Glucose Derivative Induced Vasculopathy in Children on Chronic Peritoneal Dialysis.

[Figure: see text].

Bone Allograft Acid Lysates Change the Genetic Signature of Gingival Fibroblasts.

Bone allografts are widely used as osteoconductive support to guide bone regrowth. Bone allografts are more than a scaffold for the immigrating cells as they maintain some bioactivity of the original bone matrix. Yet, it remains unclear how immigrating cells respond to bone allografts. To this end, we have evaluated the response of mesenchymal cells exposed to acid lysates of bone allografts (ALBA). RNAseq revealed that ALBA has a strong impact on the genetic signature of gingival fibroblasts, indicated by the increased expression of IL11, AREG, C11orf96, STC1, and GK-as confirmed by RT-PCR, and for IL11 and STC1 by immunoassays. Considering that transforming growth factor-ß (TGF-ß) is stored in the bone matrix and may have caused the expression changes, we performed a proteomics analysis, TGF-ß immunoassay, and smad2/3 nuclear translocation. ALBA neither showed detectable TGF-ß nor was the lysate able to induce smad2/3 translocation. Nevertheless, the TGF-ß receptor type I kinase inhibitor SB431542 significantly decreased the expression of IL11, AREG, and C11orf96, suggesting that other agonists than TGF-ß are responsible for the robust cell response. The findings suggest that IL11, AREG, and C11orf96 expression in mesenchymal cells can serve as a bioassay reflecting the bioactivity of the bone allografts.

Proteomic study of mesothelial and endothelial cross-talk: key lessons.

INTRODUCTION: 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. AREAS COVERED: 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. EXPERT OPINION: 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.

Proteomic profiling of end-stage COVID-19 lung biopsies.

The outbreak of a novel coronavirus (SARS-CoV-2) in 2019 led to a worldwide pandemic, which remains an integral part of our lives to this day. Coronavirus disease (COVID-19) is a flu like condition, often accompanied by high fever and respiratory distress. In some cases, conjointly with other co-morbidities, COVID-19 can become severe, leading to lung arrest and even death. Although well-known from a clinical standpoint, the mechanistic understanding of lethal COVID-19 is still rudimentary. Studying the pathology and changes on a molecular level associated with the resulting COVID-19 disease is impeded by the highly infectious nature of the virus and the concomitant sampling challenges. We were able to procure COVID-19 post-mortem lung tissue specimens by our collaboration with the BSL-3 laboratory of the Biobanking and BioMolecular resources Research Infrastructure Austria which we subjected to state-of-the-art quantitative proteomic analysis to better understand the pulmonary manifestations of lethal COVID-19. Lung tissue samples from age-matched non-COVID-19 patients who died within the same period were used as controls. Samples were subjected to parallel accumulation-serial fragmentation combined with data-independent acquisition (diaPASEF) on a timsTOF Pro and obtained raw data was processed using DIA-NN software. Here we report that terminal COVID-19 patients display an increase in inflammation, acute immune response and blood clot formation (with concomitant triggering of fibrinolysis). Furthermore, we describe that COVID-19 diseased lungs undergo severe extracellular matrix restructuring, which was corroborated on the histopathological level. However, although undergoing an injury, diseased lungs seem to have impaired proliferative and tissue repair signalling, with several key kinase-mediated signalling pathways being less active. This might provide a mechanistic link to post-acute sequelae of COVID-19 (PASC; "Long COVID"). Overall, we emphasize the importance of histopathological patient stratification when interpreting molecular COVID-19 data.

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

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

A Meta-Analysis of Human Transcriptomics Data in the Context of Peritoneal Dialysis Identifies Novel Receptor-Ligand Interactions as Potential Therapeutic Targets.

Peritoneal dialysis (PD) is one therapeutic option for patients with end-stage kidney disease (ESKD). Molecular profiling of samples from PD patients using different Omics technologies has led to the discovery of dysregulated molecular processes due to PD treatment in recent years. In particular, a number of transcriptomics (TX) datasets are currently available in the public domain in the context of PD. We set out to perform a meta-analysis of TX datasets to identify dysregulated receptor-ligand interactions in the context of PD-associated complications. We consolidated transcriptomics profiles from twelve untargeted genome-wide gene expression studies focusing on human cell cultures or samples from human PD patients. Gene set enrichment analysis was used to identify enriched biological processes. Receptor-ligand interactions were identified using data from CellPhoneDB. We identified 2591 unique differentially expressed genes in the twelve PD studies. Key enriched biological processes included angiogenesis, cell adhesion, extracellular matrix organization, and inflammatory response. We identified 70 receptor-ligand interaction pairs, with both interaction partners being dysregulated on the transcriptional level in one of the investigated tissues in the context of PD. Novel receptor-ligand interactions without prior annotation in the context of PD included BMPR2-GDF6, FZD4-WNT7B, ACKR2-CCL2, or the binding of EPGN and EREG to the EGFR, as well as the binding of SEMA6D to the receptors KDR and TYROBP. In summary, we have consolidated human transcriptomics datasets from twelve studies in the context of PD and identified sets of novel receptor-ligand pairs being dysregulated in the context of PD that warrant investigation in future functional studies.

Improved Alignment and Quantification of Protein Signals in Two-Dimensional Western Blotting.

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).

Effects of Alanyl-Glutamine Treatment on the Peritoneal Dialysis Effluent Proteome Reveal Pathomechanism-Associated Molecular Signatures.

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.

ECM Characterization Reveals a Massive Activation of Acute Phase Response during FSGS.

The glomerular basement membrane (GBM) and extra-cellular matrix (ECM) are essential to maintain a functional interaction between the glomerular podocytes and the fenestrated endothelial cells in the formation of the slit diaphragm for the filtration of blood. Dysregulation of ECM homeostasis can cause Focal segmental glomerulosclerosis (FSGS). Despite this central role, alterations in ECM composition during FSGS have not been analyzed in detail yet. Here, we characterized the ECM proteome changes in miR-193a-overexpressing mice, which suffer from FSGS due to suppression of Wilms' tumor 1 (WT1). By mass spectrometry we identified a massive activation of the acute phase response, especially the complement and fibrinogen pathways. Several protease inhibitors (ITIH1, SERPINA1, SERPINA3) were also strongly increased. Complementary analysis of RNA expression data from both miR-193a mice and human FSGS patients identified additional candidate genes also mainly involved in the acute phase response. In total, we identified more than 60 dysregulated, ECM-associated genes with potential relevance for FSGS progression. Our comprehensive analysis of a murine FSGS model and translational comparison with human data offers novel targets for FSGS therapy.

A combinatorial screen of the CLOUD uncovers a synergy targeting the androgen receptor.

Approved drugs are invaluable tools to study biochemical pathways, and further characterization of these compounds may lead to repurposing of single drugs or combinations. Here we describe a collection of 308 small molecules representing the diversity of structures and molecular targets of all FDA-approved chemical entities. The CeMM Library of Unique Drugs (CLOUD) covers prodrugs and active forms at pharmacologically relevant concentrations and is ideally suited for combinatorial studies. We screened pairwise combinations of CLOUD drugs for impairment of cancer cell viability and discovered a synergistic interaction between flutamide and phenprocoumon (PPC). The combination of these drugs modulates the stability of the androgen receptor (AR) and resensitizes AR-mutant prostate cancer cells to flutamide. Mechanistically, we show that the AR is a substrate for γ-carboxylation, a post-translational modification inhibited by PPC. Collectively, our data suggest that PPC could be repurposed to tackle resistance to antiandrogens in prostate cancer patients.

Complement Activation in Peritoneal Dialysis-Induced Arteriolopathy.

Cardiovascular disease (CVD) is the leading cause of increased mortality in patients with CKD and is further aggravated by peritoneal dialysis (PD). Children are devoid of preexisting CVD and provide unique insight into specific uremia- and PD-induced pathomechanisms of CVD. We obtained peritoneal specimens from children with stage 5 CKD at time of PD catheter insertion (CKD5 group), children with established PD (PD group), and age-matched nonuremic controls (n=6/group). We microdissected omental arterioles from tissue layers not directly exposed to PD fluid and used adjacent sections of four arterioles per patient for transcriptomic and proteomic analyses. Findings were validated in omental and parietal arterioles from independent pediatric control (n=5), CKD5 (n=15), and PD (n=15) cohorts. Transcriptomic analysis revealed differential gene expression in control versus CKD5 arterioles and in CKD5 versus PD arterioles. Gene ontology analyses revealed activation of metabolic processes in CKD5 arterioles and of inflammatory, immunologic, and stress-response cascades in PD arterioles. PD arterioles exhibited particular upregulation of the complement system and respective regulatory pathways, with concordant findings at the proteomic level. In the validation cohorts, PD specimens had the highest abundance of omental and parietal arteriolar C1q, C3d, terminal complement complex, and phosphorylated SMAD2/3, a downstream effector of TGF-ß Furthermore, in the PD parietal arterioles, C1q and terminal complement complex abundance correlated with the level of dialytic glucose exposure, abundance of phosphorylated SMAD2/3, and degree of vasculopathy. We conclude that PD fluids activate arteriolar complement and TGF-ß signaling, which quantitatively correlate with the severity of arteriolar vasculopathy.

A randomized controlled trial of alanyl-glutamine supplementation in peritoneal dialysis fluid to assess impact on biomarkers of peritoneal health.

In early clinical testing, acute addition of alanyl-glutamine (AlaGln) to glucose-based peritoneal dialysis (PD) fluids restored peritoneal cellular stress responses and leukocyte function. This study was designed to test the effect of extended treatment with AlaGln-supplemented PD fluid on biomarkers of peritoneal health. In a double-blinded, randomized crossover design, stable PD patients were treated with AlaGln (8 mM) or placebo added to PD fluid for eight weeks. As primary outcome measures, dialysate cancer-antigen 125 (CA-125) appearance rate and ex vivo stimulated interleukin-6 (IL-6) release were assessed in peritoneal equilibration tests. In 8 Austrian centers, 54 patients were screened, 50 randomized, and 41 included in the full analysis set. AlaGln supplementation significantly increased CA-125 appearance rate and ex vivo stimulated IL-6 release. AlaGln supplementation also reduced peritoneal protein loss, increased ex vivo stimulated tumor necrosis factor (TNF)-α release, and reduced systemic IL-8 levels. No adverse safety signals were observed. All 4 peritonitis episodes occurred during standard PD fluid treatment. A novel AlaGln-supplemented PD fluid improves biomarkers of peritoneal membrane integrity, immune competence, and systemic inflammation compared to unsupplemented PD fluid with neutral pH and low-glucose degradation. A phase 3 trial is needed to determine the impact of AlaGln supplementation on hard clinical outcomes.

Biomarker research to improve clinical outcomes of peritoneal dialysis: consensus of the European Training and Research in Peritoneal Dialysis (EuTRiPD) network.

Peritoneal dialysis (PD) therapy substantially requires biomarkers as tools to identify patients who are at the highest risk for PD-related complications and to guide personalized interventions that may improve clinical outcome in the individual patient. In this consensus article, members of the European Training and Research in Peritoneal Dialysis Network (EuTRiPD) review the current status of biomarker research in PD and suggest a selection of biomarkers that can be relevant to the care of PD patients and that are directly accessible in PD effluents. Currently used biomarkers such as interleukin-6, interleukin-8, ex vivo-stimulated interleukin-6 release, cancer antigen-125, and advanced oxidation protein products that were collected through a Delphi procedure were first triaged for inclusion as surrogate endpoints in a clinical trial. Next, novel biomarkers were selected as promising candidates for proof-of-concept studies and were differentiated into inflammation signatures (including interleukin-17, M1/M2 macrophages, and regulatory T cell/T helper 17), mesothelial-to-mesenchymal transition signatures (including microRNA-21 and microRNA-31), and signatures for senescence and inadequate cellular stress responses. Finally, the need for defining pathogen-specific immune fingerprints and phenotype-associated molecular signatures utilizing effluents from the clinical cohorts of PD patients and "omics" technologies and bioinformatics-biostatistics in future joint-research efforts was expressed. Biomarker research in PD offers the potential to develop valuable tools for improving patient management. However, for all biomarkers discussed in this consensus article, the association of biological rationales with relevant clinical outcomes remains to be rigorously validated in adequately powered, prospective, independent clinical studies.

Overexpression of Hsp70 confers cytoprotection during gliadin exposure in Caco-2 cells.

BACKGROUND: In Celiac disease (CD), cytoskeletal integrity of intestinal cells is disrupted by gliadin exposure. This study investigates the role of heat shock protein (Hsp)70 during cytoskeletal recovery in CD by assessing its induction and effects on junctional proteins. METHODS: Using an in-vitro model of CD, cytoskeletal injury and recovery was assessed in gliadin-exposed Caco-2 cells by measuring cellular distribution of ezrin, E-cadherin, and Hsp70 by differential centrifugation. Effects of Hsp70 were tested by an in-vitro repair assay, based on the incubation of injured or recovered cytoskeletal cellular fractions in noncytoskeletal supernatants containing low or high levels of Hsp70, or by transient transfection of Caco-2 cells with Hsp70. RESULTS: Cytoskeletal disruption of ezrin and E-cadherin was demonstrated in gliadin-exposed Caco-2 cells by their significant shift from the cytoskeletal pellet into the noncytoskeletal supernatant fraction. Recovery from gliadin exposure was associated with induction and cytoskeletal redistribution of Hsp70. The in-vitro repair assay delineated direct evidence for HSP-mediated repair by stabilization of junctional proteins by Hsp70. Overexpression of Hsp70 resulted in significantly increased cytoskeletal integrity. CONCLUSION: Our results establish an essential role of HSP-mediated cytoskeletal repair in Caco-2 cells during recovery from in-vitro gliadin exposure.

Dynamic O-linked N-acetylglucosamine modification of proteins affects stress responses and survival of mesothelial cells exposed to peritoneal dialysis fluids.

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.

Equalizer technology followed by DIGE-based proteomics for detection of cellular proteins in artificial peritoneal dialysis effluents.

Peritoneal dialysis effluent (PDE) represents a rich pool of potential biomarkers for monitoring disease and therapy. Until now, proteomic studies have been hindered by the plasma-like composition of the PDE. Beads covered with a peptide library are a promising approach to remove high abundant proteins and concentrate the sample in one step. In this study, a novel approach for proteomic biomarker identification in PDEs consisting of a depletion and concentration step followed by 2D gel based protein quantification was established. To prove this experimental concept a model system of artificial PDEs was established by spiking unused peritoneal dialysis (PD) fluids with cellular proteins reflecting control conditions or cell stress. Using this procedure, we were able to reduce the amount of high abundant plasma proteins and concentrate low abundant proteins while preserving changes in abundance of proteins with cellular origin. The alterations in abundance of the investigated marker for cell stress, the heat shock proteins, showed similar abundance profiles in the artificial PDE as in pure cell culture samples. Our results demonstrate the efficacy of this system in detecting subtle changes in cellular protein expression triggered by unphysiological stress stimuli typical in PD, which could serve as biomarkers. Further studies using patients' PDE will be necessary to prove the concept in clinical PD and to assess whether this technique is also informative regarding enriching low abundant plasma derived protein biomarker in the PDE.

Quantitative proteomics and phosphoproteomics profiling of meiotic divisions in the fission yeast Schizosaccharomyces pombe.

In eukaryotes, chromosomal DNA is equally distributed to daughter cells during mitosis, whereas the number of chromosomes is halved during meiosis. Despite considerable progress in understanding the molecular mechanisms that regulate mitosis, there is currently a lack of complete understanding of the molecular mechanisms regulating meiosis. Here, we took advantage of the fission yeast Schizosaccharomyces pombe, for which highly synchronous meiosis can be induced, and performed quantitative proteomics and phosphoproteomics analyses to track changes in protein expression and phosphorylation during meiotic divisions. We compared the proteomes and phosphoproteomes of exponentially growing mitotic cells with cells harvested around meiosis I, or meiosis II in strains bearing either the temperature-sensitive pat1-114 allele or conditional ATP analog-sensitive pat1-as2 allele of the Pat1 kinase. Comparing pat1-114 with pat1-as2 also allowed us to investigate the impact of elevated temperature (25 °C versus 34 °C) on meiosis, an issue that sexually reproducing organisms face due to climate change. Using TMTpro 18plex labeling and phosphopeptide enrichment strategies, we performed quantification of a total of 4673 proteins and 7172 phosphosites in S. pombe. We found that the protein level of 2680 proteins and the rate of phosphorylation of 4005 phosphosites significantly changed during progression of S. pombe cells through meiosis. The proteins exhibiting changes in expression and phosphorylation during meiotic divisions were represented mainly by those involved in the meiotic cell cycle, meiotic recombination, meiotic nuclear division, meiosis I, centromere clustering, microtubule cytoskeleton organization, ascospore formation, organonitrogen compound biosynthetic process, carboxylic acid metabolic process, gene expression, and ncRNA processing, among others. In summary, our findings provide global overview of changes in the levels and phosphorylation of proteins during progression of S. pombe cells through meiosis at normal and elevated temperatures, laying the groundwork for further elucidation of the functions and importance of specific proteins and their phosphorylation in regulating meiotic divisions in this yeast.

Identification of endophenotypes supporting outcome prediction in hemodialysis patients based on mechanistic markers of statin treatment.

Background: Statins are widely used to reduce the risk of cardiovascular disease (CVD). Patients with end-stage renal disease (ESRD) on hemodialysis have significantly increased risk of developing CVD. Statin treatment in these patients however did not show a statistically significant benefit in large trials on a patient cohort level. Methods: We generated gene expression profiles for statins to investigate the impact on cellular programs in human renal proximal tubular cells and mesangial cells in-vitro. We subsequently selected biomarkers from key statin-affected molecular pathways and assessed these biomarkers in plasma samples from the AURORA cohort, a double-blind, randomized, multi-center study of patients on hemodialysis or hemofiltration that have been treated with rosuvastatin. Patient clusters (phenotypes) were created based on the identified biomarkers using Latent Class Model clustering and the associations with outcome for the generated phenotypes were assessed using Cox proportional hazards regression models. The multivariable models were adjusted for clinical and biological covariates based on previously published data in AURORA. Results: The impact of statin treatment on mesangial cells was larger as compared with tubular cells with a large overlap of differentially expressed genes identified for atorvastatin and rosuvastatin indicating a predominant drug class effect. Affected molecular pathways included TGFB-, TNF-, and MAPK-signaling and focal adhesion among others. Four patient clusters were identified based on the baseline plasma concentrations of the eight biomarkers. Phenotype 1 was characterized by low to medium levels of the hepatocyte growth factor (HGF) and high levels of interleukin 6 (IL6) or matrix metalloproteinase 2 (MMP2) and it was significantly associated with outcome showing increased risk of developing major adverse cardiovascular events (MACE) or cardiovascular death. Phenotype 2 had high HGF but low Fas cell surface death receptor (FAS) levels and it was associated with significantly better outcome at 1 year. Conclusions: In this translational study, we identified patient subgroups based on mechanistic markers of statin therapy that are associated with disease outcome in patients on hemodialysis.

Plectin Deficiency in Fibroblasts Deranges Intermediate Filament and Organelle Morphology, Migration, and Adhesion.

Plectin, a highly versatile and multifunctional cytolinker, has been implicated in several multisystemic disorders. Most sequence variations in the human plectin gene (PLEC) cause epidermolysis bullosa simplex with muscular dystrophy (EBS-MD), an autosomal recessive skin-blistering disorder associated with progressive muscle weakness. In this study, we performed a comprehensive cell biological analysis of dermal fibroblasts from three different patients with EBS-MD, where PLEC expression analyses revealed preserved mRNA levels in all cases, whereas full-length plectin protein content was significantly reduced or completely absent. Downstream effects of pathogenic PLEC sequence alterations included massive bundling of vimentin intermediate filament networks, including the occurrence of ring-like nuclei-encasing filament bundles, elongated mitochondrial networks, and abnormal nuclear morphologies. We found that essential fibroblast functions such as wound healing, migration, or orientation upon cyclic stretch were significantly impaired in the cells of patients with EBS-MD. Finally, EBS-MD fibroblasts displayed reduced adhesion capacities, which could be attributed to smaller focal adhesion contacts. Our study not only emphasizes plectin's functional role in human skin fibroblasts, it also provides further insights into the understanding of EBS-MD-associated disease mechanisms.

Effect of cellular senescence on the response of human peritoneal mesothelial cells to TGF-ß.

Transforming growth factor ß (TGF-ß) is implicated in both mesothelial-to-mesenchymal transition (MMT) and cellular senescence of human peritoneal mesothelial cells (HPMCs). We previously showed that senescent HPMCs could spontaneously acquire some phenotypic features of MMT, which in young HPMCs were induced by TGF-ß. Here, we used electron microscopy, as well as global gene and protein profiling to assess in detail how exposure to TGF-ß impacts on young and senescent HPMCs in vitro. We found that TGF-ß induced structural changes consistent with MMT in young, but not in senescent HPMCs. Of all genes and proteins identified reliably in HPMCs across all treatments and states, 4,656 targets represented overlapping genes and proteins. Following exposure to TGF-ß, 137 proteins and 46 transcripts were significantly changed in young cells, compared to 225 proteins and only 2 transcripts in senescent cells. Identified differences between young and senescent HPMCs were related predominantly to wound healing, integrin-mediated signalling, production of proteases and extracellular matrix components, and cytoskeleton structure. Thus, the response of senescent HPMCs to TGF-ß differs or is less pronounced compared to young cells. As a result, the character and magnitude of the postulated contribution of HPMCs to TGF-ß-induced peritoneal remodelling may change with cell senescence.