#5664 PERITONEAL DIFFUSE PODOPLANIN EXPRESSION IS ASSOCIATED WITH SEVERE ARTERIOLOPATHY IN CHILDREN ON PERITONEAL DIALYSIS

Abstract

Abstract Background and Aims Podoplanin is a transmembrane glycoprotein binding chemokines and modulating inflammatory reactions. In healthy peritoneum, it is expressed in mesothelial cells and in lymphatic endothelium, expression beyond these structures (diffuse podoplanin staining, DPS) has been observed in adult patients with encapsulating peritoneal sclerosis (EPS). Method Parietal peritoneal tissues from 71 children on PD fluids with low glucose degradation product (GDP) content underwent digital histomorphometry. DPS and pathomechanisms of peritoneal membrane transformation and vasculopathy were hierarchically clustered. A panel of 13 peritoneal cell markers was selected and tissue sections were stained with metal conjugated antibodies and acquired using imaging mass cytometry (IMC; Hyperion Imaging System®). After single-cell segmentation, spillover correction, and normalization, cell types were identified using manual gating to train a random forest classifier for final cell type identifications using published workflows. Single-cell analysis was used to characterize peritoneal cells with positive podoplanin signals. Results DPS was present in 22% of the PD patients, all devoid of any clinical, radiological or histological feature of EPS. DPS positive patients were younger, had higher dialytic glucose exposure (median 103 vs. 154 g/m²/day, p = 0.002), and lower peritoneal arteriolar lumen to vessel (L/V) ratio, i.e. exhibited more arteriolar lumen narrowing. PD duration [median 20 (14.5, 48) months] and peritonitis incidence were similar as in non-DPS patients. In multivariable analysis, glucose exposure, PD duration, epithelial-to-mesenchymal transformed (EMT) cells and lower patient body surface area (BSA) independently predicted the presence of DPS. DPS and lower mesothelial surface coverage predicted lower arteriolar L/V ratio. In subgroups matched for BSA, PD duration and dialytic glucose exposure, DPS patients had a lower L/V ratio, (0.44 ± 0.13 vs. 0.31 ± 0.18, p = 0.003) and higher submesothelial CD45 leucocyte, CD68 macrophage and EMT cell count. Submesothelial lipopolysaccharide and hyaluronic acid receptor CD44 were increased in DPS positive patients, hyaluronan synthase 1 and 2 were similar. Among DPS positive patients, DPS intensity grade was higher in those with history of bacterial or fungal peritonitis (7 out of 15). Two patients had three and four peritonitis episodes respectively, their DPS pattern did not differ from patients with history of only one peritonitis episode. Hierarchical clustering demonstrated highest similarity of DPS positive areas with CD68 positive areas. In IMC analysis of DPS positive areas, highest podoplanin signals were derived from M2 classified macrophages (CD68+CD163+) and fibroblastic cells [αSMA positive/non-endothelial/non-macrophage (αSMA+PROX1-CD31-CD68-CD163-)], signals derived from M1 classified macrophages (CD68+CD163-) were lowest. Conclusion Peritoneal DPS in pediatric patients on low-GDP PD is associated with lower BSA, higher dialytic glucose exposure, history of infectious peritonitis and peritoneal inflammatory and EMT cell invasion, and predicts obliterating peritoneal vasculopathy. We identified M2 macrophages as major source of extra-lymphatic peritoneal podoplanin accumulation, suggesting their role in severe peritoneal transformation.