Glucose Degradation Products Research Articles

Detections of Chemicals and Migratory Plastics in Peritoneal Dialysis Fluids

Background: Peritoneal dialysis (PD) is an important modality of renal replacement therapy (RRT). Peritonitis and ultrafiltration failure are complications that have a long-term impact on PD patients. Besides touch contamination, procedural errors and clinical reasons of peritonitis, contaminants, and constituents of peritoneal dialysis fluids (PDFs) have been implicated in causing peritonitis and ultrafiltration failure. This study was aimed to test the PDFs in India for the presence of migratory plastics. Materials and Methods: PDFs from the two manufacturers in India were tested using liquid chromatography mass spectrometry (LCMS) and gas chromatography mass spectrometry (GCMS) with headspace analysis (volatile compounds) and pyrolysis of plastics (polymer compounds). The storage conditions and handling were uniform. Results: The results revealed impurities of acetate compounds and aldehyde derivatives of glucose degradation products (GDPs) with contaminants and leachable plastics. There were high levels of GDP derivative in the form of 5-hydroxymethylfurfural compounds (5-HMF). The analysis revealed the presence of plastic softeners in very high concentrations. Conclusion: The study unmasks the presence of chemicals and GDPs that can be implicated in pathogenesis of sterile peritonitis and ultrafiltration failure. The study demonstrated the presence of leachable plastics. In conclusion, LCMS and GCMS studies can be used to test PDFs for unwanted chemicals prior to human use.

#1288 Bioinformatic analysis of phosphate transporters in peritoneal cells and tissues and phosphate transport kinetics in vitro

Abstract Background and Aims Hyperphosphatemia is frequent in peritoneal dialysis (PD) patients due to limited dialytic clearance, and significantly increases cardiovascular risk. Molecular mechanisms of phosphate removal across the peritoneal membrane remain unclear, PD specific in vitro models for phosphate transport and modulator studies are not established. Method Phosphate transporter expression was assessed by RNAseq in mesothelial cells (HPMC, MeT5A) and human venous and microvascular endothelial cells (HUVEC, HCMEC). Transcriptome profiles of microdissected omental arterioles from 10 children with normal kidney function (NKF), 15 with chronic kidney disease (CKD5), and 10 children on PD were analyzed for phosphate transporter expression. Specific phosphate transporter localization and abundance in parietal peritoneum membranes and in the cell lines were assessed by immunostaining. In Transwell system, a phosphate transport model across polarized monolayers was established. Cell viability and integrity were verified by MTT assay and transepithelial resistance measurement (TER). Results Out of nine identified phosphate transporters, PiT1 (SLC20A1) and PiT2 (SLC20A2) exhibited high expression in all four cell lines, while SLC34A2 was specific in HPMC and SLC34A3 in MeT5A. In human arterioles, four phosphate transporters, SLC34A1, PiT1, PiT2, and SLC17A1, were detected. Arteriolar PiT1 was two-fold more abundant in PD patients treated with low glucose degradation product (GDP) fluids compared to those with CKD5 and NKF. In parietal peritoneal membranes, PiT1 was abundant in mesothelial and endothelial cells, with no difference between children with NKF, CKD5 and low GDP PD treatment. Concentration and time dependent phosphate kinetics across polarized mesothelial cells were studied in Transwells. Upon increased phosphate concentration in medium (1 mM and 2 mM vs. 0.1 mM), cells didn´t show signs of impaired viability, reduced TER or junction dysregulation (ZO-1 abundance) for up to 12 h. Independent of the added phosphate concentration, 60% of expected equilibrium was achieved after 12 h. Addition of PFA (SLC20/34 sodium-phosphate cotransporter inhibitor) alone or in combination with Tenapanor (an NHE3 blocker inhibiting paracellular phosphate transport) reduced phosphate transport across mesothelial cells by 10% and 20%, respectively. Conclusion We describe phosphate transporter expression and localization in peritoneal cells, in arterioles and parietal peritoneal tissues, and the impact of CKD5 and PD. The Transwell system allowed for quantification of phosphate transport across cell barriers and demonstrated the role of peritoneal sodium-phosphate cotransporter and of the paracellular route for phosphate transport across the mesothelial cell barrier. Further, yet unknown phosphate transport mechanisms should be active.

#1980 Past peritonitis episodes are linked to decreased peritoneal Immune Cell Function and Associated with Chronic Increased Peritoneal Inflammation

Abstract Background and Aims Chronic peritoneal inflammation and infectious complications occur in a significant proportion of PD patients. A relationship between peritoneal IL-6 levels and local immunocompetence is suspected. To analyse local immunocompetence, an ex-vivo stimulation assay of PD effluent was developed. The aim of this study was to analyse peritoneal inflammation and immunocompetence in a PD cohort in relation to clinical outcomes. Method Prospectively, 245 routinely performed 4-hour PETs (143 patients, 2679 patient-months) of patients exclusively receiving 2. generation PD fluids (two-chamber, pH neutral, low amount of glucose degradation products) were analysed. An ex-vivo cell stimulation protocol of the fresh effluents was performed of all PETs. Peritoneal immune cell function was analysed using ex-vivo Toll-like receptor (LPS/Pam3Cys) stimulated IL-6 release as an index of immunocompetence. The molecular effects of effluent components and the addition of alanyl-glutamine to PD fluid were analysed in heterologous cell stimulation experiments and using an effluent proteomics and metabolomics approach. Results IL-6 in the effluent showed no association with various patient characteristics (incl. RRF, underlying disease, peritonitis episodes, time on PD). A significant association of peritoneal IL-6 with solute transport rates and systemic markers of uremic inflammation was found. IL-6 in effluent (HR 2.54) and stimulated 1 h effluent (HR 0.34) was predictive of subsequent peritonitis episodes. An inverse association was observed with ex-vivo stimulated IL-6 release from peritoneal cells (P = .02), most pronounced in patients with a history of peritonitis (P = .008). Patients with above median elevated IL-6 levels showed reduced peritoneal immune cell function. The results were validated in an independent multi-centre PD cohort (n = 48). A proteomics (n = 17) and targeted metabolomics (patients n = 134, 1004 metabolites) analysis of PD effluents show an overrepresentation of apoptosis and neutrophil degranulation pathways in patients with elevated effluent IL-6 levels and reduced response mechanisms to pathogens when compared to effluents from patients with low effluent IL-6 levels. Heterologous donor-PBMC stimulation (4 Healthy donors) with 4h PET PD-effluents of 20 patients showed reduced immunocompetence with elevated IL-6 (P = .038) and improved immunocompetence when 8 mM alanyl-glutamine were added to the PD fluid during the dwell. These results could also be validated in a post-hoc analysis of a RCT testing alanyl-glutamine addition to PD fluid for 8 weeks in PD patients. Conclusion This is the first longitudinal study to show the relationship between peritoneal inflammation and impaired immune function of peritoneal cells, possibly predisposing to infectious complications. The possible underlying mechanistic link may represent the biological correlate (“immune-paralysis”) for PD patients at increased risk for recurrent episodes of peritonitis.

Towards a better understanding of light-glucose induced modifications on the structure and biological activity of formulated Nivolumab

In the last years, monoclonal antibodies (mAbs) have rapidly escalated as biopharmaceuticals into cancer treatments, mainly for their target specificity accompanied by less side effects than the traditional chemotherapy, and stimulation of reliable long-term anti-tumoral responses. They are potentially unstable macromolecules under shaking, temperature fluctuations, humidity, and indoor and outdoor light exposure, all stressors occurring throughout their production, transport, storage, handling, and administration steps. The chemical and physical modifications of mAbs can lead not only to the loss of their bioactivity, but also to the enhancement of their immunogenicity with increasing risk of severe hypersensitivity reactions in treated patients because of aggregation. The photostability of Nivolumab, the active principle of Opdivo®, has been here studied. The chemical modifications detected by LC-MS/MS after the light stressor showed Trp and Met mono and double oxidations as primary damage induced by light on this mAb. The oxidations were stronger when the mAb was diluted in sterile glucose solution where 5-HMF, a major heat glucose degradation product, acted as singlet oxygen producer under irradiation. However, no significant changes in the mAb conformation were found. On the contrary, formation of a significant extent of aggregates has been detected after shining high simulated sunlight doses. This again took place particularly when Nivolumab was diluted in sterile glucose, thus raising a direct correlation between the aggregation and the oxidative processes. Finally, the biological activity under light stress assessed by a blockade assay test demonstrated the maintenance of the PD-1 target recognition even under high light doses and in glucose solution, in line with the preservation of the secondary and tertiary structures of the mAb. Based on our results, as sterile glucose is mostly used for children's therapies, special warnings, and precautions for healthcare professionals should be included for their use to the pediatric population.

Associations of neutral pH, low-GDP peritoneal dialysis solutions with patient survival, transfer to haemodialysis and peritonitis.

Peritoneal dialysis (PD) solutions containing low levels of glucose degradation products (GDPs) are associated with attenuation of peritoneal membrane injury and vascular complications. However, clinical benefits associated with neutral-pH, low-GDP (N-pH/L-GDP) solutions remain unclear. Using data from the Australia and New Zealand Dialysis and Transplant Registry, we examined the associations between N-pH/L-GDP solutions and all-cause mortality, cause-specific mortality, transfer to haemodialysis (HD) for ≥30days and PD peritonitis in adult incident PD patients in Australia and New Zealand between 1 January 2005 and 31 December 2020 using adjusted Cox regression analyses. Of 12814 incident PD patients, 2282 (18%) were on N-pH/L-GDP solutions. The proportion of patients on N-pH/L-GDP solutions each year increased from 11% in 2005 to 33% in 2017. During the study period, 5330 (42%) patients died, 4977 (39%) experienced transfer to HD and 5502 (43%) experienced PD peritonitis. Compared with the use of conventional solutions only, the use of any form of N-pH/L-GDP solution was associated with reduced risks of all-cause mortality {adjusted hazard ratio [aHR] 0.67 [95% confidence interval (CI) 0.61-0.74]}, cardiovascular mortality [aHR 0.65 (95% CI 0.56-0.77)], infection-related mortality [aHR 0.62 (95% CI 0.47-0.83)] and transfer to HD [aHR 0.79 (95% CI 0.72-0.86)] but an increased risk of PD peritonitis [aHR 1.16 (95% CI 1.07-1.26)]. Patients who received N-pH/L-GDP solutions had decreased risks of all-cause and cause-specific mortality despite an increased risk of PD peritonitis. Studies assessing the causal relationships are warranted to determine the clinical benefits of N-pH/L-GDP solutions.

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

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.

#6062 EFFECT OF KIDNEY TRANSPLANTATION ON ARTERIOLAR MOLECULAR PATHWAYS IN CHILDREN WITH PRECEDING PERITONEAL DIALYSIS

Abstract Background and Aims Patients on peritoneal dialysis suffer from progressive peritoneal membrane transformation and accelerated vascular disease, underlying molecular machineries have recently been described. Molecular effects of kidney transplantation (KTx) on vascular and peritoneal pathophysiology following PD are hardly understood. Method Fat surrounded omental arterioles (i.e. not directly exposed to PD fluids) from age-matched (median 6,5 years) children with chronic kidney disease (CKD5, n = 8), 5 children on PD [neutral pH, low glucose degradation product content, median PD duration 22 (5,50) months] and 6 children, who underwent successful KTx 4-5 weeks after a median 22 (12, 36) months of PD were analyzed by digital histomorphometry. Following microdissection from fat tissue, arterioles underwent transcriptome and proteome analyses, followed by Gene Set Enrichment Analysis (GSEA), weighted gene co-expression network analysis (WGCNA), gene ontology (GO) and protein interaction analysis (string-db.org). Key pathways were validated in independent, matched cohorts by quantitative immunohistochemistry (n = 15/group) and in vitro. Results 5000 most variable arteriolar transcripts (cut off p-value<0.05 and |r| >0.5) were grouped into 23 modules by WGCNA, of which 8 significantly differed between CKD5, PD and KTx. Seven were specifically related to KTx and one module to PD. Four out of seven KTx specific modules resulted in significant GO terms summarized as fatty acid biosynthesis, negative regulation of RNA metabolism, cell cycle arrest, and apoptosis. The PD specific module was associated with muscle cell proliferation, detoxification and complement activation (with thrombospondin as most interconnected gene). Multi-omics analysis of KTx vs. PD demonstrated concordant upregulation of lipid and fatty acid biosynthesis with the hub gene fatty acid synthase (FASN) and downregulation of positive cell cycle regulation. In independent, matched cohorts, arteriolar abundance of thrombospondin and terminal complement complex were higher in children on PD, arterioles from children after KTx had levels comparable to arterioles from children with CKD5. Key drivers of fibrotic process, arteriolar TGF-ß and pSMAD2/3, were also higher in PD arterioles compared to CKD5 and persistently high after KTx. Arteriolar cell cycle arrest marker p16 and apoptosis marker cleaved Casp3 were higher after KTx as compared to PD and CKD5. FASN was abundant in intima and media layers of all arterioles, and three-fold higher after KTx then in children with PD and CKD5, respectively. Neutral lipids (oil red staining) were present in all three groups, more abundant in the media than in the intima, and two-fold increased in the media of children after KTx compared to children on PD (p = 0.018) and with CKD5 (p = 0.002). In vitro, methylprednisolone and tacrolimus, but not mycophenolate mofetil increased FASN abundance and activity in human umbilical arterial endothelial and vascular smooth muscle cells compared to media controls. Conclusion We for the first time comprehensively describe molecular pathways activated after KTx in children following a standard immunosuppressive regime. After KTx, arteriolar fatty acid biosynthesis, apoptosis and cell cycle arrest markers are increased as compared to arterioles from children with PD and CKD5; profibrotic pathways induced by PD are persistently activated. Arteriolar hubgene FASN is three-fold more abundant after KTx and neutral lipid deposition is increased, presumably due to the induction of FASN activity by methylprednisolone and tacrolimus.

Peritoneal transformation shortly after kidney transplantation in pediatric patients with preceding chronic peritoneal dialysis.

The unphysiological composition of peritoneal dialysis (PD) fluids induces progressive peritoneal fibrosis, hypervascularization and vasculopathy. Information on these alterations after kidney transplantation (KTx) is scant. Parietal peritoneal tissues were obtained from 81 pediatric patients with chronic kidney disease stage 5 (CKD5), 72 children on PD with low glucose degradation product (GDP) PD fluids, and from 20 children 4-8 weeks after KTx and preceding low-GDP PD. Tissues were analyzed by digital histomorphometry and quantitative immunohistochemistry. While chronic PD was associated with peritoneal hypervascularization, after KTx vascularization was comparable to CKD5 level. Submesothelial CD45 counts were 40% lower compared with PD, and in multivariable analyses independently associated with microvessel density. In contrast, peritoneal mesothelial denudation, submesothelial thickness and fibrin abundance, number of activated, submesothelial fibroblasts and of mesothelial-mesenchymal transitioned cells were similar after KTx. Diffuse peritoneal podoplanin positivity was present in 40% of the transplanted patients. In subgroups matched for age, PD vintage, dialytic glucose exposure and peritonitis incidence, submesothelial hypoxia-inducible factor 1-alpha abundance and angiopoietin 1/2 ratio were lower after KTx, reflecting vessel maturation, while arteriolar and microvessel p16 and cleaved Casp3 were higher. Submesothelial mast cell count and interleukin-6 were lower, whereas transforming growth factor-beta induced pSMAD2/3 was similar as compared with children on PD. Peritoneal membrane damage induced with chronic administration of low-GDP PD fluids was less severe after KTx. While peritoneal microvessel density, primarily defining PD transport and ultrafiltration capacity, was normal after KTx and peritoneal inflammation less pronounced, diffuse podoplanin positivity and profibrotic activity were prevalent.

Glucose degradation products in peritoneal dialysis solution impair angiogenesis by dysregulating angiogenetic factors in endothelial and vascular smooth muscle cells.

The accumulation of glucose degradation products (GDPs) during peritoneal dialysis (PD) can lead to immature angiogenesis in the peritoneum. However, the effect of GDPs on angiogenesis, at concentrations observed in dialysate effluent, has not been widely investigated. We do not know how the inflammation observed in PD-related peritonitis affects angiogenesis of the peritoneum. Human umbilical vessel endothelial cells (HUVEC) and human umbilical aortic smooth muscle cells (HUASMC) were used to examine the response to the three main GDPs found in peritoneal dialysate (methylglyoxal (MGO), 3-deoxyglucosone (3-DG), and 5-hydroxymethylfurfural (5-HMF). Supernatant from lipopolysaccharide (LPS)-activated murine macrophage cell lines (RAW 264.7 cells) were used to stimulate angiogenesis in the peritoneum. Changes in the expression of vascular endothelial growth factor-A (VEGF-A) and platelet-derived growth factor B (PDGFB) in HUVEC, and PDGF-receptor beta (PDGF-Rβ) in HUASMC, were examined by real-time PCR, Western blot, and ELISA. In HUVECs, the expression of PDGFB mRNA and protein were decreased by exposure to MGO, 3-DG, and 5-HMF at concentrations observed in dialysate effluent. A subsequent decrease in secreted PDGF-BB was observed. In HUASMCs, MGO and 5-HMF increased the expression of VEGF-A mRNA and protein, while 5-HMF decreased the expression of PDGF-Rβ. VEGF-A is upregulated, and PDGF-Rβ is downregulated, by conditioned medium of LPS-stimulated macrophages in HUASMCs. The GDPs of PD effluent cause an imbalance of angiogenic factors in endothelial cells and vascular smooth muscle cells that may lead to immature angiogenesis in the peritoneum.

Proteome-Wide Differential Effects of Peritoneal Dialysis Fluid Properties in an In Vitro Human Endothelial Cell Model.

To replace kidney function, peritoneal dialysis (PD) utilizes hyperosmotic PD fluids with specific physico-chemical properties. Their composition induces progressive damage of the peritoneum, leading to vasculopathies, decline of membrane function, and PD technique failure. Clinically used PD fluids differ in their composition but still remain bioincompatible. We mapped the molecular pathomechanisms in human endothelial cells induced by the different characteristics of widely used PD fluids by proteomics. Of 7894 identified proteins, 3871 were regulated at least by 1 and 49 by all tested PD fluids. The latter subset was enriched for cell junction-associated proteins. The different PD fluids individually perturbed proteins commonly related to cell stress, survival, and immune function pathways. Modeling two major bioincompatibility factors of PD fluids, acidosis, and glucose degradation products (GDPs) revealed distinct effects on endothelial cell function and regulation of cellular stress responses. Proteins and pathways most strongly affected were members of the oxidative stress response. Addition of the antioxidant and cytoprotective additive, alanyl-glutamine (AlaGln), to PD fluids led to upregulation of thioredoxin reductase-1, an antioxidant protein, potentially explaining the cytoprotective effect of AlaGln. In conclusion, we mapped out the molecular response of endothelial cells to PD fluids, and provided new evidence for their specific pathomechanisms, crucial for improvement of PD therapies.

FC088: Molecular and Functional Characterization of the Mesothelial and Endothelial Cell Barrier in Health, Ckd and Peritoneal Dialysis

Abstract BACKGROUND AND AIMS Tight junctions (TJ) and transcellular ion channels and transporters define solute transport characteristics across cellular barriers, which is of particular interest in patients on peritoneal dialysis (PD). Little is known about their cell specific expression, and regulation in PD. We studied their expression in human endothelial and mesothelial cell lines and in paediatric peritoneal tissues. METHOD In vitro, polarized primary human peritoneal mesothelial cells (HPMC), immortalized mesothelial cells (MeT-5A), human umbilical vein endothelial cells (HUVEC) and human capillary microvascular endothelial cells (HCMEC) underwent RNA sequencing, and gene enrichment analysis (GSEA, ClueGO/Cluepedia) for functional annotation. Key findings were reconfirmed by western blotting and confocal laser scanning immunofluorescence microscopy. Transepithelial electrical resistance (TER) and permeability fluxes of fluorescent 4-, 10- and 70-kDa dextran were measured in Transwells. Ex vivo, whole transcriptome and proteome data from microdissected omental arterioles were used for targeted pathway analysis in non-CKD children, chronic kidney disease (CKD5) and on PD with low and high glucose degradation product (GDP) content (n = 6/group). Mesothelial and endothelial peritoneal solute transporting proteins were quantified in parietal peritoneum of independent paediatric non-CKD, CKD5 and PD cohorts by digital immunohistochemistry. RESULTS A total of 9853 of 12 760 transcripts were common between all four cell lines. A total of 631 transcripts were MeT-5A, 366 HPMC, 99 HUVEC and 87 HCMEC specific. Next to the tissue origin and transformation status, the transcripts reflected major differences in extracellular matrix, glycocalyx and adhesion organization between HCMEC and HUVEC, and extracellular matrix, migration, growth factor and immune response between HPMC and MeT-5A cells. While total counts of cell junction, transmembrane and endocytosis related transcripts were similar among cell lines, the specific TJ, transmembrane and endocytosis related transcript patterns, differed substantially between endothelial and mesothelial cells. Of the functionally well described sealing TJs, claudin (CLDN)1 was expressed in mesothelial cells, and CLDN5 in endothelial cells. Findings were reconfirmed by western blotting and immunofluorescence staining. Functionally, transepithelial resistance (TER) was 50% lower for HCMEC compared to HPMC, MeT-5A and HUVEC; 4-, 10- and 70-kDa dextran permeability was increased in HCMEC. Ex vivo, human arteriolar pathway analysis demonstrated upregulation of paracellular transport-related pathways in CKD5 versus non-CKD patients on protein level. Compared to CKD5, low GDP PD upregulated and high GDP PD downregulated these pathways on the transcriptome and protein levels. Transcellular transporter pathway regulation was variable. In the parietal peritoneum the endothelial surface area for transport was age dependently 1.5–2-fold higher than the mesothelial surface area and the ratio markedly increased with low GDP PD. Validation in parietal peritoneal tissues, reconfirmed arteriolar sealing TJ regulation. Arteriolar CLDN2, a paracellular pore forming cation and water transporter, correlated with D/PCreatinine (r = 0.58) and D/D0Glucose (r = –0.62), mesothelial pore forming cation transporter CLDN15 with D/PCreatinine (r = 0.57) and D/D0Glucose (–0.66). Transcellular transporters for sodium, glucose and phosphate were hardly affected by PD; phosphate transporter PIT1 abundance correlated with serum phosphate (r = –0.48). CONCLUSION We provide the first comprehensive analysis of the peritoneal paracellular and transcellular determinants of solute transporters and its regulation by CKD and PD. Mesothelial and endothelial cell barrier sealing and transporter abundance differed substantially, and associated with PD membrane function, with functional data suggesting a key role of both the mesothelial and endothelial cell barrier.

MO714: PARK7—A Novel Therapeutic Target for Peritoneal Dialysis Induced Peritoneal Membrane and Vascular Transformation

Abstract BACKGROUND AND AIMS Patients with chronic kidney disease (CKD) suffer from increased oxidative stress, which is further aggravated in patients on peritoneal dialysis (PD). Parkinson disease protein 7 (PARK7) has antioxidant and antiapoptotic activity; its role in PD is unknown. METHOD Transcriptome and proteome data sets from microdissected omental arterioles obtained from age-matched non-CKD children, children with CKD5 and children on PD with fluids containing low or high concentrations of glucose degradation products (GDP; n = 6/group) underwent PARK7 related gene set analysis (FDR < 0.05). Respective parietal peritoneal tissues (n = 60) underwent digital histomorphometry analyses. PARK7 western blotting was performed in effluents of eight children on high-GDP PD. Human umbilical endothelial cells (HUVEC) viability (MTT assay) and transepithelial electrical resistance (TER, Transwells) and 4-, 10- and 70-kDa dextran transport were measured (n = 6–12/group). As an extreme phenotype approach of PD toxicity, C57/BL6J mice were treated with chlorhexidine digluconate (CG) and PARK7 activator for 7 days and parietal peritoneal damage was quantitated (n = 6–8/group). RESULTS Arteriolar transcriptome analyses in children on low-GDP PD demonstrated the enrichment of PARK7 related GO terms of oxidant detoxification as compared to CKD5 and in children on high-GDP PD that of reactive oxygen species-, mitochondria- and apoptosis-related processes. On the proteome level the DNA repair/organization, catabolic and mitochondria associated processes were enriched in children on low-GDP PD, and mitochondrial processes in children on high-GDP PD. PARK7 was detected in the parietal peritoneal tissues in mesothelial, endothelial and inflammatory cells, in myocytes and fibroblasts and was present in the PD effluents. Total peritoneal and submesothelial PARK7 abundance was similar in controls, patients with CKD5 and in patients on low-GDP PD, but 2-fold increased in patients on high GDP PD compared to controls and CKD5. Mesothelial PARK7 was 2-fold increased in children on low-GDP PD versus CKD5, endothelial PARK7 abundance was similar in all four groups. In low-GDP PD patients endothelial PARK7 abundance correlated with vessel lumen/vessel diameter ratio (r = 0.53, P = 0.06), i.e. inversely with lumen obliteration. Submesothelial PARK7 correlated with microvessel density (r = 0.55, P = 0.05), with submesothelial hypoxia inducible factor-1 and angiopoietin-1 and -2 (ρ = 0.63, P = 0.023; r = 0.91, P < 0.0001; r = 0.60, P = 0.03) but not with VEGF. Submesothelial and endothelial PARK7 correlated with respective caspase 3 abundances (r = 0.74/0.68, P = 0.009/0.015). In HUVEC methylglyoxal (MG) dose- and time-dependently reduced viability, coincubation with PARK7 activator partially preserved endothelial cell viability. In Transwells, MG treatment decreased TER and increased dextran transport, but none of them was improved by PARK7 activation. In mice treated with CG submesothelial thickness was 2-fold increased, microvessel density was unchanged; PARK7 protein abundance was 5-fold reduced. Co-treatment of CG with PARK7 activator prevented the submesothelial thickening. CONCLUSION PD modifies arteriolar PARK7 related biological processes of oxidant detoxification, mitochondria- and apoptosis-related processes. PARK7 is ubiquitously expressed in the parietal peritoneum and regulated by the GDP content of PD fluids. In patients on low-GDP PD, PARK7 abundance correlated with the degree of arteriolar lumen narrowing, and VEGF-independent angiogenesis. Activation of PARK7 preserves endothelial cell viability in vitro and prevents CG induced peritoneal membrane damage in mice and thus represents a potential novel therapeutic approach.

In Vitro Reactivity of the Glucose Degradation Product 3,4-Dideoxyglucosone-3-ene (3,4-DGE) towards Abundant Components of the Human Blood Circulatory System.

3,4-Dideoxyglucosone-3-ene (3,4-DGE) is a glucose degradation product present in processed foods and medicinal products. Additionally, its constant formation from 3-deoxyglucosone in plasma has been suggested. Due to its α,β-unsaturated dicarbonyl moiety, 3,4-DGE is highly reactive and has shown harmful effects in vitro. Here, we investigated the impact of major components of the human blood circulatory system on 3,4-DGE in vitro. Under physiological conditions, plasma concentrations of human serum albumin (HSA) reacted efficiently with 3,4-DGE, resulting in only 8.5% of the initial 3,4-DGE concentration after seven hours (vs. 83.4% without HSA, p < 0.001). Thereby, accessible thiol groups were reduced from 0.121 to 0.064 mol/mol HSA, whereas ketoprofen binding and esterase-like activity of HSA were not affected. Plasma concentrations of glutathione (GSH) reacted immediately and completely with 3,4-DGE, leading to two stereoisomeric adducts. Plasma concentrations of immunoglobulin G (IgG) bound to 3,4-DGE to a lower extent, resulting in 62.6% 3,4-DGE after seven hours (vs. 82.2% in the control, p < 0.01). Immobilized human collagen type IV did not alter 3,4-DGE concentrations. The results indicated that particularly HSA, GSH, and IgG readily scavenge 3,4-DGE after its appearance in the blood stream, which may be associated with a reduced antioxidative and cytoprotective activity for the living cells and, thus, the human organism by blocking free thiol groups.

Degradation and de novo formation of nine major glucose degradation products during storage of peritoneal dialysis fluids

Reactive glucose degradation products (GDPs) are formed during heat sterilization of glucose-containing peritoneal dialysis fluids (PDFs) and may induce adverse clinical effects. Long periods of storage and/or transport of PDFs before use may lead to de novo formation or degradation of GDPs. Therefore, the present study quantified the GDP profiles of single- and double-chamber PDFs during storage. Glucosone, 3-deoxyglucosone (3-DG), 3-deoxygalactosone (3-DGal), 3,4-dideoxyglucosone-3-ene (3,4-DGE), glyoxal, methylglyoxal (MGO), acetaldehyde, formaldehyde, and 5-hydroxymethylfurfural (5-HMF) were quantified by two validated UHPLC-DAD methods after derivatization with o-phenylenediamine (dicarbonyls) or 2,4-dinitrophenylhydrazine (monocarbonyls). The PDFs were stored at 50 °C for 0, 1, 2, 4, 13, and 26 weeks. The total GDP concentration of single-chamber PDFs did not change considerably during storage (496.6 ± 16.0 µM, 0 weeks; 519.1 ± 13.1 µM, 26 weeks), but individual GDPs were affected differently. 3-DG (− 82.6 µM) and 3-DGal (− 71.3 µM) were degraded, whereas 5-HMF (+ 161.7 µM), glyoxal (+ 32.2 µM), and formaldehyde (+ 12.4 µM) accumulated between 0 and 26 weeks. Acetaldehyde, glucosone, MGO, and 3,4-DGE showed time-dependent formation and degradation. The GDP concentrations in double-chamber fluids were generally lower and differently affected by storage. In conclusion, the changes of GDP concentrations during storage should be considered for the evaluation of clinical effects of PDFs.

Quantification of Degradation Products Formed during Heat Sterilization of Glucose Solutions by LC-MS/MS: Impact of Autoclaving Temperature and Duration on Degradation.

Heat sterilization of glucose solutions can lead to the formation of various glucose degradation products (GDPs) due to oxidation, hydrolysis, and dehydration. GDPs can have toxic effects after parenteral administration due to their high reactivity. In this study, the application of the F0 concept to modify specific time/temperature models during heat sterilization and their influence on the formation of GDPs in parenteral glucose solutions was investigated using high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). Glucose solutions (10%, w/v) were autoclaved at 111 °C, 116 °C, and 121 °C for different durations. The GDPs glyoxal, methylglyoxal, glucosone, 3-deoxyglucosone/3-deoxygalactosone, 3,4-dideoxyglucosone-3-ene, and 5-hydroxymethylfurfural were quantified after derivatization with o-phenylenediamine by an optimized LC-MS/MS method. For all GDPs, the limit of detection was <0.078 μg/mL, and the limit of quantification was <0.236 μg/mL. The autoclaving time of 121 °C and 15 min resulted in the lowest levels of 3-DG/3-DGal and 5-HMF, but in the highest levels of GO and 2-KDG. The proposed LC-MS/MS method is rapid and sensitive. So far, only 5-HMF concentrations are limited by the regulatory authorities. Our results suggest reconsidering the impurity limits of various GDPs, especially the more toxic ones such as GO and MGO, by the Pharmacopoeias.

The Effect of Far-Infrared Therapy on the Peritoneal Expression of Glucose Degradation Products in Diabetic Patients on Peritoneal Dialysis

The Effect of Far-Infrared Therapy on the Peritoneal Expression of Glucose Degradation Products in Diabetic Patients on Peritoneal Dialysis

The Effect of Far-Infrared Therapy on the Peritoneal Membrane Transport Characteristics of Uremic Patients Undergoing Peritoneal Dialysis: An Open-Prospective Proof-of-Concept Study.

Long-term peritoneal dialysis (PD) can lead to detrimental changes in peritoneal membrane function, which may be related to the accumulation of glucose degradation products. A previous study demonstrated that 6 months of far-infrared (FIR) therapy may decrease glucose degradation products in PD dialysate. Due to limited literature on this matter, this study aims to investigate the effect of FIR therapy on the peritoneal membrane transport characteristics of PD patients. Patients were grouped according to baseline peritoneal transport status: lower transporters (low and low-average) and higher transporters (high-average and high). Both groups underwent 40 min of FIR therapy twice daily for 1 year. In lower transporters, FIR therapy increased weekly dialysate creatinine clearance (6.91 L/wk/1.73 m2; p = 0.04) and D/P creatinine (0.05; p = 0.01). In higher transporters, FIR therapy decreased D/P creatinine (−0.05; p = 0.01) and increased D/D0 glucose (0.05; p = 0.006). Fifty percent of high transporter patients shifted to high-average status after FIR therapy. FIR therapy may decrease D/P creatinine for patients in the higher transporter group and cause high transporters to shift to high-average status, which suggests the potential of FIR therapy in improving peritoneal membrane function in PD patients.

In This Issue

HomeCirculation ResearchVol. 129, No. 5In This Issue Free AccessIn BriefPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessIn BriefPDF/EPUBIn This Issue Ruth Williams Ruth WilliamsRuth Williams Search for more papers by this author Originally published19 Aug 2021https://doi.org/10.1161/RES.0000000000000499Circulation Research. 2021;129:511is related toElectronic and Tobacco Cigarettes Alter Polyunsaturated Fatty Acids and Oxidative Biomarkersis related toGlucose Derivative Induced Vasculopathy in Children on Chronic Peritoneal DialysisSilencing Myeloid Netrin-1 Induces Inflammation Resolution and Plaque RegressionElectronic and Tobacco Cigarettes Alter Polyunsaturated Fatty Acids and Oxidative Biomarkers (p 514)Gupta et al measure oxidized lipid biomarkers to assess health risks of electronic cigarettes.E-cigarettes have surged in popularity in the last decade. While they are generally viewed as a safe alternative to smoking tobacco, studies suggest that E-cigarettes, like tobacco ones, cause oxidative stress, inflammation and endothelial dysfunction in users. Indeed, the aerosols produced during vaping contain comparable levels of reactive oxygen species (ROS) to those in tobacco smoke. Data on the extent to which E-cigarette ROS influence cardiovascular health is lacking, however. To address this, Gupta and colleagues recruited 32 chronic users of E-cigarettes, 29 chronic tobacco smokers, and 45 individuals that used neither, and measured their plasma levels of oxidative biomarkers. The team found similarities and differences between E-cigarette and tobacco users. Both, for example, had increased plasma antioxidant capacity and decreased levels of oxidized linoleic acid compared with the levels seen in non-users, while arachidonic acid levels were raised in tobacco smokers and reduced in the E-cigarette users. Overall, however, biomarker levels were deemed to be intermediate for E-cigarette users between non-users and tobacco users. The study suggests, therefore, that while E-cigarettes carry a lower health risk than tobacco ones, they are by no means safe.Download figureDownload PowerPointGlucose Derivative Induced Vasculopathy in Children on Chronic Peritoneal Dialysis (p e102)Glucose degradation products damage blood vessels during dialysis, report Bartosova et al.Diabetes, high blood pressure and obesity are risk factors for both cardiovascular disease (CVD) and chronic kidney disease (CKD). Worse still, loss of kidney function and even dialysis itself are thought to exacerbate cardiovascular issues. In the case of dialysis, it’s thought that high levels of glucose degradation products (GDPs) in the dialysis fluids can promote the addition of sugar moieties to vascular proteins and lipids (glycation), causing vessel damage. To investigate this theory, Bartosova and colleagues studied vascular tissue from children with CKD receiving dialysis fluid with either high levels or low levels of GDPs, or that were not on dialysis at all. Proteome and transcriptome analyses of the vessel tissues revealed that, compared with patients on no or low-GDP fluids, patients receiving high-GDP fluids had higher levels of damaging glycation, increased transcription of genes involved in cell death, and decreased transcription of genes involved in cell survival and cytoskeleton organization. In line with these findings, vessels from high-GDP patients displayed considerable evidence of damage (apoptosis, cytoskeletal disintergration, thickened intimas). The results thus confirm GDPs can cause vasculopathy and suggest low-GDP fluids should be favored for dialysis.Download figureDownload PowerPointTargeting Netrin-1 in Established Atherosclerosis Promotes Inflammation Resolution and Plaque Regression (p 530)Blocking netrin-1 diminishes atherosclerotic plaques in mice, say Schlegel et al.While excess lipids in the blood spur the development of atherosclerosis, the disease quickly becomes one of chronic inflammation that encourages the persistence of the lipid-filled plaques in vessel walls. Indeed, sustained inflammation is thought to explain why lipid-lowering drugs often aren’t enough to combat the condition. Inflammation-suppressing drugs are also being investigated, but because these may cause susceptibility to infections, Schlegel and colleagues reasoned that promoting resolution of inflammation may be a preferable goal. To that end, the team tested blocking nectrin-1 in atherosclerosis-prone mice. Originally identified as a factor essential for nerve cells, Netrin-1 has also been found in macrophages where it promotes persistence and survival of the cells in atherosclerotic plaques. The team showed that in mice with advanced atherosclerosis, turning off netrin-1 in macrophages—via an inducible genetic switch—led to significant plaque regression. Not only did plaque macrophages exhibit reduced retention, proliferation and survival, those that remained were more likely to be pro-resolving. While susceptibility to infection was not investigated in this study, the results suggest in principle that netrin-1 blockade could be a strategy for atherosclerosis treatment.Download figureDownload PowerPoint Previous Back to top Next FiguresReferencesRelatedDetailsRelated articlesElectronic and Tobacco Cigarettes Alter Polyunsaturated Fatty Acids and Oxidative BiomarkersRajat Gupta, et al. Circulation Research. 2021;129:514-526Glucose Derivative Induced Vasculopathy in Children on Chronic Peritoneal DialysisMaria Bartosova, et al. Circulation Research. 2021;129:e102-e118Silencing Myeloid Netrin-1 Induces Inflammation Resolution and Plaque RegressionMartin Schlegel, et al. Circulation Research. 2021;129:530-546 August 20, 2021Vol 129, Issue 5 Advertisement Article InformationMetrics © 2021 American Heart Association, Inc.https://doi.org/10.1161/RES.0000000000000499 Originally publishedAugust 19, 2021 PDF download Advertisement

Ongoing Exposure to Peritoneal Dialysis Fluid Alters Resident Peritoneal Macrophage Phenotype and Activation Propensity.

Peritoneal dialysis (PD) is a more continuous alternative to haemodialysis, for patients with chronic kidney disease, with considerable initial benefits for survival, patient independence and healthcare costs. However, long-term PD is associated with significant pathology, negating the positive effects over haemodialysis. Importantly, peritonitis and activation of macrophages is closely associated with disease progression and treatment failure. However, recent advances in macrophage biology suggest opposite functions for macrophages of different cellular origins. While monocyte-derived macrophages promote disease progression in some models of fibrosis, tissue resident macrophages have rather been associated with protective roles. Thus, we aimed to identify the relative contribution of tissue resident macrophages to PD induced inflammation in mice. Unexpectedly, we found an incremental loss of homeostatic characteristics, anti-inflammatory and efferocytic functionality in peritoneal resident macrophages, accompanied by enhanced inflammatory responses to external stimuli. Moreover, presence of glucose degradation products within the dialysis fluid led to markedly enhanced inflammation and almost complete disappearance of tissue resident cells. Thus, alterations in tissue resident macrophages may render long-term PD patients sensitive to developing peritonitis and consequently fibrosis/sclerosis.

Safety of electrooxidation for urea removal in a wearable artificial kidney is compromised by formation of glucose degradation products

A major challenge for the development of a wearable artificial kidney (WAK) is the removal of urea from the spent dialysate, as urea is the waste solute with the highest daily molar production and is difficult to adsorb. Here we present results on glucose degradation products (GDPs) formed during electrooxidation (EO), a technique that applies a current to the dialysate to convert urea into nitrogen, carbon dioxide, and hydrogen gas. Uremic plasma and peritoneal effluent were dialyzed for 8 hours with a WAK with and without EO‐based dialysate regeneration. Samples were taken regularly during treatment. GDPs (glyoxal, methylglyoxal, and 3‐deoxyglucosone) were measured in EO‐ and non‐EO‐treated fluids. Glyoxal and methylglyoxal concentrations increased 26‐ and 11‐fold, respectively, in uremic plasma (at [glucose] 7 mmol/L) and 209‐ and 353‐fold, respectively, in peritoneal effluent (at [glucose] 100 mmol/L) during treatment with EO, whereas no change was observed in GDP concentrations during dialysate regeneration without EO. EO for dialysate regeneration in a WAK is currently not safe due to the generation of GDPs which are not biocompatible.