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The peritoneum, pleura, and pericardium are yet understudied multicellular systems where mesothelial cells (MCs) and endothelial cells (ECs) are in close proximity. Crosstalk between these cell types likely plays role in molecular transport, immunological reactions, and metabolic processes in health, disease, and therapeutic intervention. In this review, we discuss recent proteomic efforts to characterize the crosstalk between MC and EC. We describe the proteomic methods necessary for investigation of crosstalk between MC and EC, as well as the in-vitro models that can be employed. Potential experimental approaches range from conditioned medium, via co-culture on semi-permeable membranes, to 3D cell culture based organoid models. While the biological and clinical relevance of the models may increase with their ability to mimic close cell communication, the practicality of these complex experiments corresponds vice versa, making standardization more difficult and expensive. Currently, data and reports on mesothelial-to-endothelial crosstalk are still very scarce. In our opinion, the in-vitro model using semi-permeable cell culture inserts will allow to establish a basic understanding of cellular crosstalk that may occur between those cell types. Later-on, more sophisticated 3D cell cultures may be better able to simulate the transport dynamics within the peritoneal membrane.

Most individuals with rare diseases initially consult their primary care physician. For a subset of rare diseases, efficient diagnostic pathways are available. However, ultra-rare diseases often require both expert clinical knowledge and comprehensive genetic diagnostics, which poses structural challenges for public healthcare systems. To address these challenges within Germany, a novel structured diagnostic concept, based on multidisciplinary expertise at established university hospital centers for rare diseases (CRDs), was evaluated in the three year prospective study TRANSLATE NAMSE. A key goal of TRANSLATE NAMSE was to assess the clinical value of exome sequencing (ES) in the ultra-rare disease population. The aims of the present study were to perform a systematic investigation of the phenotypic and molecular genetic data of TRANSLATE NAMSE patients who had undergone ES in order to determine the yield of both ultra-rare diagnoses and novel gene-disease associations; and determine whether the complementary use of machine learning and artificial intelligence (AI) tools improved diagnostic effectiveness and efficiency.ES was performed for 1,577 patients (268 adult and 1,309 pediatric). Molecular genetic diagnoses were established in 499 patients (74 adult and 425 pediatric). A total of 370 distinct molecular genetic causes were established. The majority of these concerned known disorders, most of which were ultra-rare. During the diagnostic process, 34 novel and 23 candidate genotype-phenotype associations were delineated, mainly in individuals with neurodevelopmental disorders.To determine the likelihood that ES will lead to a molecular diagnosis in a given patient, based on the respective clinical features only, we developed a statistical framework called YieldPred. The genetic data of a subcohort of 224 individuals that also gave consent to the computer-assisted analysis of their facial images were processed with the AI tool Prioritization of Exome Data by Image Analysis (PEDIA) and showed superior performance in variant prioritization.The present analyses demonstrated that the novel structured diagnostic concept facilitated the identification of ultra-rare genetic disorders and novel gene-disease associations on a national level and that the machine learning and AI tools improved diagnostic effectiveness and efficiency for ultra-rare genetic disorders.

Deep brain stimulation of the subthalamic nucleus is effective to alleviate motor symptoms in advanced Parkinson's disease. Using a novel conditioning paradigm, it has been shown that deep brain stimulation pulses from electrodes in the subthalamic nucleus modulate corticospinal excitability as determined with transcranial magnetic stimulation applied to the motor cortex. The mechanism of action is unclear.To investigate the effects of subthalamic nucleus and dorsal premotor cortex conditioning on corticospinal excitability as a function of interstimulus intervals between target areas and deep brain stimulation frequencies.In 19 patients with Parkinson's disease with subthalamic nucleus deep brain stimulation, the premotor-motor interaction was investigated in four different deep brain stimulation conditions (off, clinically used settings, 3 Hz, 20 Hz). Transcranial magnetic pulses were applied to the premotor and motor cortex and paired at certain intervals with deep brain stimulation pulses. The volume of tissue activated by deep brain stimulation was correlated with neurophysiological findings.There was distinct motor cortex inhibition by premotor cortex conditioning at an interstimulus interval of 1 ms before the motor cortex stimulation. Subthalamic nucleus conditioning with deep brain stimulation frequencies of 3 and 20 Hz at an interstimulus interval of 10 ms between subthalamic nucleus and primary motor cortex reduced premotor-motor inhibition. The volume of tissue activated by deep brain stimulation correlated positively with this effect. Corticospinal excitability was not affected by subthalamic nucleus conditioning as used here.Premotor-motor inhibition is modulated by subthalamic nucleus conditioning, presumably through the monosynaptic hyperdirect pathway.

Pathogenic variants in 5 genes (GCH1, TH, PTS, SPR, and QDPR), involved in dopamine/tetrahydrobiopterin biosynthesis or recycling, have been linked to Dopa-responsive dystonia (DRD). Diagnosis and treatment are often delayed due to high between- and within-group variability.Comprehensively analyzed individual genotype, phenotype, treatment response, and biochemistry information.734 DRD patients and 151 asymptomatic GCH1 mutation carriers were included using an MDSGene systematic literature review and an automated classification approach to distinguish between different forms of monogenic DRDs.Whereas dystonia, L-Dopa responsiveness, early age at onset, and diurnal fluctuations were identified as red flags, parkinsonism without dystonia was rarely reported (11%) and combined with dystonia in only 18% of patients. While sex was equally distributed in autosomal recessive DRD, there was female predominance in autosomal dominant DYT/PARK-GCH1 patients accompanied by a lower median age at onset and more dystonia in females compared to males. Accordingly, the majority of asymptomatic heterozygous GCH1 mutation carriers (8 years of age) were males. Multiple other subgroup-specific characteristics were identified, showing high accuracy in the automated classification approach: Seizures and microcephaly were mostly seen in DYT/PARK-PTS, autonomic symptoms appeared commonly in DYT/PARK-TH and DYT/PARK-PTS, and sleep disorders and oculogyric crises in DYT/PARK-SPR. Biochemically, homovanillic acid and 5-hydroxyindoleacetic acid in CSF were reduced in most DRDs, but neopterin and biopterin were increased only in DYT/PARK-PTS and DYT/PARK-SPR. Hyperphenylalaninemia was seen in DYT/PARK-PTS, DYT/PARK-QDPR, and rarely reported in autosomal recessive DYT/PARK-GCH1.Our indicators will help to specify diagnosis and accelerate start of treatment. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

In vitro studies are essential in pre-clinical research. While choice of cell lines is often driven by handling and cost-effectiveness, in-depth knowledge on specific characteristics is scant. Mesothelial cells, which interact with endothelial cells, are widely used in research, including cancer and drug development, but have not been comprehensively profiled. We therefore performed RNA sequencing of polarized, primary peritoneal (HPMC) and immortalized pleural mesothelial cells (MeT-5A), and compared them to endothelial cells from umbilical vein (HUVEC) and cardiac capillaries (HCMEC). Seventy-seven per cent of 12,760 genes were shared between the 4 cell lines, 1003 were mesothelial and 969 were endothelial cell specific. The transcripts reflected major differences between HPMC and MeT-5A in DNA-related processes, extracellular matrix, migration, proliferation, adhesion, transport, growth factor- and immune response, and between HUVEC and HCMEC in DNA replication, extracellular matrix and adhesion organization. Highly variable shared genes were related to six clusters, cell tissue origin and immortalization, but also cell migration capacity, cell adhesion, regulation of angiogenesis and response to hypoxia. Distinct, cell type specific biological processes were further described by cellular component-, molecular function- and Reactome pathway analyses. We provide crucial information on specific features of the most frequently used mesothelial and endothelial cell lines, essential for appropriate use.

Transcranial current stimulation (tCS) techniques have been shown to induce cortical plasticity. As an important relay in the motor system, the cerebellum is an interesting target for plasticity induction using tCS, aiming to modulate its excitability and connectivity. However, until now it remains unclear, which is the most effective tCS method for inducing plasticity in the cerebellum. Thus, in this study, the effects of anodal transcranial direct current stimulation (tDCS), 50 Hz transcranial alternating current stimulation (50 Hz tACS), and high frequency transcranial random noise stimulation (tRNS) were compared with sham stimulation in 20 healthy subjects in a within-subject design. tCS was applied targeting the cerebellar lobe VIIIA using neuronavigation. We measured corticospinal excitability, short-interval intracortical inhibition (SICI), short-latency afferent inhibition (SAI), and cerebellar brain inhibition (CBI) and performed a sensor-based movement analysis at baseline and three times after the intervention (post1 = 15 min; post2 = 55 min; post3 = 95 min). Corticospinal excitability increased following cerebellar tACS and tRNS compared to sham stimulation. This effect was most pronounced directly after stimulation but lasted for at least 55 min after tACS. Cortico-cortical and cerebello-cortical conditioning protocols, as well as sensor-based movement analyses, did not change. Our findings suggest that cerebellar 50 Hz tACS is the most effective protocol to change corticospinal excitability.

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.

BACKGROUND AND AIMS Due to chronic damage of the peritoneal membrane on long-term treatment, peritoneal dialysis (PD) is a time-limited therapy. Effective PD is achieved by adequate peritoneal ultrafiltration (UF) and small solute clearance while maintaining an appropriate fluid and salt homeostasis. Availability of remote patient monitoring on automated PD (APD) allows clinicians to remotely retrieve data for treatment analysis, proposed to improve clinical outcomes. However, machine-derived data are vastly different from traditionally observed data (e.g. quantity, noise, missing normative values). Recently correlation of daily UF changes on APD with changing peritoneal membrane function in association with clinical risk factors and outcomes has been observed. High intra- and interpatient variability of UF remains not fully explained. We aim at a better understanding of UF variability by submitting APD machine readouts to spectral analysis in association with clinical risk factors of membrane dysfunction. METHOD This was a secondary analysis of the Medical University of Vienna APD cycler treatment data from patients using Homechoice Pro cyclers (Baxter, IL, USA), extracted from the cycler management software (PD link, Baxter). Analysis was conducted using R software (R Core Team 2020). Daily UF was processed using the Lomb-Scargle periodogram (LSP), a method commonly used in astrophysics and neurophysiology to detect rhythms in (unevenly sampled) time series. Peaks of power spectrum, frequencies, and periodicities were compared between patients with and without peritonitis episodes during their PD treatment utilizing a Mann–Whitney U-test and associated with the total number of peritonitis episodes using Kendall rank correlation. A P-value RESULTS LSP analysis was performed on daily UF data from n = 129 APD patients with a mean observation time of 598 days (SD ± 517), male: female 61:39% (n = 79:50), incident: prevalent patients 77:23% (n = 99:30), and diabetic: non-diabetic patients 25:75% (n = 32:97). Observation time extended from the introduction of APD until kidney transplantation (43%, n = 55), transfer to hemodialysis (23%, n = 29), death (27%, n = 35), kidney function recovery (8%, n = 1), or loss to follow-up (7%, n = 9). After inspection of LSPs n = 9 patients were excluded from further analysis as their periodicity matched the time of their clinical endpoint. A total of 45 of the remaining 120 patients (38%) never experienced a peritonitis episode, and a total of 0.3 (SD ± 0.7) peritonitis episodes/patient-year were observed. LSP computation revealed cyclic periodicity in UF in all patients with an overall median peak period length of 329 days (IQR 62–882 days), median peak power of 21 (IQR 7–91), and median peak frequency of 0.42 (IQR 0.36–0.46). Differences between patients with and without peritonitis episodes during PD treatment are displayed in Table 1. Correlation analysis revealed significant association between the number of peritonitis episodes and peak periodicity of UF (tau=0.14, P = 0.04), and peak power (tau = 0.21, P = 0.003). Peak frequency of UF was not significantly correlated. CONCLUSION Patients on APD cycler treatment seem to display cyclic periodicity of UF, being significantly associated with the number of peritonitis episodes and significantly different between patients with and without peritonitis episodes. Smaller-scale periodicity (median 216 days) in patients without peritonitis seems to be disrupted by peritonitis resulting in significantly higher periodicity (median 670 days). The causes and implications of these results remain unclear and need further investigation. However, as peritonitis is a known risk factor for peritoneal membrane dysfunction, prolongation of UF periodicity might reflect disruption of peritoneal membrane function.

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.

BACKGROUND AND AIMS Infectious complications and peritoneal inflammation occurring in a significant proportion of PD patients lead to progressive peritoneal membrane injury and account for most peritoneal dialysis (PD) technique failures. Reduced peritoneal immune-competence, caused by the continuous exposure to PD fluids, has been described as a therapy-related pathomechanism. We therefore hypothesized a relationship between dialysate interleukin 6 (IL-6) concentrations and peritoneal host defense. We established an ex vivo stimulation assay to test host defense mechanisms in only 9 mL of PD effluent. The aim of this study was to analyse basal inflammation and immune-competence in the PD population at routine conditions to evaluate the assay as surrogate parameter of immune competence and linking it to PD vintage and clinical outcome parameters. METHOD We prospectively analysed 195 serial routine peritoneal equilibration tests (PETs) of 123 stable PD patients treated exclusively with low-GDP, multi-chamber PD fluids during the glucose dwells and compared the data to routine follow-up clinical data. The cohort represents 76% of all eligible PD patients treated between April 2013 and September 2020 at the local Department of Nephrology. The study was approved by the local ethics committee and was conducted in accordance with the Declaration of Helsinki. All participants underwent a standardized 4-h PET with 3.86% glucose PDF as routine follow-up to test peritoneal membrane transport function, with an additional ex vivo cell stimulation protocol. PD effluent samples were obtained at the 1- and 4-h PET timepoints and immediately processed. Effluent samples were collected directly from the drainage bags into standard 9 mL additive-free sample tubes. For ex vivo stimulation, 100 ng/mL toll-like receptor (TLR) 4 agonist LPS and TLR2 agonist Pam3Cys were added to the effluent in the 9 mL collection tubes in duplicates and incubated at 37°C for 24 h. Unstimulated samples kept in parallel were used as controls. IL-6 concentrations were measured with ELISA in the supernatants. RESULTS Patients were stratified into two cohorts (incident and prevalent) at 120 days of continuous PD therapy. The cohorts were statistically indistinguishable except for stratification-specific variables. During a total follow-up period of 2499 patient-months, 55 patients underwent repeated PETs. Ex vivo stimulation of peritoneal cells significantly increased the IL-6 release 20-fold compared to unstimulated controls and resulted in a dwell-time dependent increase, with a significant lower cytokine released at the 1 h PET timepoint. To assess local inflammation, IL-6 concentrations in PD effluent of the 4 h PET were analysed. PD effluent IL-6 concentrations from the entire study cohort or from incident and prevalent patient subcohorts correlated neither with patient characteristics, RRF, disease background (including prior peritonitis) nor with time on PD. Effluent IL-6 concentrations correlated significantly with markers of systemic inflammation, serum CRP and albumin, with PSTR and with peritoneal protein loss. Interestingly, effluent IL-6 concentrations also had predictive value for risk of a subsequent peritonitis episode during follow-up. Effluent IL-6 levels were, in contrast, negatively correlated with ex vivo–stimulated IL-6 release from PD effluent cells, most remarkably in PD patients with history of prior peritonitis. CONCLUSION This longitudinal study provides the first direct evidence in PD patients of a correlation between peritoneal inflammation and impaired peritoneal immune cell function, likely driving infectious complications. Our study results suggest a mechanistic link between peritoneal inflammation and host defense that may foster innovative therapeutic approaches to improve clinical outcomes of chronic PD.

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 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 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.

BACKGROUND AND AIMS Paediatric patients with chronic kidney disease (CKD) develop significant atherosclerosis and vascular calcifications until early adulthood. Largely devoid of confounding lifestyle related factors and underlying disease mostly limited to congenital abnormalities of the kidneys and urinary tract, these patients provide highly sensitive and specific information on early CKD-induced molecular mechanisms of vascular disease and on putative therapeutic targets. METHOD Standardized omental and parietal peritoneal tissue samples from 95 non-CKD individuals [median age 9.2 (interquartile range, IQR 15), 110 children with CKD5 (median age 8.6 (IQR 12)] underwent digital histomorphometry. Omental arterioles microdissected from surrounding fat tissue underwent whole-exome and proteome analyses, followed by gene set enrichment and Ingenuity pathway analysis. Vascular calcification pathway analysis was performed for 370 biological processes and molecular functions associated with vascular calcification extracted from Gene Ontology database. Key regulated pathways were validated by quantitative immunostaining. The effects of uraemic toxins on endothelial integrity were studied in vitro in human umbilical arterial and vein endothelial cells in Transwells. RESULTS Lumen to vessel diameter (L/V) ratio was reduced in patients with CKD5. Parietal peritoneal arteriole L/V ratio was 0.54 (0.2) versus 0.63 (0.1) in non-CKD controls, omental arteriole L/V ratio 0.58 (0.1) versus 0.76 (0.1) in controls (both P < 0.001), indicating significant CKD5 related vascular disease. These findings were independent of underlying disease entities and gender. The parietal peritoneal submesothelial space exhibited infiltration of single CD45 positive lymphocytes, mesothelial cells which had undergone epithelial-to-mesenchymal-transition, and isolated peritoneal fibrin deposits. Submesothelial TGF-ß induced pSMAD was 4-fold increased and IL-17A 2-fold, while VEGF was not different compared to non-CKD controls. Gene set enrichment analysis of omental arteriolar multi-omics identified enrichment of pathways including telomere extension by telomerase, chromatin histone methylation, actin cytoskeleton, integrin- and tight junction signaling, and focal adhesion in children with CKD5 children compared to controls (P < 0.05). Vascular calcification pathway analysis identified 16/370 pathways significantly enriched on arteriolar transcriptome (P < 0.01), related to Wnt signalling, extracellular matrix organization, complement activation, autophagy and ossification. Applying the same threshold on proteome level, 10 calcification-related arteriolar pathways were identified and included DNA damage, fatty acid metabolism, calcium ion binding, extracellular matrix organization and complement activation. In independent age-matched cohorts, CKD5 children had shorter endothelial telomere and less endothelial methylated histone 3. The endothelial complement system was activated and arteriolar actin cytoskeleton interacting proteins gamma actin and profilin-1 were reduced in CKD5, cofilin-1 remained unchanged. In vitro, methylglyoxal and 3.4-di-deoxyglucosone-3-ene reduced transendothelial resistance, increased endothelial monolayer permeability and induced cytoskeleton disassembly (zonula occludens-1 and F-actin). These effects were prevented by co-incubation with anserine, 3-methylhistidine and alanyl-glutamine, but not by carnosine, L-histidine, 1-methylhistidine and methyl-alanyl. CONCLUSION CKD5 results in major vascular ageing already in early childhood. Multi-omics analysis of omental arterioles identified specific mechanisms of CKD-induced vascular ageing and of vascular calcification. Endothelial cell barrier integrity is impaired, and in vitro reversed by specific dipeptides.

BACKGROUND AND AIMS Peritoneal dialysis effluent (PDE) is a rich but underexplored source of molecular markers for therapy monitoring and investigation of deregulated processes during PD. Modern high performance mass spectrometry (MS) and sequencing methods allow monitoring of hundreds of analytes in parallel. For understanding PD related processes on a systems biology level, a multilevel omics approach is particularly attractive. Here, we investigate the cellular transcriptome and cell-free proteome of PDE samples in combination with the publicly available Human Plasma Proteome Database to investigate the origin of proteins found in peritoneal dialysis effluent. METHOD Samples were obtained from clinically stable patients on chronic peritoneal dialysis during a highly standardized clinical routine check. The effluent material was separated into a cellular and cell-free component. Soluble proteins in the cell-free compartment were processed using our equalizing and TMT-labeling workflow followed by LC-MS. The cellular material was subjected to RNA sequencing. The Human Plasma Proteome database (peptideatlas.org/hupo/hppp) was used for referencing plasma proteins and estimating plasma concentration. A bioinformatic workflow conjoined information from the datasets to reveal novel insights into the ‘PD effluentome,’ especially clarifying the source of proteins found in PDE. RESULTS Combining two targeted metabolomics methods enabled detecting 207 unique metabolites in cell-free PDE. Metabolites not detected in our samples were included in the panel for in vitro studies of cellular systems. A mixed-effect ANOVA of all metabolites demonstrated dwell time-dependent concentration changes in 173 metabolites. Post-hoc testing revealed most metabolites to be changed between 1 and 16 h [ON] of fluid dwell (160), followed by 114 and 46 differently concentrated metabolites between 4 and 16 h and 1 and 4 h of dwell, respectively. We quantified 9797 transcripts in PD-effluent cells and 2729 solved proteins in PD effluent. A total of 342 proteins were filtered from plasma, while 800 proteins were attributable to local production. A quantitative analysis of the interaction proteome and cellular transcripts of roughly 1700 protein-transcript pairs showed clusters of proteins explained by overexpression in peritoneal cells compared to plasma concentrations. CONCLUSION Multi-omic profiling of PD effluent proved to be a valuable approach for revealing small molecule related changes during PD treatment. The exploitation of PD effluent information on multiple omics levels as identified by our bioinformatic approach has been shown to improve our understanding of the molecular processes in the peritoneal cavity and their role in development of complications for ultimately improving PD therapy. The combinatorial investigation of proteome and transcriptome of PD effluent represents a first step in identifying locally produced proteins for further validation as biomarkers of peritoneal health in peritoneal dialysis patients. Proteins of plasma origin could be tested for their value as diagnostic tools in monitoring treatment success and protein transport over the peritoneal barrier. Our work suggests feasibility of multi-omics approaches to investigate cell-derived biomarkers for their involvement in pathomechanisms relevant in PD.

BACKGROUND AND AIMS Long-term usage of peritoneal dialysis (PD) leads to morphological and functional changes—such as progressive thickening, vascularization and vasculopathy—of the peritoneum, thereby limiting PD efficiency. Vasculopathy induces changes in permeability of the endothelial barrier, which has been suggested to influence the gut microbiome (gMB). Microbial dysbiosis in turn can potentiate inflammation, a risk factor for cardiovascular disease (CVD). The complex interplay of molecules associated with CKD and PD with the gMB is hypothesized to be in causal relationship with occurrences of cardiovascular events and progression of CVD. Novel PD fluids that slow or prevent these processes are in need. The immunomodulatory and cytoprotective compound alanyl-glutamine (AG) has been shown in other model systems to improve the endothelial barrier function, supposedly protecting the gMB from the extraintestinal environment. Our aim is to identify changes of the gMB and the systemic metabolome caused by the addition of AG to PD under conditions of CKD. METHOD Mice (C57/Bl6 NCrl) underwent a 5/6 nephrectomy to induce uraemia. Via a subcutaneously implanted catheter the animals received five times per week a daily injection of 2 mL PD fluid (3.86% glucose with/without 8 mM AG) for six weeks to a total of 30 injections. During the experiment, mice received standard chow and tap water ad libitum. At the end of the experiment, fecal samples from three different intestinal sites and stool were collected and a serum sample was drawn from each mouse. Tissue samples from parietal and visceral peritoneum and relevant organs were preserved for paraffin and cryo sections. Peritoneal effluent cells were spun down and snap-frozen in liquid nitrogen and the supernatant was stored on −80°C. Bacterial ribosomal RNA (rRNA) was isolated from each fecal sample and the V4 region of the 16S rRNA was amplified. Assessment of the plasma metabolome was performed by targeted mass spectrometry (MxP Quant 500, Biocrates, Austria). The experiment was approved by the institutional animal ethics committee. RESULTS Elevated creatinine levels after subtotal nephrectomy confirmed the uremic status of the mice. Descriptive analysis of the gMB showed decreased Shannon diversity, indicating reduced bacterial diversity, in uremic and PD fluid receiving mice over time, whereas AG receiving and control mice retained a stable Shannon diversity. Furthermore, the gMB of mice exposed to PD fluid with AG showed five bacterial classes and 13 bacterial orders to be differently abundant versus mice exposed to PD fluid without AG. In the colon of uremic mice Clostridia was decreased versus control. Serum concentrations of 630 metabolites were assessed by our metabolomics approach. A significant increase in alanine and glutamine serum concentrations was seen in mice exposed to AG supplemented PD fluid. Further, 17 amino acids and four biogenic amines showed to be differently abundant in mice exposed to AG supplemented PD fluid versus without AG. Lastly, plasma of uremic mice showed a significant increase of the toxic amino acid symmetric dimethyl arginine (SDMA) and citrulline. CONCLUSION Exposure to PD fluid and having chronic kidney disease both affect the gut microbiome and systemic metabolome. Supplementation of AG to PD fluid showed changes at overall microbiome diversity and serum metabolites. Further analysis of these changes in the context of tissue alterations will clarify functional differences of the cytoprotective additive AG. Preservation of the mesothelial and endothelial barrier function may represent a mechanistic explanation for the observed improvement in protein loss and systemic inflammation in a recent phase II clinical trial in PD patients. Better understanding of the gut-peritoneal barrier may open up new therapeutic targets for improving clinical outcome of PD treatment and its long-term efficacy.

BACKGROUND AND AIMS Peritoneal dialysis (PD) is a life-saving renal replacement therapy. The composition of all currently available peritoneal dialysis fluids (PDF) triggers morphological and functional changes in the peritoneal membrane. Periodic exposure leads to vasculopathy, hypervascularization and diabetes-like damage of vessels, eventually leading to failure of the technique. In vitro and in vivo studies have shown that cytoprotective additives [e.g. dipeptide alanyl-glutamine (AlaGln) or kinase inhibitor lithium chloride (LiCl)] to PDF reduce peritoneal damage in mesothelial (MC) and endothelial (EC) cells. However, it is yet unclear if potential cross talk between cells present in the peritoneal membrane mediates the observed modulation of PD-associated changes. Currently, there is no model system available to study relevant processes in these cells when in close proximity. Here, we aimed to develop a co-culture model for investigating cell-to-cell communication in a PD-relevant setting using cellular proteome and secretome analyses and investigate the effects of cytoprotective additives. METHOD For modelling the peritoneal membrane in vitro, MCs and ECs were co-cultured in transwell plates. MCs were grown in the upper compartment and primary microvascular ECs were grown in the lower compartment. PDF with or without additives, was added to the upper compartment to expose MCs directly, while the ECs below were kept in medium. Cellular proteome and secretome profiles were analysed for both cell types cultured individually or together by a quantitative mass spectrometry approach. Prior to analysis of the secretome, a combinatorial peptide ligand library coupled to beads was employed to enrich low abundant proteins and deplete high abundant proteins. RESULTS Proteome analysis revealed perturbation of major cellular processes including cytoskeleton reorganization, stress response, and regulation of cell death that characterize PDF cytotoxicity. Co-cultured cells yielded differently regulated pathways following PDF exposure compared to individual cultures. Several pathways relevant for PD, such as VEGF signaling in ECs or oxidative stress response in MCs were found only under co-culture conditions. Combined analysis of proteome and secretome showed specific ligand–receptor pairs expressed only under co-culture conditions regulating some of these pathways. Differentially regulated cellular and secreted proteins after PDF exposure were related to processes like angiogenesis, immune response, or and extracellular processes such as integrin signaling. Addition of 10 mM LiCl to PDF led to modulation of VEGF and oxidative stress response pathways in ECs as well as ligand–receptor pairs involved in these pathways, possibly explaining MC–EC crosstalk during PDF exposure of these cells and the cytoprotective effect of LiCl. CONCLUSION Harmful effects of PDF on MCs may also affect ECs, as demonstrated by our data. Interestingly, both cells type react differently when co-cultured compared to individual culture models, showing the importance of models that allow cell type interaction to mimic the in vivo conditions. We identified potential signaling axes between the cell types that could explain pathophysiological changes of the peritoneal membrane during PD treatment. We have also elucidated potential mechanisms by which the cytoprotective additive LiCl may modulate crosstalk between MCs and ECs to counteract adverse PDF effects in the local peritoneal environment. Linking local peritoneal damage with systemic vasculopathy through perturbations of PD-induced cellular crosstalk, may identify therapeutic targets to reduce the cardiovascular risk of PD patients and current limitations of the therapy.

Complex dystonias are defined as dystonias that are accompanied by neurologic or systemic manifestations beyond movement disorders. Many syndromes or diseases can present with complex dystonia, either as the cardinal sign or as part of a multi-systemic manifestation. Complex dystonia often gradually develops in the disease course, but can also be present from the outset. If available, the diagnostic workup, disease-specific treatment, and management of patients with complex dystonias require a multi-disciplinary approach. This article summarizes current knowledge on complex dystonias with a particular view of recent developments with respect to advances in diagnosis and management, including causative treatments.

Western blotting is widely used for protein identification and quantification in research applications, but different protein species, resulting from alternative splicing and post-translational modifications, can often only be detected individually by two-dimensional gel electrophoresis and immunodetection by Western blotting (2D-WB). The additional separation by isoelectric focusing enables the detection of different protein species with the same specific antibody. Reliable assignment of signals from antibody-based detection to the total protein spot pattern of the original gel image is a challenge in 2D-WB, often resulting in ambiguous results. We therefore propose a reliable strategy for assignment of antibody signals from 2D-WB to the total protein spot pattern, using an imaging workflow in combination with a straightforward and easily reproducible image alignment strategy. The strategy employs vector-based alignment of protein spots and image contours in a stepwise manner. Our workflow is compatible with various protein visualization techniques, including prelabeling of proteins and poststaining of gels and membranes, as well as with chemiluminescent and fluorescent detection of bound antibody. Here, we provide a detailed description of potential applications and benefits of our workflow. We use experimental test settings with gold-standard stressors in combination with multiple staining and detection methods, as well as spike-in recombinant proteins. Our results demonstrate reliable attribution of signals to very similar heat shock proteins, phosphorylation patterns, and global analysis of proteins modified with O-linked N-acetylglucosamine (O-GlcNAc).

Used hemodialysis membranes (HD-M) are a valuable reservoir of biological information. Proteins bind to HD-M, but whether this process depends on the type of membrane or patient factors or selectively affects specific protein classes has not been adequately elucidated. State-of-the-art proteomics techniques are capable of identifying and quantifying this therapy-specific subproteome to enable the analysis of disease- or membrane-induced pathophysiologies. We demonstrate the feasibility of the deep proteomic characterization of the extracorporeal proteome adsorbed to HD-M. A shotgun proteomics approach using nano-flow liquid chromatography coupled to mass-spectrometry identified 1648 unique proteins eluted by a chaotropic buffer from the HD-M of eight patients. In total, 995 proteins were present in all eluates; a more stringent approach showed that a core proteome of 310 proteins could be identified independently in all samples. Stability of the dialyzer proteome was demonstrated by a >90% re-identification rate on longitudinal samples of a single patient. The core proteome showed an overrepresentation of pathways of hemostasis and the immune system, and showed differences in membrane materials (polysulfone vs. helixone). This study demonstrates that optimized conditions combined with high-performance proteomics enable the in-depth exploration of the subproteome bound to HD-M, yielding a stable core proteome that can be exploited to study patient-specific factors and improve hemodialysis therapy.

In medical tasks such as human motion analysis, computer-aided auxiliary systems have become the preferred choice for human experts for their high efficiency. However, conventional approaches are typically based on user-defined features such as movement onset times, peak velocities, motion vectors, or frequency domain analyses. Such approaches entail careful data post-processing or specific domain knowledge to achieve a meaningful feature extraction. Besides, they are prone to noise and the manual-defined features could hardly be re-used for other analyses. In this paper, we proposed probabilistic movement primitives (ProMPs), a widely-used approach in robot skill learning, to model human motions. The benefit of ProMPs is that the features are directly learned from the data and ProMPs can capture important features describing the trajectory shape, which can easily be extended to other tasks. Distinct from previous research, where classification tasks are mostly investigated, we applied ProMPs together with a variant of Kullback-Leibler (KL) divergence to quantify the effect of different transcranial current stimulation methods on human motions. We presented an initial result with 10 participants. The results validate ProMPs as a robust and effective feature extractor for human motions.

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.

Background and Aims Patients with chronic kidney disease patients (CKD) have an exceedingly high cardiovascular risk. While vasculopathy is further accelerated during peritoneal dialysis (PD), the pathophysiological role of reactive metabolites such as glucose degradation products (GDP) is uncertain. Method Omental and parietal peritoneal tissues from 100 non-CKD individuals, 107 children with CKD5, 60 children treated with neutral pH, low GDP, and 30 children treated with acidic pH, high GDP PD fluids underwent standardized digital histomorphometry. Omental arterioles localized within the fat tissue, protected from direct PD fluid exposure were microdissected for multi-omics analysis. Key regulated pathways were validated by quantitative immunostaining, with localization microscopy in peritoneal tissues of matched cohorts and in vitro in human umbilical vein endothelial cells. Results Arterioles from children with CKD5 exhibited reduced lumen to vessel ratio (L/V) and reduced endothelial telomere length compared to non-CKD individuals; gene ontology analysis identified enrichment of arteriolar genes associated with nuclear telomere cap complex and focal adhesion. Pathway analysis of arteriolar cross-omics identified top canonical pathways including telomere extension by telomerase, actin cytoskeleton, integrin and tight junction signalling. Peritoneal vasculopathy progressed with PD vintage and was more pronounced with high versus low GDP exposure (p Quantitative validation in PD cohorts with similar PD vintage, dialytic glucose exposure and age (n=15 / group) verified increased proapoptotic activity and cytoskeleton disintegration with high-GDP exposure; single-molecule-localization microscopy demonstrated arteriolar endothelial zonula occludens-1 (ZO-1) disruption. Absolute and relative to endoluminal surface length, arteriolar endothelial cell counts were inversely correlated with GDP exposure, with apoptosis marker caspase-3, TGF-ß induced pSMAD2/3, interleukin-6, ZO-1 protein abundance and the degree of vasculopathy. In vitro, exposure to GDP 3,4-dideoxyglucosone-3-ene dose-dependently reduced nuclear endothelial lamin-A/C and membrane ZO-1 assembly. Transendothelial electrical resistance was decreased. ZO-1 and sealing tight junction claudin-5 protein abundance were decreased in cells after incubation with high GDP compared to low GDP PD fluid and culture media. On nanoscale level GDP reduced junction cluster formation in the membrane area. Conclusion Multi-omics analysis of omental arterioles from children without pre-existing vasculopathy and life-style related confounders identified key mechanisms of vascular aging in CKD5 and the major contribution of GDP to accelerated vasculopathy during PD, i.e. disruption of endothelial cell junctions and cytoskeleton and induction of apoptosis.

Background and Aims Renal replacement therapy by peritoneal dialysis (PD) is limited in use and duration by progressive impairment of peritoneal membrane integrity and homeostasis. Preservation of peritoneal membrane integrity during chronic PD remains an urgent but long-unmet medical need. PD therapy failure results from peritoneal fibrosis and angiogenesis caused by hypertonic PD fluid (PDF)-induced mesothelial cytotoxicity. The incompletely defined pathophysiological mechanisms involved confound informed selection of therapeutic targets. Addition of cytoprotective agents to PDF have been shown to counteract pathophysiological mechanisms induced by current PDF. Lithium is a well described inhibitor of glycogen synthase kinase 3β and has recently been shown to also have nephroprotective effects in low doses. Here, we aim to characterize icodextrin-based, PDF-induced cellular injury with a combined omics approach and to investigate the effects of LiCl on the PD-induced observed molecular perturbations. Method To investigate mechanisms of acute cellular damage by PDF we chose an in vitro model of primary omental-derived peritoneal mesothelial cells with direct exposure to icodextrin-based PDF, followed by short-term or extended recovery for detection of short-term and long-term changes in transcriptome, proteome, and cell injury. 0, 2.5 or 10 mM LiCl were added to the PDF. In-vitro findings were validated in peritoneal biopsies (n=41) from pediatric PD and CDK5 patients or healthy controls and peritoneal effluents from adult and pediatric PD patients (n=27) or ascites samples (n=4) as control. For in-vivo experiments, healthy and uremic mice (C57/Bl6, female) were chronically exposed to PD-fluid without or with the addition of 5 mM LiCl via an implanted catheter. In-vivo overexpression of CRYAB was induced by i.p. injection of an adenoviral vector. All animal experiments and use of patient samples were approved by the local ethics committees and performed according to animal protection laws or the Declaration of Helsinki, respectively. Results LiCl significantly improved mesothelial cell survival in a dose-dependent manner. Combined transcriptomic and proteomic characterization of icodextrin-based PDF-induced mesothelial cell injury identified αB-crystallin as the mesothelial cell protein most significantly and consistently counter-regulated by LiCl. In-vitro and in-vivo overexpression of αB-crystallin triggered a fibrotic phenotype and PDF-like upregulation of vascular endothelial growth factor (VEGF), CD31-positive cells, and TGFβ-independent activation of TGFβ-regulated targets. In contrast, αB-crystallin knock-down decreased VEGF expression and early mesothelial-to-mesenchymal transition (MMT). LiCl reduced VEGF release and counteracted fibrosis- and angiogenesis-associated processes. αB-crystallin in patient-derived mesothelial cells was specifically upregulated in response to PDF and increased in peritoneal mesothelial cells from pediatric PD patient biopsies, correlating with markers of angiogenesis and fibrosis. Conclusion The cytoprotective effects of LiCl-supplemented PDF may be explained by counter-regulation of PD-induced angiogenesis via the novel target αB-crystallin. Reduction of mesothelial cell damage, peritoneal fibrosis and VEGF suggests therapeutic potential of this intervention. Repurposing LiCl as a cytoprotective PDF additive may offer a translatable therapeutic strategy to combat peritoneal membrane deterioration during PD therapy. Further study of LiCl-supplemented PDF is merited as a realistic approach to improving treatment longevity and patient outcomes during PD treatment.

Background and Aims Due to the unphysiological composition of PD fluids, chronic peritoneal dialysis (PD) induces progressive peritoneal fibrosis, hypervascularization, and vasculopathy. The evolution of the PD membrane and vasculopathy following kidney transplantation (KTx) is largely unknown. Method Arteriolar and peritoneal tissues were obtained from 107 children with chronic kidney disease (CKD5), 72 children on PD (treated with neutral pH PD fluids, with low glucose degradation product content, GDP) and 21 children, who underwent KTx 4-5 weeks after a median 21 months of PD. Specimen underwent standardized digital quantitative histomorphometry. Molecular mechanisms were studied in omental arterioles microdissected from surrounding fat by multi-omics followed by Gene Set Enrichment Analysis (GSEA); key findings were validated in parietal tissues of independent, matched cohorts by quantitative immunohistochemistry (n=15/group). Results Arteriolar transcriptome and proteome GSEA revealed suppression of leucocyte migration and T-cell activation / secretory pathways regulation, of sprouting angiogenesis biological processes and of epithelial proliferation and cell cycle after KTx as compared to PD. Lipid / fatty acid metabolism, autophagy and ATP synthesis pathways were activated. Transcriptome analysis including KTx, PD and CKD5 specifically attributed regulation of arteriolar lipid and fatty acid metabolism to transplantation and comprised 140 transcripts; their regulation was confirmed on the proteome level. Hub gene fatty acid synthase was identified by protein interaction analysis (string-db.org). 15 arteriolar genes activated by PD were inactivated after KTx and included glucose metabolisms and cytoskeleton related transcripts. 24 transcripts and 10 corresponding proteins induced by PD were still active after KTx and associated with biological processes related to TGF-ß signaling, fibrosis and mineral absorption. In line with arteriolar multi-omics findings, peritoneal hypervascularization induced by chronic PD was reversed after Tx to CKD5 level. CD45 positive tissue infiltrating leucocytes count was reduced by 40% and was independently associated with microvessel density in multivariable analysis including PD vintage, daily GDP exposure and recent KTx. Peritoneal lymphatic vessel density, submesothelial thickness, activated fibroblast, fibrin deposit, macrophage and EMT cell counts remained unchanged after KTx compared to PD. Arteriolar lumen to vessel ratios (a marker of vasculopathy) were similar in both groups. Vessel-homeostasis-related proteins in independent, matched cohorts demonstrated increased caspase-3 abundance in peritoneal arterioles after KTx. Arteriolar VEGF-A, thrombospondin, angiopoietin1/2, and hypoxia-inducible factor-1 (HIF-1a) were unchanged, while submesothelial HIF-1a and angiopoietin1/2 were decreased after Tx, favoring vessel maturation. The abundance of the key driver of fibrosis, TGF-ß-effector pSMAD2/3, was unchanged in the peritoneum and arterioles after Tx. Conclusion Our multi-omics analyses of fat covered omental arterioles, not directly exposed to PD fluids, demonstrate inhibition of PD induced immune response and angiogenesis pathways, of glucose metabolism and cytoskeleton regulation to levels similar as seen in children with CKD5. Arteriolar lipid and fatty acid metabolism is selectively altered after KTx. Reversal of low GDP PD induced hypervascularization and inflammation of the parietal peritoneum after KTx, mirror molecular changes in omental arterioles, while profibrotic activity persists after KTx in omental arterioles and in the parietal peritoneum.

Background and Aims Infectious complications occur in a significant proportion of PD patients, limiting long-term applicability. Reduced peritoneal immune-competence, caused by the continuous exposure to PD-fluids, has been described as a therapy-related pathomechanisms, prompting the need for a tool to assess the functional peritoneal immune status. We established an ex-vivo stimulation assay to test host defence mechanisms in only 9ml of PD-effluent. The aim of this study was to analyse basal inflammation and immune-competence in the general PD population at routine conditions to evaluate the assay as surrogate parameter of immune competence and linking it to PD vintage and clinical outcome parameters. Method 147 of 284 (51.8%) adult and paediatric PD patients treated between April 2013 and September 2020 at the local Department of Nephrology were included in the analysis. The study was approved by the local ethics committee and was conducted in accordance with the Declaration of Helsinki. Patients were exclusively treated with neutral pH/multi-chamber PD fluids during the glucose dwells. The majority of the 558 included PD-effluent samples were obtained during standard 4-hours peritoneal equilibration tests (PET) with 3.86% glucose containing PDF. Samples from the pre-PET dwell and at PET time points 1-hour and 4-hours were collected and immediately processed. Additional effluent samples were obtained during unscheduled hospitalization and in the event of an acute peritonitis. Effluent samples were collected directly from the drainage bags into standard 9 ml additive-free sample tubes. For ex-vivo stimulation, 100 ng/ml toll-like receptor (TLR) 4 agonist LPS and TLR2 agonist Pam3Cys were added to the effluent in the 9 ml collection tubes in duplicates and incubated at 37°C for 24h. Unstimulated samples kept in parallel were used as controls. IL-6 and TNF-α concentrations were measured with ELISA in the supernatants. Results Ex-vivo stimulation of peritoneal cells significantly increased the IL-6 and TNF-α release compared to unstimulated controls and resulted in a dwell-time dependent increase, with a significant lower cytokine released at the 1h PET time point. To assess local inflammation IL-6 levels of crude effluent were determined. IL-6 concentrations remained stable over time on PD. Interestingly, we were able to show higher IL-6 levels in CAPD patients in comparison to APD. As chronic exposure to PD-fluids has been shown to dampen the peritoneal immune competence, consecutive peritoneal effluent bags, obtained from patients were analysed. In this subcohort of 183 4h-PET effluents we found a decline in cytokine secretion with time on PD (IL-6 r=-0.27, p=0.00015, TNFa r=-0.25, p=0.00071). In a subgroup the ex-vivo cytokine release of effluent samples from patients with an acute peritonitis was assessed. IL-6 levels of acute peritonitis effluent samples did not differ from the stimulated IL-6 levels of effluent samples without acute peritonitis (2.45 pg/mL vs 2.31 pg/mL, p=0.85, t-test) suggesting that the assay seemingly represents the in-vivo host-defence cytokine release accurately. Conclusion The study provides evidence of a correlation of declining local host defence and duration of PD-therapy. It supports the hypothesis of PD duration-dependent progressive impairment of the ability of the peritoneal immune cells to secrete cytokines in response to a pathogenic stimulus and thereby dampening the global peritoneal immuno-competence. This suggests the utility of this clinically feasible ex-vivo induced cytokine-release assay in peritoneal effluent as a surrogate of the functional peritoneal immune competence. Future analyses need to evaluate the assay as a tool to predict common clinical outcomes and define reference values to facilitate stratification of patient populations, clinical staging and to guide novel therapeutic interventions.

Background and Aims Reactive oxygen species (ROS) in the peritoneal cavity may result both from CKD and the specific composition of peritoneal dialysis fluids (PDF). Elevated cellular oxidative stress is defined as a cellular oxidant/antioxidant imbalance which impairs not only peritoneal cell viability but also contributes to progression of local and systemic PD-related pathomechanisms. So far only single targets or mediators of oxidative stress were investigated in mesothelial cells exposed to PD fluids. Here, we aim to analyze the broad impact and also identify individual targets of ROS during PD. Using the developed technique the anti-oxidative effect of alanyl-glutamine (AlaGln) supplementation of PDF was characterized on the proteome level. Method To establish a redox-proteomics workflow for studying oxidative stress in peritoneal mesothelial cells we used a gold-standard model of redox-stress (100 µm hydrogen peroxide (H2O2)) and PD-fluid induced stress. Levels of oxidative stress were first evaluated by intracellular ROS levels and superoxide dismutase activity. Oxidative stress levels induced by PDF were titrated to comparable levels of H2O2 treatment to be able to characterize redox modifications and the effect of addition of 8 mM AlaGln. To detect alterations of the redox proteome we adapted and refined an approach combining redox-sensitive isobaric mass tags and high-performance liquid chromatography coupled to mass spectrometry (LC/MS). We used a sequential combination of direct and indirect labeling of redox-sensitive cysteine residues. Results Exposure to PDF increased intracellular ROS production and accumulation as well as cell damage assessed by LDH-release compared to control cells. Cells exposed to AlaGln supplemented PDF showed less cell damage compared to PDF alone. Addition of AlaGln not only reduced the overall redox status (intracellular ROS and superoxide dismutase activity) but also led to different proteins being affected by redox modifications. The carefully optimized highly sensitive LC/MS-based redox proteomics workflow allowed identification of 5537 proteins of which 2614 contained a labeled cysteine. H2O2 treatment resulted in a shift of median oxidation from 11% under control conditions to 36%. While PDF alone increased the oxidation level to 31%, AlaGln supplemented PDF only led to 15% oxidation. Pathway analysis of proteins that changed their oxidation level >50% following the treatment were subjected to molecular pathway analysis revealing distinct differences. PDF exposure leads to regulation of general cell processes like regulation of glucokinase, RNA-binding and SUMOylation, addition of AlaGln regulated more specific signaling pathways for example fibrosis related pathways like TGF-ß and SMAD signaling. Conclusion Redox proteomics of peritoneal cells could represent a novel tool for the identification of mediators of uraemia and PD-induced pathomechanisms, and also to evaluate anti-oxidant pharmacological interventions to improve PD outcomes.

Background and Aims Peritoneal dialysis (PD) is associated with morphological and also functional changes to the peritoneum limiting the long-term use. PD and CKD lead to vasculopathy, disrupt the endothelial and peritoneal barrier, but also influence the gut microbiome. Microbial dysbiosis in return is speculated to drive inflammation as an additional risk factor for cardiovascular disease. New PD-fluids that could slow or prevent these processes are needed. Alanyl-glutamine (AlaGln) has immunomodulatory and cytoprotective properties and has been shown to also improve endothelial barrier functions. Our aim is to investigate the effects of AlaGln-supplementation to PD-fluid on the gut microbiome and plasma and effluent metabolome and their interplay. Method Mice (C57/BI6N) underwent a subtotal nephrectomy (5/6 nephrectomy) to induce uraemia. Chronic exposure to PD-fluid was performed for 9 weeks via subcutaneously implanted peritoneal catheters. Mice were exposed daily to 2 ml of commercially available glucose-based PD-fluid (3.86% glucose) without or with the addition of 8 mM AlaGln. Uremic and healthy mice, kept in parallel were used as controls. All mice were fed standard chow and tap water ad libitum. On the last day, blood and an effluent (after a 30 minute dwell) were collected and faecal matter was collected from 3 different sites of the gut (ileum, caecum and colon). The plasma and effluent metabolome were analysed using a targeted approach. 180 metabolites were analysed with a mass spectrometry based kit (Biocrates) in both samples types. Following microbial DNA isolation, the microbiome has been analysed by 16S rRNA sequencing. The experiment was approved by the local animal ethics committee. Results Significantly elevated creatinine values in mice following 5/6 nephrectomy confirmed their uremic status. Significantly different plasma and effluent levels of alanine and glutamine were found in mice exposed to AlaGln supplemented PD-fluid. A correlation analysis of the plasma revealed the uremic status of the mice as main driver of differences whereas the effluent metabolome was mainly changed by PD fluid exposure. Plasma of uremic mice also showed significantly increased levels of a toxic non-proteinogenic amino acid symmetric dimethyl arginine (SDMA) and citrulline. Microbiome analysis yielded over 2800 amplicon sequence variants. Microbiome composition was location specific and influenced by the different treatments. The microbiome data were also correlated with over 130 metabolites in both plasma and PD effluent. Our data showed increased abundance in bacterial family Pseudomonadacea in caecum of uremic mice and positive correlation with the plasma level of trans-tetra-hydroxyproline. Mice exposed to conventional PD fluid had higher abundance of the bacterial class Clostridia in the colon compared to mice with no PD exposition. Conclusion CKD itself and PD-fluid exposure both affect the gut microbiome and the AlaGln-supplementation effects are likely reflected at the level of microbial diversity and functional interaction with metabolites. As next step specific cellular and molecular mechanisms of these effects are analysed. Preservation of mesothelial and also endothelial cellular barrier function could be a clinically important benefit for patients treated with PD, by preventing increased PD-associated pathomechanisms.

Background and Aims Tight junction (TJ) proteins have been suggested as molecular correlates for peritoneal semi-permeability and dialytic transport function in patients on peritoneal dialysis. Junction abundance in healthy individuals, in those with CKD5 and in patients on PD has not been described yet, the relation with peritoneal solute transport is unknown. Method Junction and transporter expression was analysed in multi-omics data sets from microdissected omental arterioles in children with normal renal function, CKD5 and on PD with low and high glucose degradation product (GDP) content (n=6/group). Parietal peritoneal tight junction proteins CLDN-1,-2,-3,-4,-5,-15, the adapter protein of claudins to actin cytoskeleton protein, zonula occludens-1 (ZO-1), the tricellular junction protein tricellulin (TriC), and transcellular transporters for sodium (ENaC), glucose (SGLT-1) and phosphate (PIT-1) were quantified in 40 non-CKD individuals, 20 children with CKD5 and 20 and 15 children on low- and high-GDP PD by quantitative, digital immunohistochemistry. Findings were correlated to 2-hour peritoneal equilibration test data obtained within 6 months of biopsy sampling (n=23). Primary human umbilical vein endothelial cells (HUVEC) were used to study the effects of single PD compounds on transepithelial electrical resistance (TER) and molecular size-dependent paracellular transport capacity. Co-stained monolayers were visualized by confocal microscopy. Single junction molecule localization and clustering were analysed by super resolution microscopy. Results Transcriptome and proteome pathway enrichment analysis of arteriolar junction and membrane protein demonstrated regulation in CKD5 versus health, and differential regulation by low- and high-GDP PD versus CKD5. In the parietal peritoneum all junctions and cellular transporters were expressed in endothelial and mesothelial cells. Pore forming CLDN-2, -4 and -15 were localized also in submesothelial immune cells. Parietal peritoneal junction abundance was age-dependent and also modified by CKD5 and PD. Mesothelial and endothelial abundance of the selective cation/water channel CLDN-2 increased in patients on low- and high-GDP PD fluids. Adaptor protein ZO-1 was upregulated in low GDP-PD versus CKD5, while sealing proteins CLDN -3 and -5 were downregulated. D/P creatinine, D/P phosphate, D/D0 glucose were similar in CKD5 and PD groups. D/P creatinine correlated with mesothelial CLDN-15, with arteriolar CLDN-2 and TriC and with endothelial ENaC. D/P phosphate correlated with endothelial CLDN-15, D/D0 glucose with mesothelial CLDN-4 and arteriolar CLDN-2. Capillary ZO-1 correlated with 24-h ultrafiltration standardized to body surface area and dialytic glucose exposure. In vitro, TER was decreased by low pH, glucose and 0.5µM methylglyoxal after 5h. Alanyl-glutamine (AlaGln) dose-dependently increased TER, and reduced 10kDa and 70kDa solute at 24mM, increased the abundance of ZO-1 and CLDN5 at cell-cell contacts, and on nanoscale clustering of the pore-forming CLDN2 and CLDN5. Conclusion Abundance of parietal peritoneal sealing and pore forming junctions and transcellular solute transporters varies with cell type and age and is differentially regulated by PD and associated with dialytic transport function. Our preliminary analyses illustrate the role of junctions and cellular transporters for solute transport across the peritoneal mesothelial and endothelial cell barrier. In-depth understanding of specific molecular functions should provide targets for modulation to improve efficacy of PD.

Pantala flavescens is the world’s most abundant and widely distributed dragonfly and with its outstanding migratory capacity an important model system to study insect migration at the evolutionary base of winged insects. We here report on the first complete mitochondrial genome (mitogenome) of P. flavescens sampled from a population in Rufiji River, Tanzania. The mitogenome is 14,853 bp long with an AT-biased base composition (72.7% A + T) and encodes a typical set of 13 protein-coding genes (PCGs), 22 tRNAs, and two rRNAs. The control region (CR) (171 bp) is the shortest reported in any anisopteran odonate, so far. Phylogenetic analyses support the placement of P. flavescens within the Libellulidae.

Odonata (dragonflies and damselflies) is a small order at the base of flying insects (Pterygota). Resolving family-level phylogenetic relationships within this order receives great attention. Hereby, genetic data already resulted in various changes, which are however still under discussion. Mitochondrial genomes may further enhance such phylogenies. This study presents the complete mitochondrial genome of the Neotropical damselfly Megaloprepus caerulatus based on next generation sequencing (NGS) data on total genomic DNA. The total length comprises 16,094 bp and includes the standard metazoan set of 37 genes together with a 1376 bp long A + T rich (control) region. Gene content, gene arrangement and base frequency are consistent with other odonate mitochondrial genomes. It further contains four intergenic spacer regions, indicating a possible family specific feature for the Coenagrionidae and its close relatives.

Background and Aims Recent evidence suggests a bidirectional relationship between the intestinal microbiome and CKD. CKD and concomitant uremia lead to loss of integrity of the intestinal barrier, causing increased intestinal permeability and potentially contributing to systemic inflammation. Uremia itself has been speculated to cause a selection of certain microorganisms, known as dysbiosis. The alteration of the intestinal microbiome may favor the formation of specific uremic toxins. We hypothesized that the composition of the microbiome is altered between patients with CKD and AKI. The aim was to investigate the effect of CKD (with already established uremic dysbiosis) and AKI (without established uremic dysbiosis) and the exposure to PD-fluids on the intestinal microbiome and on a local (peritoneal) and systemic metabolome level of patients treated with PD. Method Patients on PD therapy with CKD and AKI were included. Stool samples were analyzed by 16S rRNA sequencing, 188 systemic (serum) and local (PD-effluent) metabolites as well as 10 uremic toxins (including p-cresyl sulfate and indoxyl sulfate) were analyzed using a mass spectrometric approach. Clinical standard parameters in serum and PD-effluent were measured by routine laboratory techniques. Results Changes in relative abundances of intestinal bacteria and differences in gut microbiome composition at the phylum level were associated with chronic disease. Patients with CKD and AKI had significantly increased levels of uremic toxins and revealed a distinct metabolomic pattern. Cross-omics correlation between bacterial phyla and metabolites was performed to obtain functional information on differentially abundant species and molecules. Conclusion Patients with CKD on PD are characterized by an altered intestinal microbiome, local and systemic metabolome changes in contrast to patients with AKI. PD-patients with CKD and AKI have increased serum levels of bacterially-derived uremic toxins. Further investigations are required to obtain deeper understanding of reciprocal influences of the uremic status and intestinal microbiome.

Background and Aims The composition of all currently available peritoneal dialysis (PD) fluids triggers morphological and functional changes in the peritoneal membrane. Periodic exposure leads to vasculopathy, hypervascularization, and diabetes-like damage of vessels, eventually leading to failure of the technique. Patients undergoing dialysis generally, have a high risk of cardiovascular events. It is currently unclear if there is a mechanistic link between peritoneal membrane failure and cardiovascular risk. In vitro and in vivo studies have shown that cytoprotective additives (e.g. dipeptide alanyl-glutamine (AlaGln) or kinase inhibitor lithium chloride (LiCl)) to PDF reduce peritoneal damage. Here, we developed an experimental model for investigating effects of these cytoprotective additives in PDF in the cardiovascular context. Method For modelling the peritoneal membrane in vitro, mesothelial and endothelial cells were co-cultured in transwell plates. Mesothelial cells were grown in the upper compartment and primary human umbilical vein endothelial cells (HUVEc) or primary microvascular cells were grown in the lower compartment. PDF with or without cytoprotective compounds, was added to the upper compartment to only expose mesothelial cells directly to different dilutions of the fluid. Effects on cell damage was assessed by quantification of lactate-dehydrogenase (LDH) release and live-dead staining of cells. Proteome profiles were analysed for both cell-types separately and in combination using two-dimensional difference gel electrophoresis (2D-DiGE) and liquid chromatography coupled to mass spectrometry (LC-MS). In vitro findings were related to PD-induced arteriolar changes based on abundance profiles of micro-dissected omental arterioles of children treated with conventional PD-fluids and age-matched controls with normal renal function. Results Marked cellular injury of HUVEc after PD-fluid exposure was associated with a molecular landscape of the enriched biological process clusters ‘glucose catabolic process’, ‘cell redox homeostasis’, ‘RNA metabolic process’, ‘protein folding’, ‘regulation of cell death’, and ‘actin cytoskeleton reorganization’ that characterize PD-fluid cytotoxicity and counteracting cellular repair process respectively. PDF-induced cell damage was reduced by AlaGln and LiCl both in mesothelial and endothelial cells. Proteome analysis revealed perturbation of major cellular processes including regulation of cell death and cytoskeleton reorganization. Selected markers of angiogenesis, oxidative stress, cell junctions and transdifferentiation were counter-regulated by the additives. Co-cultured cells yielded differently regulated pathways following PDF exposure compared to separate culture. Comparison to human arterioles confirmed overlapping protein regulation between endothelial cells in vitro and in vivo, proving harmful effects of PD-fluids on endothelial cells leading to drastic changes of the cellular process landscape. Conclusion In summary, this study shows harmful effects of PD-fluids also effecting endothelial cells and elucidates potential mechanisms by which cytoprotective additives may counteract the signalling axis between local peritoneal damage and systemic vasculopathy. An in vitro co-culture system may be an attractive approach to simulate the peritoneal membrane for testing direct and indirect effects of cytoprotective additives in PDF. When cultured and stressed in close proximity cells may respond differently. Characterisation of PD-induced perturbations will allow identifying molecular mechanisms linking the peritoneal and cardiovascular context, offering therapeutic targets to reduce current limitations of PD and ultimately decreasing cardiovascular risk of dialysis patients.

Background and Aims Infectious complications occur in a significant proportion of patients treated with peritoneal dialysis (PD), limiting its long-term applicability. Reduced peritoneal immune-competence, caused by the continuous exposure to PD-fluids, has been described as a therapy-related pathomechanisms, prompting the need for a tool to assess the functional peritoneal immune status. We established an ex-vivo stimulation assay to test host defence mechanisms in only 9ml of crude PD-effluent. The assay was used in two randomized clinical trials investigating immune-modulatory effects of a novel additive (alanyl-glutamine) to PD-fluid. Here we aimed to evaluate the assay as a method to assess peritoneal immune-competence in the general PD-population and linking it to PD vintage and outcome parameters. Method 462 PD-effluent samples, originating from 148 adult and paediatric PD patients, were stimulated ex-vivo (24h, 37°C) with toll-like receptor (TLR)-agonists lipopolysaccharide (LPS) and Pam3Cys. Unstimulated samples of each effluent were used as controls. Interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) concentrations and the predictive value of the assay for clinical outcome parameters were analyzed using Wilcoxon matched-pairs signed rank test and a mixed model logistic regression analysis. Results Ex-vivo stimulation of peritoneal cells resulted in a dwell-time dependent increase of cytokine release, with a significant lower cytokine released at the 1h PET time point (normalized to unstimulated controls). Ratios between stimulated PDE-samples and unstimulated controls, accounting for a potential increase in basal inflammation, significantly declined with PD therapy duration. Correlation of the cytokine levels with the predefined clinical parameters revealed predictive potential of the assay for the occurrence of infectious complications, as well as of ultrafiltration and technique failure. Conclusion In summary, the current study provides evidence of a correlation of declining local host defence and duration of PD-therapy. These data support the hypothesis of PD-duration dependent progressive impairment of the ability of the peritoneal immune cells to secrete cytokines in response to a pathogenic stimulus and thereby dampening the global peritoneal immuno-competence. The results suggest the utility of this clinically feasible ex-vivo induced cytokine-release assay in crude peritoneal effluent as a surrogate of the functional peritoneal immune competence. Future analyses will have to evaluate the assay as a tool to predict common clinical outcomes and define reference values to facilitate stratification of patient populations, clinical staging and to guide novel therapeutic interventions.

Background and Aims PD-fluids lead to generation of reactive oxygen species (ROS) in the peritoneal cavity. The caused oxidative stress, defined as a cellular oxidant-antioxidant imbalance impairs not only peritoneal cell viability but also contributes to progression of local and systemic PD-related pathomechanisms. We aim to analyze the impact and specific targets of ROS during PD and the anti-oxidative mechanism of supplementation of PD-fluid with alanyl-glutamine (AlaGln) on a global proteome-wide level. Method To establish a redox-proteomics workflow for studying oxidative stress in peritoneal mesothelial cells we used a gold-standard model of redox-stress (H2O2) and PD-fluid induced stress. Levels of oxidative stress were first validated by increased intracellular ROS and superoxide dismutase activity with PD-fluid and H2O2 treatment and a reduction of these parameters by the addition of AlaGln. To detect alterations of the redox proteome, cysteine residues were either directly or indirectly labeled with fluorescent dyes (redox-2D-DiGE) or isobaric tags (iodo-TMT). Results: The gel-based approach allowed global visualization of the reduced and oxidized cysteines and revealed redox profiles of 540 protein spots. Compared to control, we found an increase in oxidized and decrease in reduced cysteines in all PD treatments. The development of a highly sensitive LC/MS-based redox proteomics workflow allowed identification of ∼950 proteins affected by redox-stress in mesothelial cells and confirmed the quantitative levels seen on cysteine oxidation. The addition of AlaGln reduced the overall redox status (intracellular ROS and superoxide dismutase activity) but further showed different proteins to be affected by redox modifications. Conclusion: Redox proteomics of peritoneal cells could represent a novel approach for the identification of mediators of PD-induced pathomechanisms, but also to evaluate effects of novel anti-oxidant therapeutical or pharmacological interventions.

Long-term clinical outcome of peritoneal dialysis (PD) depends on adequate removal of small solutes and water. The peritoneal endothelium represents the key barrier and peritoneal transport dysfunction is associated with vascular changes. Alanyl-glutamine (AlaGln) has been shown to counteract PD-induced deteriorations but the effect on vascular changes has not yet been elucidated. Using multiplexed proteomic and bioinformatic analyses we investigated the molecular mechanisms of vascular pathology in-vitro (primary human umbilical vein endothelial cells, HUVEC) and ex-vivo (arterioles of patients undergoing PD) following exposure to PD-fluid. An overlap of 1813 proteins (40%) of over 3100 proteins was identified in both sample types. PD-fluid treatment significantly altered 378 in endothelial cells and 192 in arterioles. The HUVEC proteome resembles the arteriolar proteome with expected sample specific differences of mainly immune system processes only present in arterioles and extracellular region proteins primarily found in HUVEC. AlaGln-addition to PD-fluid revealed 359 differentially abundant proteins and restored the molecular process landscape altered by PD fluid. This study provides evidence on validity and inherent limitations of studying endothelial pathomechanisms in-vitro compared to vascular ex-vivo findings. AlaGln could reduce PD-associated vasculopathy by reducing endothelial cellular damage, restoring perturbed abundances of pathologically important proteins and enriching protective processes.

Alterations in the nitric oxide (NO) pathway play a major role in pulmonary arterial hypertension (PAH). L-arginine (LA) and tetrahydrobiopterin (BH4) are main substrates in the production of NO, which mediates pulmonary vasodilation. Administration of either LA or BH4 decrease pulmonary artery pressure (PAP). A combined administration of both may have synergistic effects in the therapy of PAH. In a telemetrically monitored model of unilateral pneumonectomy and monocrotaline-induced PAH, male Sprague-Dawley rats received either LA (300 mg/kg; n = 15), BH4 (20 mg/kg; n = 15), the combination of LA and BH4 (300 mg/kg, 20 mg/kg; n = 15), or vehicle (control group; n = 10) from day 28 after monocrotaline induction. Therapy was orally administered once daily over consecutive 14 days. LA, BH4, or both equally lowered PAP, increased pulmonary vascular elasticity, restored spontaneous locomotoric activity, prevented body weight loss and palliated small vessel disease of severely pulmonary hypertensive rats. BH4 substitution lowered asymmetric dimethylarginine levels sustainably at 60 min after administration and downregulated endothelial NO synthase mRNA expression. No significant survival, macro- and histomorphologic or hemodynamic differences were found between therapy groups at the end of the study period. Administration of LA and BH4 both mediated a decrease of mean PAP, attenuated right ventricular hypertrophy and small vessel disease in monocrotaline-induced pulmonary hypertensive rats, though a combined administration of both substances did not reveal any synergistic therapy effects in our animal model.

Life-saving renal replacement therapy by peritoneal dialysis (PD) is limited in use and duration by progressive impairment of peritoneal membrane integrity and homeostasis. Preservation of peritoneal membrane integrity during chronic PD remains an urgent but long unmet medical need. PD therapy failure results from peritoneal fibrosis and angiogenesis caused by hypertonic PD fluid (PDF)-induced mesothelial cytotoxicity. However, the pathophysiological mechanisms involved are incompletely understood, limiting identification of therapeutic targets. We report that addition of lithium chloride (LiCl) to PDF is a translatable intervention to counteract PDF-induced mesothelial cell death, peritoneal membrane fibrosis, and angiogenesis. LiCl improved mesothelial cell survival in a dose-dependent manner. Combined transcriptomic and proteomic characterization of icodextrin-based PDF-induced mesothelial cell injury identified αB-crystallin as the mesothelial cell protein most consistently counter-regulated by LiCl. In vitro and in vivo overexpression of αB-crystallin triggered a fibrotic phenotype and PDF-like up-regulation of vascular endothelial growth factor (VEGF), CD31-positive cells, and TGF-β-independent activation of TGF-β-regulated targets. In contrast, αB-crystallin knockdown decreased VEGF expression and early mesothelial-to-mesenchymal transition. LiCl reduced VEGF release and counteracted fibrosis- and angiogenesis-associated processes. αB-crystallin in patient-derived mesothelial cells was specifically up-regulated in response to PDF and increased in peritoneal mesothelial cells from biopsies from pediatric patients undergoing PD, correlating with markers of angiogenesis and fibrosis. LiCl-supplemented PDF promoted morphological preservation of mesothelial cells and the submesothelial zone in a mouse model of chronic PD. Thus, repurposing LiCl as a cytoprotective PDF additive may offer a translatable therapeutic strategy to combat peritoneal membrane deterioration during PD therapy.

Summary The enigmatic phylum Placozoa is harboring an unknown number of cryptic species and has become a challenge for modern systematics. Only recently, a second species has been described [1] , while the presence of more than a hundred additional species has been suggested [2] . The original placozoan species Trichoplax adhaerens [3] , the second species Hoilungia hongkongensis [1] and all yet undescribed species are morphologically indistinguishable (i.e. no species diagnostic characters are available [4] ). Here, we report on a new placozoan species, Polyplacotoma mediterranea gen. nov., spec. nov., which differs from other placozoans in its completely different morphological habitus, including long polytomous body branches and a maximum body length of more than 10 mm. Polyplacotoma mediterranea also necessitates a different view of placozoan mitochondrial genetics. P. mediterranea harbors a highly compact mitochondrial genome with overlapping mitochondrial tRNA and protein coding genes. Furthermore, the new species lacks typical placozoan features, including the cox1 micro exon and cox1 barcode intron. As phylogenetic analyses suggest a sister group relationship of P. mediterranea to all other placozoans, this new species may also be relevant for studies addressing the relationships at the base of the metazoan tree of life.

Readily available peritoneal dialysis (PD) effluents from PD patients in the course of renal replacement therapy are a potentially rich source for molecular markers for predicting clinical outcome, monitoring the therapy, and therapeutic interventions. The complex clinical phenotype of PD patients might be reflected in the PD effluent metabolome. Metabolomic analysis of PD effluent might allow quantitative detection and assessment of candidate PD biomarkers for prognostication and therapeutic monitoring. We therefore subjected peritoneal equilibration test effluents from 20 stable PD patients, obtained in a randomized controlled trial (RCT) to evaluate cytoprotective effects of standard PD solution (3.86% glucose) supplemented with 8 mM alanyl-glutamine (AlaGln) to targeted metabolomics analysis. One hundred eighty eight pre-defined metabolites, including free amino acids, acylcarnitines, and glycerophospholipids, as well as custom metabolic indicators calculated from these metabolites were surveyed in a high-throughput assay requiring only 10 μl of PD effluent. Metabolite profiles of effluents from the cross-over trial were analyzed with respect to AlaGln status and clinical parameters such as duration of PD therapy and history of previous episodes of peritonitis. This targeted approach detected and quantified 184 small molecules in PD effluent, a larger number of detected metabolites than in all previous metabolomic studies in PD effluent combined. Metabolites were clustered within substance classes regarding concentrations after a 4-h dwell. PD effluent metabolic profiles were differentiated according to PD patient sub-populations, revealing novel changes in small molecule abundance during PD therapy. AlaGln supplementation of PD fluid altered levels of specific metabolites, including increases in alanine and glutamine but not glutamate, and reduced levels of small molecule indicators of oxidative stress, such as methionine sulfoxide. Our study represents the first application of targeted metabolomics to PD effluents. The observed metabolomic changes in PD effluent associated with AlaGln-supplementation during therapy suggested an anti-oxidant effect, and were consistent with the restoration of important stress and immune processes previously noted in the RCT. High-throughput detection of PD effluent metabolomic signatures and their alterations by therapeutic interventions offers new opportunities for metabolome-clinical correlation in PD and for prescription of personalized PD therapy.

The phosphodiesterase (PDE)-5 inhibitor sildenafil is a commonly used drug to treat erectile dysfunction. As its vascular relaxation effects might affect not only the corpus cavernosum, but as well vasculature in general, an association with bleeding incidents has been discussed. We report on a 55-year-old patient presenting with thunderclap headache due to perimesencephalic subarachnoid hemorrhage (SAH) after ingestion of a very high dose (200 mg) of sildenafil. No aneurysm or other vascular abnormality was found as a cause of the bleeding. We discuss possible mechanisms by which sildenafil might alter intracranial blood coagulation, e.g. via acting on the PDE-1 and PDE-2 enzymes and via affecting platelet aggregation. In conclusion, Sildenafil may cause or intensify extra- and intracranial hemorrhage. Thus, careful discussion and indication are mandatory. On the other hand, sildenafil is also addressed as a potential option in the prevention and treatment for cerebral vasospasm as complication of SAH.

PurposePeritoneal dialysis fluids (PDFs) may induce inadequate heat-shock protein (HSP) expression and injury-related inflammation in exposed mesothelial cells. The aim of this study was to relate cellular injury to these cellular responses in mesothelial cells following repeated exposure to 3 commercial PDFs with different biocompatibility profiles.MethodsPrimary cultures of human peritoneal mesothelial cells (HPMC) were exposed to a 1:2 mixture of cell culture medium and CAPD2 (single-chamber bag PDF; Fresenius, Bad Homburg, Germany), Physioneal (dual-chamber bag PDF; Baxter, Deerfield, IL, USA) or Balance (dual-chamber bag PDF, Fresenius) for up to 10 days exposure time (4 dwells). Supernatant was analyzed for LDH, IL-6, and IL-8, cells for HSP-72 expression, and protein content.ResultsPDF exposure resulted in a biphasic pattern of cell damage switching from an earlier phase with increased injury by single-chamber PDF to a delayed phase with increased susceptibility to dual-chamber PDF. Sterile inflammation was related to LDH release over time and could be reproduced by exposure to necrotic cellular material. PDF exposure resulted in low HSP-72 expression in all tested PDFs.ConclusionsExposure to single-chamber as well as to dual-chamber bag PDFs induce increased vulnerability of mesothelial cells to repeated exposure of the same solution. These effects were delayed with dual-chamber PDFs. Injury-induced inflammation and impaired HSP expression upon PDF exposure might initiate a vicious cycle with progredient mesothelial cell damage upon repeated PDF exposure. Certainly, interventional studies and translation of these results into the in vivo system is needed.

Peritonitis remains a major cause of morbidity and mortality during chronic peritoneal dialysis (PD). Glucose-based PD fluids reduce immunological defenses in the peritoneal cavity. Low concentrations of peritoneal extracellular glutamine during PD may contribute to this immune deficit. For these reasons we have developed a clinical assay to measure the function of the immune-competent cells in PD effluent from PD patients. We then applied this assay to test the impact on peritoneal immune-competence of PD fluid supplementation with alanyl-glutamine (AlaGln) in 6 patients in an open-label, randomized, crossover pilot trial (EudraCT 2012-004004-36), and related the functional results to transcriptome changes in PD effluent cells. Ex-vivo stimulation of PD effluent peritoneal cells increased release of interleukin (IL) 6 and tumor necrosis factor (TNF) α. Both IL-6 and TNF-α were lower at 1 h than at 4 h of the peritoneal equilibration test but the reductions in cytokine release were attenuated in AlaGln-supplemented samples. AlaGln-supplemented samples exhibited priming of IL-6-related pathways and downregulation of TNF-α upstream elements. Results from measurement of cytokine release and transcriptome analysis in this pilot clinical study support the conclusion that suppression of PD effluent cell immune function in human subjects by standard PD fluid is attenuated by AlaGln supplementation.

Peritoneal dialysis (PD) is a modality of renal replacement therapy in which the high volumes of available PD effluent (PDE) represents a rich source of biomarkers for monitoring disease and therapy. Although this information could help guide the management of PD patients, little is known about the potential of PDE to define pathomechanism-associated molecular signatures in PD. We therefore subjected PDE to a high-performance multiplex proteomic analysis after depletion of highly-abundant plasma proteins and enrichment of low-abundance proteins. A combination of label-free and isobaric labeling strategies was applied to PDE samples from PD patients (n = 20) treated in an open-label, randomized, two-period, cross-over clinical trial with standard PD fluid or with a novel PD fluid supplemented with alanyl-glutamine (AlaGln). With this workflow we identified 2506 unique proteins in the PDE proteome, greatly increasing coverage beyond the 171 previously-reported proteins. The proteins identified range from high abundance plasma proteins to low abundance cellular proteins, and are linked to larger numbers of biological processes and pathways, some of which are novel for PDE. Interestingly, proteins linked to membrane remodeling and fibrosis are overrepresented in PDE compared with plasma, whereas the proteins underrepresented in PDE suggest decreases in host defense, immune-competence and response to stress. Treatment with AlaGln-supplemented PD fluid is associated with reduced activity of membrane injury-associated mechanisms and with restoration of biological processes involved in stress responses and host defense. Our study represents the first application of the PDE proteome in a randomized controlled prospective clinical trial of PD. This novel proteomic workflow allowed detection of low abundance biomarkers to define pathomechanism-associated molecular signatures in PD and their alterations by a novel therapeutic intervention.

Modern conservationists call for long term genetic monitoring datasets to evaluate and understand the impact of human activities on natural ecosystems and species on a global but also local scale. However, long-term monitoring datasets are still rare but in high demand to correctly identify, evaluate and respond to environmental changes. In the presented study, a population of the riverine dragonfly, Orthetrum coerulescens (Odonata: Libellulidae), was monitored over a time period from 1989 to 2013. Study site was an artificial irrigation ditch in one of the last European stone steppes and "nature heritage", the Crau in Southern France. This artificial riverine habitat has an unusual high diversity of odonate species, prominent indicators for evaluating freshwater habitats. A clearing of the canal and destruction of the bank vegetation in 1996 was assumed to have great negative impact on the odonate larval and adult populations. Two mitochondrial markers (CO1 & ND1) and a panel of nuclear microsatellite loci were used to assess the genetic diversity. Over time they revealed a dramatic decline in diversity parameters between the years 2004 and 2007, however not between 1996 and 1997. From 2007 onwards the population shows a stabilizing trend but has not reached the amount of genetic variation found at the beginning of this survey. This decline cannot be referred to the clearing of the canal or any other direct anthropogenic impact. Instead, it is most likely that the populations' decay was due to by extreme weather conditions during the specific years. A severe drought was recorded for the summer months of these years, leading to reduced water levels in the canal causing also other water parameters to change, and therefore impacting temperature sensitive riverine habitat specialists like the O. coerulescens in a significant way. The data provide important insights into population genetic dynamics and metrics not always congruent with traditional monitoring data (e.g. abundance); a fact that should be regarded with caution when management plans for developed landscapes are designed.

The ability of cells to respond and survive stressful conditions is determined, in part, by the attachment of O-linked N-acetylglucosamine (O-GlcNAc) to proteins (O-GlcNAcylation), a post-translational modification dependent on glucose and glutamine. This study investigates the role of dynamic O-GlcNAcylation of mesothelial cell proteins in cell survival during exposure to glucose-based peritoneal dialysis fluid (PDF). Immortalized human mesothelial cells and primary mesothelial cells, cultured from human omentum or clinical effluent of PD patients, were assessed for O-GlcNAcylation under normal conditions or after exposure to PDF. The dynamic status of O-GlcNAcylation and effects on cellular survival were investigated by chemical modulation with 6-diazo-5-oxo-L-norleucine (DON) to decrease or O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino N-phenyl carbamate (PUGNAc) to increase O-GlcNAc levels. Viability was decreased by reducing O-GlcNAc levels by DON, which also led to suppressed expression of the cytoprotective heat shock protein 72. In contrast, increasing O-GlcNAc levels by PUGNAc or alanyl-glutamine led to significantly improved cell survival paralleled by higher heat shock protein 72 levels during PDF treatment. Addition of alanyl-glutamine increased O-GlcNAcylation and partly counteracted its inhibition by DON, also leading to improved cell survival. Immunofluorescent analysis of clinical samples showed that the O-GlcNAc signal primarily originates from mesothelial cells. In conclusion, this study identified O-GlcNAcylation in mesothelial cells as a potentially important molecular mechanism after exposure to PDF. Modulating O-GlcNAc levels by clinically feasible interventions might evolve as a novel therapeutic target for the preservation of peritoneal membrane integrity in PD.