Project description:Loss of ultrafiltration capacity in peritoneal dialysis (PD) patients is often associated with peritoneal vascular changes, manifest as diabetes-like vasculopathy and angiogenesis. The endothelial monolayer lining the vessel lumen controls vessel barrier function and thereby influences peritoneal substrate transport and ultrafiltration. In this study, we first characterized the response to conventional PD fluids of endothelial cells isolated from peritoneal biopsies of children undergoing PD. 9 omental biopsies from the pediatric biopsy biobank (mean age 5.7 years) were included in this study. PD patients were treated with conventional PD fluid (mean PD vintage 12.5 months). Age-matched controls (n=5) with normal renal function undergoing elective surgery were also included. In patients on PD, the biopsy sampling site was at least 5 cm away from the PD catheter entry site. Written informed consent was obtained from parents, and from patients as appropriate. The study was performed according to the Declaration of Helsinki and registered at www.clinicaltrials.gov (NCT01893710). The study was part of the International Pediatric Dialysis Network (IPDN; www.pedpd.org). Patients with a BMI of >35 kg/m2 and with chronic inflammatory diseases were excluded. Specimens obtained during surgery were instantaneously frozen with liquid nitrogen and stored at -80°C. Arterioles macroscopically located within fat tissue and microscopically at least 1mm distant from the peritoneal surface were micro-dissected. Elastica van Gieson stainings of the arteriolar elastic lamina were used as templates for microdissection of arterioles from cresol violet–stained neighboring sections. 100 µm thick tissue slices were cut with a cryotome and 30 deep-frozen arteriolar rings per patient were micro-dissected using a stereo microscope and a microneedle. Arteriolar rings were lysed in 100 µl lysis buffer (30 mM Tris, pH 8.5, 7M urea, 2M thiourea, 4% 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), 1 mM EDTA, 1 tablet of Complete Protease Inhibitor (Roche, Basel, Switzerland) and 1 tablet of phosphatase inhibitor (PhosSTOP, Roche) per 100 ml). The resulting lysates were stored at -80°C until further processing. Total protein concentration was determined with the 2D-Quant kit (GE). Quality control and verification of protein concentrations was performed by SDS-PAGE. We investigated the molecular mechanisms of endothelial cell pathology during in vivo and in vitro PD fluid exposure by proteomic and bioinformatic analysis of the endothelial cell response to hyperglycemic stress, integrating current understanding of PD-related cellular injury and stress responses (Bender et al., Nephrol Dial Transplant, 2011, doi:10.1093/ndt/gfq484; Kratochwill et al., BioMed research international, 2015, doi:10.1155/2015/628158; Kratochwill et al., J Proteome Res, 2009, doi:10.1021/pr800916s; Lechner et al., J Proteome Res, 2010, doi:10.1021/pr9011574).

Project description:Loss of ultrafiltration capacity in peritoneal dialysis (PD) patients is often associated with peritoneal vascular changes, manifest as diabetes-like vasculopathy and angiogenesis. The endothelial monolayer lining the vessel lumen controls vessel barrier function and thereby influences peritoneal substrate transport and ultrafiltration. The dipeptide alanyl-glutamine (AlaGln) has recently shown cytoprotective effects when added to PD fluid, including preservation of mesothelial cells in vitro (Kratochwill et al., Nephrol Dial Transplant, 2012, doi:10.1093/ndt/gfr459) and attenuation of the exuberant angiogenesis seen in long-term rodent models of PD (Ferrantelli et al., Kidney international, 2016, doi:10.1016/j.kint.2015.12.005). These effects have been reflected in early phase clinical trials as restoration of effluent cell stress responses and improved biomarkers of peritoneal health (Kratochwill et al., PLoS One, 2016, doi:10.1371/journal.pone.0165045; Vychytil et al., Kidney international, 2018, doi:10.1016/j.kint.2018.08.031). In a randomized clinical phase II trial, 8 weeks treatment with AlaGln in PD fluid decreased peritoneal protein loss. This clinically important effect might be explained by preserved peritoneal membrane and vessel integrity, but the role of AlaGln in preservation of peritoneal endothelial cell function remains unclear. In this study, we investigated to what extent in vivo observed biological processes and pathways are replicated by in vitro exposure of human umbilical vein endothelial cells (HUVEC) to conventional PD fluid. HUVECs were exposed to experimental solutions for up to 24 h. All test ﬂuids were sterile-ﬁltered before usage. Each experiment consisted of three independent samples in biological replicates on separate culture plates. For PD fluid incubation, cells were first exposed for 1 h to pure glucose-based PD fluid (Dianeal PD4 3.86% glucose, Baxter, Castlebar, Ireland), or to the same PD fluid supplemented with AlaGln dipeptide (8 mM, Dipeptiven; Fresenius Kabi, Bad Homburg, Germany), or to normal medium without growth factors as control. Cells were subsequently exposed for 24 h to the above solutions diluted 1:1 with culture medium and brought to 2% FCS. Cells were then washed three times (250 mM sucrose, 10 mM Tris/HCl, pH 7) and lysed in 125 µL per well of lysis buffer (30 mM Tris, pH 8.5, 7 M urea, 2 M thiourea, 4% CHAPS, 1 mM EDTA, one tablet of Complete Protease Inhibitor (Roche, Basel; Switzerland) per 100 ml, and one tablet of PhosStop Protease Inhibitor (Roche) per 100 mL). Total protein concentration was determined with the 2D-Quant kit (GE Healthcare, Uppsala, Sweden) per manufacturer’s manual. Lysates were stored at -80°C until further processing. We investigated the molecular mechanisms of endothelial cell pathology during in vivo and in vitro PD fluid exposure by proteomic and bioinformatic analysis of the endothelial cell response to hyperglycemic stress, integrating current understanding of PD-related cellular injury and stress responses (Bender et al., Nephrol Dial Transplant, 2011, doi:10.1093/ndt/gfq484; Kratochwill et al., BioMed research international, 2015, doi:10.1155/2015/628158; Kratochwill et al., J Proteome Res, 2009, doi:10.1021/pr800916s; Lechner et al., J Proteome Res, 2010, doi:10.1021/pr9011574). We then evaluated the effect of AlaGln addition to conventional PD fluid on these parameters in endothelial cells. This strategy allowed characterization of endothelial cell injury and stress responses during exposure to conventional hyperglycemic PD fluid, and their modulation by added AlaGln.

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Transcriptome of isolated mouse peritoneal endothelial cells

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