Morphological Retrospective Study of Peritoneal Biopsies from Patients with Encapsulating Peritoneal Sclerosis: Underestimated Role of Adipocytes as New Fibroblasts Lineage?

Abstract

Background. Encapsulating peritoneal sclerosis (EPS) is a rare but serious complication of peritoneal dialysis (PD). Besides the endothelial-to-mesenchymal transition (EMT), recently peritoneal adipocytes emerged as a potential source of fibrosis. We performed immunohistochemistry to approach EMT and to localize peritoneal adipocytes in peritoneal biopsies from PD-related EPS patients. Material and Methods. We investigated tissue expression of podoplanin, cytokeratin AE1/AE3 (mesothelium), calretinin (adipocytes), alpha-smooth muscle actin [α-SMA] (mesenchymal cells), interstitial mononuclear cell inflammation, and neoangiogenesis (CD3, CD4, CD8, CD20, CD68, and CD31 immunostainings, resp.). Results. Three patients (1 man/2 women; 17, 64, and 39 years old, resp.) developed EPS after 21, 90, and 164 months of PD therapy. In patients with EPS, we observed (1) loss of AE1/AE3 cytokeratin+ mesothelial cells without any evidence of migration into the interstitium, (2) disappearance of adipose tissue, (3) diffuse infiltration of calretinin+ cells in the areas of submesothelial fibrosis with a huge number of α-SMA and calretinin+ fusiform cells, and (4) increased vascular density. Conclusion. We report that the involvement of EMT in peritoneal fibrosis is difficult to demonstrate and that the calretinin+ adipocytes might be an underestimated component and a new source of myofibroblasts in peritoneal remodeling during PD-related EPS.

Figure 1

Radiological findings suggesting encapsulating peritoneal sclerosis (EPS). (a, c and b, d correspond to cases EPS 2 and EPS 3, resp.) Abdominal computed tomography without contrast agent showed massive nonloculated fluid and bowel loops drowned into the center of the abdominal cavity suggestive of bowel adhesions. As compared with EPS case  2, EPS case  3 presented more abundant abdominal ascites, marked narrowing of bowel lumen and thickening of visceral and parietal peritoneum, absence of dilated bowel loop, air-fluid levels or entrapped fluid collections, and several calcifications in both parietal and visceral peritoneum. (e and f, case EPS 3) Fluorodeoxyglucose¹⁸ positron emission tomography (F¹⁸ PET) scan showed a mild increase in the tracer uptake in the peritoneal areas.

Figure 2

Peritoneal biopsies representative photomicrographs of haematoxylin-eosin and Masson's trichrome stainings in studied cases (a, d, and g). Mesothelium (→) and adipocytes (#) in the submesothelial area in normal peritoneal membrane (b, e, and h). Well preserved mesothelium with hyperplasic mesothelial cells and increase in conjunctive tissue (∗) associated with interstitial submesothelial infiltrate mainly polymorphonuclear neutrophils (++) in acute peritonitis case. Superficial black lining related to the surgery technique (use of Indian ink). (c, f, and i) Disappearance of mesothelium (arrowhead), major submesothelial fibrosis containing mainly mononuclear cells, and sever fibrosis (←→) in case of EPS. Original magnifications: (a–f) ×10, (g–i) ×40.

Figure 3

Peritoneal biopsy representative photomicrographs of mesothelial phenotype markers expression: podoplanin (a–d), cytokeratin AE1/AE3 (e–h), and calretinin (i–l). Positive control of immunostainings for used antibodies (internal controls) (a, e, and i), control case: normal peritoneum (b, f, and j), case of acute peritonitis (control 3, c, g, and k), and case  1 of encapsulating peritoneal sclerosis (EPS) (d, h, and l). Physiologic expression of podoplanin (cytoplasm of endothelium in lymphatics (↠)), AE1/AE3 cytoplasm in epithelial cells of stomach and calretinin (cytoplasm in epithelial cells) (a, e, and i, resp.). (b, f, and j) Intact peritoneal membrane biopsy in controls; mesothelial cells (→) with obvious expression of adipocytes (#). (c, g, and k) Acute peritonitis case: hyperplasia of mesothelial cells. Superficial black lining related to the surgery technique (use of Indian ink). (d, h, and l) EPS case: loss of mesothelial cells (arrowheads) as attested by absence of podoplanin, AE1/AE3, and calretinin expressions. Note hyperplasia of mesothelial cells expressed all mesothelial markers podoplanin, cytokeratin AE1/AE3, and calretinin located in interstitial areas. The architecture of interstitium is strongly modified and contains several calretinin+ fusiform (fibroblasts-like) cells (arrow). Immunoperoxidase staining counterstained with haematoxylin. Original magnification: (a–l) ×20.

Figure 4

Representative photomicrographs demonstrating histopathological data of interstitial inflammatory cells infiltration in parietal peritoneal tissues biopsy using immunostaining of CD8 (a–d), CD4 (e–h), CD20 (i–l), and CD68 (m–p). Positive control of immunostainings for used antibodies (internal controls) (a, e, i, and m), control: normal peritoneum (b, f, j, and n), case of acute peritonitis (control 3; c, g, k, and o), and case  1 of encapsulating peritoneal sclerosis (EPS) (d, h, l, and p). Absence of mononuclear cells in normal peritoneum. Weak expression of all CD8, CD4, CD20, and CD68 in conjunctive tissue areas adjacent to mesothelium in case with acute peritonitis characterized mainly by polymorphonuclear cells infiltration. Marked interstitial inflammatory cells identified (d) several CD8+ cells (T lymphocytes), (h) few CD4+ cells (T lymphocytes), and (l) CD20+ (B cells), which were accompanied by several (f) CD68+ cells (macrophages) diffusely infiltrating fibrotic areas. Immunoperoxidase staining counterstained with hematoxylin. Original magnification: (a–p) ×20. Small pictures (a, e, i, and m): ×40.

Figure 5

Representative photomicrographs demonstrating histopathological data of mesenchymal markers expression in parietal peritoneal tissues biopsy using immunostaining of vimentin (a–d) and alpha-smooth muscle actin (α-SMA) (e–h). Positive control of immunostainings for used antibodies (internal controls) (a, e), control: normal peritoneum (b, f), case of acute peritonitis (control 3; c and g), and case  1 of encapsulating peritoneal sclerosis (EPS) (d and h). (a, e) Normal peritoneum, expression of vimentin (→) limited to few interstitial cells and of α-SMA expression to vascular walls. (c, g) Acute peritonitis: interstitial vimentin+ cells, lack of expression of vimentin in mesothelial cell layer; α-SMA found only in the vessels. (d, h) Case of encapsulating peritoneal sclerosis (EPS): absence of expression of both markers in the mesothelial cell layer. Note diffuse accumulation of vimentin+ cells identifying interstitial mesenchymal cells and α-SMA immunostaining of numerous interstitial cells entrapped in the fibrotic areas reflecting the presence of myofibroblasts. Immunoperoxidase staining counterstained with haematoxylin. Original magnification: (a–g) ×20. (h) ×4.

Figure 6

Representative photomicrographs demonstrating histopathological data of endothelial cells marker expression in parietal peritoneal tissues biopsy using immunostaining of CD31 (a). Positive control of immunostainings for used antibodies (internal controls) (a), control: normal peritoneum (b), case of acute peritonitis (control 3; c), and case  1 of encapsulating peritoneal sclerosis (EPS) (d). As compared with normal and acute peritonitis cases, increases in endothelial CD31 expression identifying nonlymphatic vascular network were localized in the deep areas of peritoneum and reflected increased vessels density. Immunoperoxidase staining counterstained with hematoxylin. Original magnification: (a–d) ×20.