell layer was absent from the specimens, the distinction between the former and the latter was sometimes ambiguous. The calcifications were extremely diverse in size and shape. Massive amorphous deposits were conspicuous in the SEP cases (Figure 1A
). Calcifications clearly associated with the collagenous matrix were noted in 3 specimens (Figure 1G
). Some deposits were granular (not shown), sandy (Figure 1C
) or crystalline (Figures 1B
and 1H
). Another type of calcification had a characteristic oval or round shape (Figures 1D
and 1E
) and the oval calcifications were surrounded by highly hyalinized blood vessels that sometimes showed partial calcification (Figures 1D
and 1E
), suggesting that the oval calcifications were the terminal form of calcified vessels. Scattered perivascular granular deposits (Figure 1I
) and sandy deposits within vessel walls (Figure 1F
) and visceral smooth muscle (Figure 1J
) were also found in a few specimens. Each of the forms of calcification predominated in hypocellular regions. To summarize the findings, the calcium deposits occurred as (a) various sized calcifications facing the peritoneal cavity (8 out of 18), (b) scattered small granular or needle-shaped calcifications in submesothelial fibrous tissue (10/18), (c) oval-shaped deposits formed from hyalinized vessels (4/18), (d) scattered granules surrounding blood vessels (2/18), (e) fine granules in vessel walls (5/18) and (f) dispersed powdery deposits within visceral smooth muscle (1/18). As shown in Table 3
, calcification was observed more frequently in peritoneal specimens from patients maintained for a longer time CAPD therapy. Re-evaluation of CT images taken around the time of catheter removal surgery also revealed the rather frequent presence of small and sometimes indistinct calcifications along the intestinal loop in patients on long-term CAPD. No surface or submesothelial calcifications were detected in the autopsy specimens of 5 HD patients or the biopsy specimens of 3 pre-CAPD patients.
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Extracellular osteopontin deposition
The immunohistochemical study revealed OPN antigenicity both within cells and extracellularly. In most instances, the extracellular OPN staining corresponded to the sites that stained positive for calcium by von Kossa's method. Figure 2 shows OPN staining of the sections adjacent to those shown in Figure 1
(Figures 1A
F
correspond to Figures 2A
F
). Large calcium deposits were always accompanied by strong OPN staining and it was particularly prominent at the calcification front, but occasionally there was no clear co-localization of OPN with smaller calcium deposits. For example, the calcifications shown in Figures 1C
, 1H
and 1I
were not accompanied by corresponding OPN staining. There were also a few sections on which the superficial peritoneal stroma specifically stained with anti-OPN antibody in the absence of adjacent calcium deposition (figures not shown). In all, we observed some extracellular OPN deposition in the tissue sections from 11 CAPD patients (Table 3
).
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Cellular osteopontin staining
In addition to its extracellular localization, OPN protein was detected in the cytoplasm of several cell types. Four of the specimens examined exhibited obvious leukocyte infiltration, which was probably caused by abdominal infection. As shown in Figure 3A, and also in Figure 2A
, infiltrating neutrophils and some mononuclear cells stained strongly for OPN. Areas of dense inflammatory cell infiltration often exhibited extracellular OPN staining unassociated with calcium deposits, probably reflecting deposition of secreted OPN in the surrounding matrix. Six biopsy specimens contained granulation tissue that stained red with Azan stain and presumably had developed in response to physical contact with the peritoneal catheter. The cells in the granulation tissue were mostly OPN-positive (Figure 3B
), but they showed variable immunostaining for macrophages (CD68) and
-SMA. Small numbers of macrophages were detected by CD68 immunostaining in the majority of specimens and macrophages in some areas expressed OPN. One specimen from a patient who had experienced severe peritonitis showed heavy macrophage infiltration. Serial sections from the specimen were double-stained with OPN (red) and either CD68 or
-SMA (brown) (Figures 3C
and 3D
). Because the OPN signal was overlaid by the CD68 signal, but distinct from the
-SMA signal, as shown in Figures 3C
and 3D
, most OPN-positive cells in this region appeared to be macrophages. In addition to macrophages, many peritoneal specimens contained dispersed OPN-positive fibroblast-like cells, and the majority of them also stained for
-SMA with variable intensity (Figures 3E
and 3F
). Such cellular OPN staining in macrophages and fibroblast-like cells was also observed in the specimens from patients without active peritonitis. A few unidentified cells in small vessels (Figure 3H
) and microvascular endothelial cells in selected areas (Figure 3G
) also appeared to be OPN-positive. Many mast cells were present in the submesothelial stroma of the visceral peritoneum, and specific OPN immunoreactivity was detected in a subset of mast cells in 2 of the 3 autopsy specimens from CAPD patients (Figures 2E
and 3G
). Cellular OPN was mostly detectable in the tissue preparations that exhibited extracellular OPN deposition and corresponding advanced sclerotic changes (Table 3
). However, there was no clear topological association between calcium/OPN deposition and cellular OPN expression within a tissue preparation, because highly sclerosed lesions are often devoid of living cells. The control peritoneal tissue of 5 HD and 3 pre-CAPD patients did not contain any OPN-positive leukocytes, smooth muscle cells, fibroblasts or endothelial cells.
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Discussion |
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Judging from the CT scan images, microscopic calcification should be more frequent in the visceral peritoneum than in the parietal peritoneum, although biopsy specimens can only be taken from the parietal peritoneum. The sections are small and usually stained with haematoxylin and eosin, and small areas of calcification or incomplete calcification in such sections are difficult to detect unless a special stain for calcium is used. Because of the high rate of peritonitis in the past, patients used to be maintained on CAPD for shorter periods. These circumstances might explain the underestimation of the occurrence of microscopic peritoneal calcifications in CAPD.
As shown in Table 3, CT-detectable peritoneal calcification was present in 7 of our 16 biopsy cases with a clear relation to the duration of CAPD therapy. Among the nine biopsy cases without CT-detectable calcification, calcium deposits were demonstrated by von Kossa staining in four. However, in one case (patient 18), histological examination of the biopsied parietal peritoneum failed to reveal calcium deposits despite their presence in the visceral peritoneum. These results show the high sensitivity of histological examination, but also seem to indicate the limitations of histological surveys of small biopsy specimens.
As the number of our control cases was limited, the absence of peritoneal calcification in HD and pre-CAPD patients was not fully substantiated. However, the high incidence of peritoneal calcification demonstrated by CT or histological examination in our CAPD patients was quite a contrast. Even if taking into account the selection bias in the biopsy examinations, our results indicate that sub-clinical peritoneal calcification often develops after extended CAPD therapy for more than 10 years. Consistent with our results, Araki et al. [6] reported prevalent CT-detected peritoneal calcifications in paediatric patients on long-term PD. As microscopic peritoneal calcification was mostly detected in highly degenerated area, it may represent an advanced pathological change following fibrosis. Our cases 3 and 5 are noteworthy for the presence of microscopic calcification in spite of a relatively short duration of therapy. Both had peritonitis at CAPD withdrawal, although its relevance to the calcification is not clear.
The majority of the CAPD patients in this study had high PTH values and were classified in the high or high average PET category. However, the incidence of peritoneal calcification was not associated clearly with total number of peritonitis episodes, extent of ultrafiltration failure, PET category or the dosage of supplemental calcium and vitamin D (Tables 13
). Other biochemical parameters, including serum levels of calcium, phosphorus, the calciumxphosphorus product, albumin and PTH, did not differ between patient groups with and without peritoneal calcification (Table 2
). Other ectopic calcifications, except of arteries and the kidneys, were rare and they appeared not to be associated with peritoneal calcification (Table 3
).
Ahmed et al. [7] have recently reported a clinical and histological study on calcific uraemic arteriolopathy. As in our study, they observed OPN immunostaining and decreased -SMA staining in calcified vessels. They, however, demonstrated higher serum phosphorus values and calciumxphosphorus products in such patients. Similar results have been published in regard to dialysis patients with cardiac valve calcification. The absence of any clear association with such biochemical parameters of peritoneal calcification might be a reflection of the small number of subjects and having a variety of backgrounds. Or, it may suggest that peritoneal injury caused by CAPD itself has a far greater impact on peritoneal calcification than systemic metabolic deviation.
Dystrophic calcification is generally thought to be associated with inflammation, infection or tissue necrosis, although its exact cause is still unknown [8]. An extremely varied conformation of peritoneal calcifications implies the contribution of multiple factors and several potential mechanisms can be considered. A high free calcium ion concentration in the dialysate and the high serum phosphate levels of CAPD patients may play a part in hydroxyapatite formation in the peritoneum. Several natural inhibitors of hydroxyapatite formation, such as phosphocitrate, citrate, pyrophosphate and anionic polypeptides, may be lost in drained dialysate. CAPD therapy with acidic dialysate has been reported to impair macrophage function and it, therefore, may decrease macrophage removal of hydroxyapatite microcrystals by phagocytosis [9]. Degenerated matrix proteins or deposited nonphysiological proteins, such as advanced glycation endproducts and fibrin, may have increased affinity for hydroxyapatite or serve as cores for initial hydroxyapatite crystal formation, as argued in regard to calcification of aldehyde-fixed prosthetic heart valves [8]. Acidic dialysate can release ferric ions from transferrin and may cause peritoneal iron deposition and tissue injury (our unpublished observation). Iron deposition may cause calcification, because a previous study has shown that intraperitoneal injection of iron solution can induce calcification in experimental animals [10].
The OPN molecule contains a polyaspartic acid sequence that mediates hydroxyapatite binding, an arginineglycineaspartate (RGD) cell binding motif, a calcium binding site and two heparin binding sites [11]. Because of possessing these amino acid sequences, OPN has been reported to promote adhesion of vascular endothelial cells and smooth muscle cells, and to be a potent chemotactic factor for macrophages, T cells and smooth muscle cells. It also seems to facilitate bone resorption through its binding to the vß3 integrin on the surface of osteoclasts.
Osteopontin synthesis has been demonstrated in vivo in osteoblasts, osteoclasts and various epithelial cells. Osteopontin is also expressed in T cells, macrophages and polymorphonuclear cells at sites of inflammation. In addition, smooth muscle cells with the synthetic phenotype in arterial intimal lesions have been reported to synthesize OPN as well as fibronectin, collagen and tenascin, etc. Osteopontin is regarded as a good marker of the smooth muscle cell phenotypic transition. In vitro experiments have revealed that angiotensin II, basic fibroblast growth factor (bFGF), transforming growth factor ß (TGFß), platelet-derived growth factor (PDGF), high glucose medium, etc., stimulate OPN expression by cultured vascular smooth muscle cells or cardiac fibroblasts [1113]. Studies on several injury models have suggested that transient expression of OPN by macrophages or other cells might represent a generalized response to tissue injury [9,14]. Consistent with the results of such studies, we observed strong cytoplasmic OPN staining in infiltrating macrophages and neutrophils, stellate fibroblast-like cells, and even in mast cells, in the peritoneal tissue of CAPD patients, but never of control HD/pre-CAPD patients. Some of the specimens from the patients without episodes of peritonitis also exhibited OPN-positive fibroblastic cells, suggesting that the pathological changes and OPN expression may occur independent of bacterial infection in CAPD patients.
It is noteworthy that most OPN-positive fibroblast-like cells also expressed -SMA. Expression of
-SMA by fibroblast-like cells is regarded as a feature of myofibroblasts or activated myofibroblasts (reviewed in [15] and [16]). Their origin is assumed to be resident fibroblasts, smooth muscle cells or progenitor stem cells, and amplified
-SMA expression in response to TGFß, PDGF, angiotensin II, etc. has been reported. Giachelli et al. [11] observed OPN/
-SMA double-positive cells in human coronary atherosclerotic plaques as well as in rat arterial neointima formed following balloon-injury. Although the authors did not identify these cells as myofibroblasts, they clearly have a myofibroblast phenotype. In fact, several recent studies have indicated a contribution to neointima formation by adventitial myofibroblasts, possibly of non-muscle cell origin [15]. Since common mediators, i.e. TGFß, PDGF and angiotensin II, can stimulate
-SMA as well as OPN expression, OPN can be used as an additional marker of myofibroblasts or at least of a subset of myofibroblasts. Importantly, no OPN expression was observed in morphologically intact vascular or visceral smooth muscle, except in a few unidentified cells present in small vessels (Figure 3H
). Therefore, combined immunodetection of OPN and
-SMA may have the advantage of distinguishing myofibroblasts from smooth muscle cells.
There is some controversy as to the role of OPN in calcification. Osteopontin expression has been demonstrated in the granulomas of tuberculosis and sarcoidosis, diseases in which tissue calcification often develops. Studies on atherosclerosis have demonstrated local OPN expression by macrophages and SMC-derived form cells in close association with atheromatous calcification [3]. In low-density lipoprotein receptor-deficient mice, a high-fat diet has been found to induce aortic calcification along with expression of osteoblast transcription factors, Msx2 and Msx1, and of OPN by periaortic adventitial cells, suggesting acquisition of osteogenic potential by these cells [17]. While these findings support a positive contribution of OPN to dystrophic calcification, other studies have shown it to possess protective activity against tissue calcification. Osteopontin inhibits hydroxyapatite crystal growth in calcium phosphate solution [18] and phosphorylated OPN inhibits hydroxyapatite formation in aortic smooth muscle culture [19]. An ultrastructural immunohistochemical study on wound healing in mineralized tissues showed both OPN secretion and phagocytosis of OPN-coated calcified debris by macrophages [9], suggesting that OPN can act as an opsonin and facilitate removal of hydroxyapatite. Expression of OPN may therefore represent an adaptive mechanism to limit calcification.
As already stated, extracellular OPN protein was found to be mostly co-localized with calcium deposits. Large calcium deposits, in particular, were always accompanied by solid OPN immunostaining that was most prominent on their surface. Some small calcium deposits in a few regions were unaccompanied by OPN staining. Since calcification is often observed in sclerosed hypocellular areas with little OPN expression and mineralizing tissues can take up serum OPN [20], such OPN staining may reflect passive adsorption of OPN onto the precipitated hydroxyapatite rather than an active contribution by locally expressed OPN to calcification. On the other hand, OPN deposition unassociated with calcification was observed on some peritoneal surfaces and areas of dense inflammatory cell infiltration, and in some regions the distribution of calcium and OPN deposition differed (compare Figure 1C with Figure 2C
). In such regions, OPN must have localized in the peritoneum by mechanisms other than adsorption to hydroxyapatite, and OPN has in fact been shown to bind to collagen type I and fibronectin in vitro. We were, therefore, unable to exclude the possibility that locally released and precipitated OPN in the early stages of tissue injury may somehow contribute to calcium deposition.
In summary, this study showed that microscopic calcium deposition and associated OPN precipitation are not exceptional events in the peritoneum of patients maintained on CAPD for a long period. It also demonstrated local OPN expression in damaged peritoneum and supported a proposed connection between OPN and tissue repair/fibrosis. Experiments in animal models seem indispensable to clarifying the mechanisms of peritoneal calcification and especially the actual roles of OPN. In addition, a greater number of visceral and parietal peritoneum specimens collected from multiple centres should be examined to make our results more conclusive.
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Acknowledgments |
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Notes |
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References |
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