Understanding and targeting the molecular basis of peritoneal solute and protein transport is essential to improve peritoneal dialysis (PD) efficacy and patient outcome. Supplementation of PD fluids (PDF) with alanyl-glutamine (AlaGln) increased small solute transport and reduced peritoneal protein loss in a recent clinical trial. Transepithelial resistance and 10 kDa and 70 kDa dextran transport were measured in primary human endothelial cells (HUVEC) exposed to conventional acidic, glucose degradation products (GDP) containing PDF (CPDF) and to low GDP containing PDF (LPDF) with and without AlaGln. Zonula occludens-1 (ZO-1) and claudin-5 were quantified by Western blot and immunofluorescence and in mice exposed to saline and CPDF for 7 weeks by digital imaging analyses. Spatial clustering of ZO-1 molecules was assessed by single molecule localization microscopy. AlaGln increased transepithelial resistance, and in CPDF exposed HUVEC decreased dextran transport rates and preserved claudin-5 and ZO-1 abundance. Endothelial clustering of membrane bound ZO-1 was higher in CPDF supplemented with AlaGln. In mice, arteriolar endothelial claudin-5 was reduced in CPDF, but restored with AlaGln, while mesothelial claudin-5 abundance was unchanged. AlaGln supplementation seals the peritoneal endothelial barrier, and when supplemented to conventional PD fluid increases claudin-5 and ZO-1 abundance and clustering of ZO-1 in the endothelial cell membrane.

Rationale: Patients with chronic kidney disease (CKD) have an exceedingly high cardiovascular risk; which further increases in patients on peritoneal dialysis (PD). The pathophysiological role of reactive metabolites accumulating in CKD such as glucose degradation products (GDP) is uncertain. Objective: Delineating the impact of GDP present in PD fluids in accelerated vasculopathy development in patients with CKD. Methods and Results: Omental and parietal peritoneal tissues were obtained from 107 children with CKD before dialysis and 90 children on chronic PD with PD fluids containing very low or high concentrations of GDP. Omental arterioles, protected from local PD fluid exposure by surrounding fat, were microdissected for multiomics analyses. High-GDP exposed omental arterioles exhibited 3-fold higher advanced glycation endproduct concentrations and upregulated genes involved in cell death/apoptosis and suppressed genes related to cell viability/survival, cytoskeleton organization, and immune response biofunctions. Vasculopathy-associated canonical pathways concordantly regulated on gene and protein level with high-GDP exposure included cell death/proliferation, apoptosis, cytoskeleton organization, metabolism and detoxification, cell junction signaling, and immune response. Parietal peritoneal arterioles of patients exposed to high-GDP fluids exhibited lumen narrowing compared to patients with CKD stage 5 (end-stage kidney disease) and patients on low-GDP PD, intima thickness was increased. Protein quantification verified increased proapoptotic activity and cytoskeleton disintegration, single-molecule-localization microscopy demonstrated arteriolar endothelial ZO-1 (zonula occludens-1) disruption. Absolute and per endoluminal surface length, arteriolar endothelial cell counts inversely correlated with GDP exposure, caspase-3, TGF (transforming growth factor)-β–induced pSMAD2/3 (phosphorylated SMAD2/3), interleukin-6, ZO-1 abundance, and lumen narrowing. In vitro, 3,4-dideoxyglucosone-3-ene reduced lamin-A/C and membrane ZO-1 assembly, increased pSMAD2/3, and ionic and 4 and 10 kDa permeability of arterial endothelial cells. Conclusions: Our findings indicate a fundamental role of GDP in PD-associated vasculopathy, exerted by endothelial cell junction and cytoskeleton disruption, and induction of apoptosis. They should redirect the focus of research and intervention on targeting reactive metabolite overload in CKD and PD. Registration: URL: https://www.clinicaltrials.gov ; Unique identifier: NCT01893710.

Endothelial and epithelial barrier function is crucial for the maintenance of physiological processes. The barrier paracellular permeability depends on the composition and spatial distribution of the cell-to-cell tight junctions (TJ). Here, we provide an experimental workflow that yields several layers of physiological data in the setting of a single endothelial cell monolayer. Human umbilical vein endothelial cells were grown on Transwell filters. Transendothelial electrical resistance (TER) and 10 kDa FITC dextran flux were measured using Alanyl-Glutamine (AlaGln) as a paracellular barrier modulator. Single monolayers were immunolabelled for Zonula Occludens-1 (ZO-1) and Claudin-5 (CLDN5) and used for automated immunofluorescence imaging. Finally, the same monolayers were used for single molecule localization microscopy (SMLM) of ZO-1 and CLDN5 at the nanoscale for spatial clustering analysis. The TER increased and the paracellular dextran flux decreased after the application of AlaGln and these functional changes of the monolayer were mediated by an increase in the ZO-1 and CLDN5 abundance in the cell–cell interface. At the nanoscale level, the functional and protein abundance data were accompanied by non-random increased clustering of CLDN5. Our experimental workflow provides multiple data from a single monolayer and has wide applicability in the setting of paracellular studies in endothelia and epithelia.

Fibroblasts play a central role in diseases associated with excessive deposition of extracellular matrix (ECM), including idiopathic pulmonary fibrosis. Investigation of different properties of fibroblasts, such as migration, proliferation, and collagen-rich ECM production is unavoidable both in basic research and in the development of antifibrotic drugs. In the present study we developed a cost-effective, 96-well plate-based method to examine the migration of fibroblasts, as an alternative approach to the gold standard scratch assay, which has numerous limitations. This article presents a detailed description of our transient agarose spot (TAS) assay, with instructions for its routine application. Advantages of combined use of different functional assays for fibroblast activation in drug development are also discussed by examining the effect of nintedanib—an FDA approved drug against IPF—on lung fibroblasts.

Background/aim: Children on dialysis are under increased risk of influenza and invasive pneumococcal disease. Although vaccination against these microorganisms are recommended in dialysis patients and despite the fact that these vaccines can reduce disease burden and rates of hospitalization due to infection, vaccination rates are below expected and desired. We aimed to evaluate influenza and pneumococcal vaccination and infection rates in European pediatric dialysis centers. Materials and methods: In 16 centers from 11 countries, 357 pediatric dialysis patients were evaluated retrospectively during 1 year of observation period between 01.01.2014 and 01.01.2015. Results: In all centers, vaccination policy included immunization of dialysis patients with inactive influenza vaccine and pneumococcal conjugate vaccine (PCV). Fifty percent of the centers recommended pneumococcal polysaccharide vaccine following routine PCV series. A significantly higher pneumococcal vaccination rate (43.9%) was seen in peritoneal dialysis (PD) patients compared to those on hemodialysis (HD) (32.9%) (p = 0.035), while the rates for influenza were similar (42.4% and 46.1% respectively, p = 0.496). Among all dialysis patients, 2.2% (n = 8) developed pneumonia and 6.4% (n = 23) was infected by Influenza. Pneumococcic pneumonia rate was 5% for 140 patients who received antipneumococcal vaccine, while only one pneumonia episode was recorded out of 217 unvaccinated patients (p = 0.007). The influenza virus infection rates were similar for patients vaccinated and nonvaccinated (7 % and 6 %, respectively). Conclusions: Although influenza and pneumococcal vaccines are highly recommended in pediatric dialysis patients, vaccination rates were lower than expected. Pneumococcal vaccination rates were higher in PD compared to the patients on HD. The rate of children with influenza infection was higher than pneumonia. The efficacy of influenza and pneumococcal vaccines was highlighted by the low infection rates. Higher pneumonia rates in patients vaccinated against pneumococcus compared to unvaccinated ones might be due to coexisting risk factors.

Absztrakt: Bevezetés, célkitűzés, módszer: A Semmelweis Egyetem I. Gyermekgyógyászati Klinikája hazánk legnagyobb, krónikus veseelégtelen gyermekeket ellátó centruma, ahol kiemelt szerepet kap a peritonealis dialízis alkalmazása. 2010 és 2017 között a Nemzetközi Gyermek Peritonealis Dialízis Regiszter keretében prospektív adatgyűjtés kapcsán elemeztük kezelési gyakorlatunkat. Eredmények: A fenti időszak 39 betegének átlagéletkora a kezelés indításakor 9,3 ± 6,6 év, a dialíziskezelésen töltött idő 1,3 ± 1,3 év. Veseelégtelenségük hátterében álló vezető okok a glomerulopathiák és a vese/húgyutak fejlődési rendellenességei voltak. Centrumunkban magas arányban megfigyelhetők komorbiditások, szindrómák. 18 betegünknél diagnosztizáltunk peritonitist. Laborértékeinkben eltérést az ajánlott tartományoktól parathormon- és foszfátértékekben tapasztaltunk. Gyógyszerelési statisztikáink megfeleltek az ajánlásoknak, bár bizonyos szerek hazánkban gyermek betegek számára nem elérhetőek. Betegeink rövid dialízisen töltött ideje a gyors transzplantációnak köszönhető. A ritka betegségek és számos komorbiditás megjelenésének oka, hogy klinikánk kiemelt gyermeknefrológiai centrum. Következtetés: Gyakorlatunk megfelel a nemzetközi irányelvekben foglaltaknak, az eltérések esetében kezeléseink egyéni átgondolására volt szükség. A nemzetközi regiszterben való részvétel saját gyakorlatunk elemzése és a terület fejlődése szempontjából kiemelt jelentőségű. Orv Hetil. 2017; 158(46): 1831–1840. Introduction, aim and method: At the 1st Department of Paediatrics, Semmelweis University – the largest center for treating children with chronic kidney disease in Hungary – peritoneal dialysis has a leading role. Between 2010–2017, while being part of the International Pediatric Dialysis Network, we collected and analyzed our data in a prospective study. Beside our patients’ basic data, we registered medication statistics, treatment- and laboratory results every 6 months. Results: During this period we had 39 patients on peritoneal dialysis, the average age of dialysis-initiation was 9.3 ± 6.6 years, with 1.3 ± 1.3 years spent on dialysis. The most common causes of ESRD were glomerulopathies and renal-/urinary malformations. A high rate of comorbidities was found, and in 13 cases complex syndromes. Many patients’ height- and BMI SDS values differed from the normal for their age. Average glucose-monohydrate concentration was 1.6 ± 0.2%. During our investigation we used lactate-based, acidic or neutral (balanced) dialysates. Between 2010–2017 we diagnosed peritonitis in 18 of our cases. The patients were hospitalized average 70.5 ± 97.7 days. There were differences from the normal range for age in PTH- and phosphate values. Drug-statistics met the requirements of recommendations, though some medications are not available for children in Hungary. The short time spent on dialysis is due to fast- and also living-donor transplantations and efficient management of the transplantation list. The high rates of special diseases and comorbidities are due to being a tertiary referral center. Conclusion: Our practice is suitable for the international guidelines, any differences are results of individual considerations. Being a part of the Registry should be maintained. Orv Hetil. 2017; 158(46): 1831–1840.