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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.

Mutations in pre-mRNA processing factors (PRPFs) cause 40% of autosomal dominant retinitis pigmentosa (RP), but it is unclear why mutations in ubiquitously expressed PRPFs cause retinal disease. To understand the molecular basis of this phenotype, we have generated RP type 11 (PRPF31-mutated) patient-specific retinal organoids and retinal pigment epithelium (RPE) from induced pluripotent stem cells (iPSC). Impaired alternative splicing of genes encoding pre-mRNA splicing proteins occurred in patient-specific retinal cells and Prpf31 /- mouse retinae, but not fibroblasts and iPSCs, providing mechanistic insights into retinal-specific phenotypes of PRPFs. RPE was the most affected, characterised by loss of apical-basal polarity, reduced trans-epithelial resistance, phagocytic capacity, microvilli, and cilia length and incidence. Disrupted cilia morphology was observed in patient-derived-photoreceptors that displayed progressive features associated with degeneration and cell stress. In situ gene-editing of a pathogenic mutation rescued key structural and functional phenotypes in RPE and photoreceptors, providing proof-of-concept for future therapeutic strategies.

SummaryMutations in pre-mRNA processing factors (PRPFs) cause 40% of autosomal dominant retinitis pigmentosa (RP), but it is unclear why mutations in ubiquitously expressed PRPFs cause retinal disease. To understand the molecular basis of this phenotype, we have generated RP type 11 (PRPF31-mutated) patient-specific retinal organoids and retinal pigment epithelium (RPE) from induced pluripotent stem cells (iPSC). Impaired alternative splicing of genes encoding pre-mRNA splicing proteins occurred in patient-specific retinal cells and Prpf31+/− mouse retinae, but not fibroblasts and iPSCs, providing mechanistic insights into retinal-specific phenotypes of PRPFs. RPE was the most affected, characterised by loss of apical-basal polarity, reduced trans-epithelial resistance, phagocytic capacity, microvilli, and cilia length and incidence. Disrupted cilia morphology was observed in patient-derived-photoreceptors that displayed progressive features associated with degeneration and cell stress. In situ gene-editing of a pathogenic mutation rescued key structural and functional phenotypes in RPE and photoreceptors, providing proof-of-concept for future therapeutic strategies.eTOCPRPF31 is a ubiquitously expressed pre-mRNA processing factor that when mutated causes autosomal dominant RP. Using a patient-specific iPSC approach, Buskin and Zhu et al. show that retinal-specific defects result from altered splicing of genes involved in the splicing process itself, leading to impaired splicing, loss of RPE polarity and diminished phagocytic ability as well as reduced cilia incidence and length in both photoreceptors and RPE.HighlightsSuccessful generation of iPSC-derived RPE and photoreceptors from four RP type 11 patientsRPE cells express the mutant PRPF31 protein and show the lowest expression of wildtype proteinPRPF31 mutations result in altered splicing of genes involved in pre-mRNA splicing in RPE and retinal organoidsPrpf31 haploinsufficiency results in altered splicing of genes involved in pre-mRNA splicing in mouse retinaRPE cells display loss of polarity, reduced barrier function and phagocytosisPhotoreceptors display shorter and fewer cilia and degenerative featuresRPE cells display most abnormalities suggesting they might be the primary site of pathogenesisIn situ gene editing corrects the mutation and rescues key phenotypes

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.

The aim of study is to visualize not only the primary and active element but also sensors, returning the current state of the system. The work shows the use of piezoelectric accelerometer sensors in the active vibration reduction. In addition to the elements reduction of vibrations also are necessary elements with which it will be possible to constantly test and measure vibration. It is necessary to generate the force to appropriate executive to reduce the effects of vibration. It will be also shows how to design basic system in something special way which is the synthesis of mechanical systems.

Recent research suggests that cytoprotective responses, such as expression of heat-shock proteins, might be inadequately induced in mesothelial cells by heat-sterilized peritoneal dialysis (PD) fluids. This study compares transcriptome data and multiple protein expression profiles for providing new insight into regulatory mechanisms. Two-dimensional difference gel electrophoresis (2D-DIGE) based proteomics and topic defined gene expression microarray-based transcriptomics techniques were used to evaluate stress responses in human omental peritoneal mesothelial cells in response to heat- or filter-sterilized PD fluids. Data from selected heat-shock proteins were validated by 2D western-blot analysis. Comparison of proteomics and transcriptomics data discriminated differentially regulated protein abundance into groups depending on correlating or noncorrelating transcripts. Inadequate abundance of several heat-shock proteins following exposure to heat-sterilized PD fluids is not reflected on the mRNA level indicating interference beyond transcriptional regulation. For the first time, this study describes evidence for posttranscriptional inadequacy of heat-shock protein expression by heat-sterilized PD fluids as a novel cytotoxic property. Cross-omics technologies introduce a novel way of understanding PDF bioincompatibility and searching for new interventions to reestablish adequate cytoprotective responses.