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Background: Malignant pleural mesothelial cells are affected by the extracellular milieu while such data on benign cells are scarce. Benign cells sense the extracellular environment with the Primary Cilium (PC) and its molecular complex, the BBSome, is critical for this process. Here we aimed at assessing the changes in BBSome genes expression in ordinary 2D and spheroid 3D cell cultures after incubation with pleural effusion fluids (PF) of several etiologies., Methods: Benign human mesothelial cells MeT-5A were incubated with PF from patients with mesothelioma (Meso-PF), breast cancer (BrCa-PF), hemothorax (Hemo-PF) and congestive heart failure (CHF-PF). Gene expression of BBS1, 2, 4, 5, 7, 9, 18 was assessed by quantitative real-time PCR (qRT-PCR) to monitor PF-induced gene expression changes. MeT-5A cell migration using the PC-modulating drugs ammonium sulfate (AS) and lithium chloride (LC) during PF incubation was also determined., Results: BBSome gene expression upon influence of BrCa-PF and Hemo-PF was more pronounced in 2D compared to 3D, inducing global changes in 2D. CHF-PF and Meso-PF also induced changes in 2D but not as many, while in all cases MeT-5A grown in 3D were more resistant to the effects of the PF. Meso-PF decreased 2D cell migration, while the disturbance of PC in all PF cases resulted in decreased cell migration., Conclusions: These data suggest distinct BBSome molecular profile changes in benign mesothelial cells exposed to malignant and benign PF, in each case, in both 2D and 3D. Cell migration is sensitive to drug disturbance with PC modulators in PF-exposed cells.

Introduction: Peritoneal dialysis (PD) is a life maintaining treatment in patients with end-stage renal disease. Its chronic application leads to peritoneal mesothelial layer denudation and fibrotic transformation along with vascular activation of inflammatory pathways. The impact of different PD fluids (PDF) on mesothelial and endothelial cell function and repair mechanisms are not comprehensively described., Materials and Methods: Mesothelial (MeT-5A) and endothelial cells (EA.hy926) were cultured in 1:1 ratio with cell medium and different PDF (icodextrin-based, amino acid-based, and glucose-based). Cell adhesion, cell migration, and cell proliferation in 2D and spheroid formation and collagen gel contraction assays in 3D cell cultures were performed., Results: Cell proliferation and cell-mediated gel contraction were both significantly decreased in all conditions. 3D spheroid formation was significantly reduced with icodextrin and amino acid PDF, but unchanged with glucose PDF. Adhesion was significantly increased by amino acid PDF in mesothelial cells and decreased by icodextrin and amino acid PDF in endothelial cells. Migration capacity was significantly decreased in mesothelial cells by all three PDF, while endothelial cells remained unaffected., Conclusions: In 3D phenotypes the effects of PDF are more uniform in both mesothelial and endothelial cells, mitigating spheroid formation and gel contraction. On the contrary, effects on 2D phenotypes are more uniform in the icodextrin and amino acid PDF as opposed to glucose ones and affect mesothelial cells more variably. 2D and 3D comparative assessments of PDF effects on the main peritoneal membrane cell barriers, the mesothelial and endothelial, could provide useful translational information for PD studies., (© 2023 The Authors. Artificial Organs published by International Center for Artificial Organ and Transplantation (ICAOT) and Wiley Periodicals LLC.)

Peritoneal dialysis (PD) and prolonged exposure to PD fluids (PDF) induce peritoneal membrane (PM) fibrosis and hypervascularity, leading to functional PM degeneration. 2-deoxy-glucose (2-DG) has shown potential as PM antifibrotic by inhibiting hyper-glycolysis induced mesothelial-to-mesenchymal transition (MMT). We investigated whether administration of 2-DG with several PDF affects the permeability of mesothelial and endothelial barrier of the PM. The antifibrotic effect of 2-DG was confirmed by the gel contraction assay with embedded mesothelial (MeT-5A) or endothelial (EA.hy926) cells cultured in Dianeal® 2.5 % (CPDF), BicaVera® 2.3 % (BPDF), Balance® 2.3 % (LPDF) with/without 2-DG addition (0.2 mM), and qPCR for αSMA, CDH2 genes. Moreover, 2-DG effect was tested on the permeability of monolayers of mesothelial and endothelial cells by monitoring the transmembrane resistance (R TM ), FITC-dextran (10, 70 kDa) diffusion and mRNA expression levels of CLDN-1 to -5, ZO1, SGLT1, and SGLT2 genes. Contractility of MeT-5A cells in CPDF/2-DG was decreased, accompanied by αSMA (0.17 ± 0.03) and CDH2 (2.92 ± 0.29) gene expression fold changes. Changes in αSMA, CDH2 were found in EA.hy926 cells, though αSMA also decreased under LPDF/2-DG incubation (0.42 ± 0.02). Overall, 2-DG mitigated the PDF-induced alterations in mesothelial and endothelial barrier function as shown by R TM , dextran transport and expression levels of the CLDN-1 to -5, ZO1, and SGLT2. Thus, supplementation of PDF with 2-DG not only reduces MMT but also improves functional permeability characteristics of the PM mesothelial and endothelial barrier., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)