Quality of design and reporting of animal research in peritoneal dialysis: A scoping review.

The concerns about reproducibility and validity of animal studies are partly related to poor experimental design and reporting. Here, we undertook a scoping review of the literature to determine the extent and quality of reporting of animal studies on peritoneal dialysis (PD). Online databases were searched to identify 567 relevant original articles published between 1979 and 2018. These were analyzed with respect to bibliographic parameters and general aspects of animal experimentation. A subgroup of 120 studies was analyzed in detail in terms of the impact on the reporting quality of the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines for animal studies. The number of animal studies on PD increased continuously over the years with a thematic shift toward long-term preservation of the peritoneum as a dialyzing organ. There were significant deficiencies in research design with the lack of sample size estimation, randomization, and blinding being the commonest shortcomings. The description of animal numbers, housing conditions, use of medication, and statistical analysis was incomplete. The introduction in 2010 of the ARRIVE guidelines produced very little improvement in the completeness of reporting regardless of journal impact factor. The animal studies on PD suffer from deficits in experimental protocols and transparent reporting. These drawbacks need to be corrected to ensure high-quality and much-needed animal research in PD.

Epithelial-to-Mesenchymal Transition and Migration of Human Peritoneal Mesothelial Cells Undergoing Senescence.

BACKGROUND: Epithelial-to-mesenchymal transition (EMT) of human peritoneal mesothelial cells (HPMCs) contributes to fibrotic thickening of the peritoneum that develops in patients on peritoneal dialysis (PD). The process is thought to be largely mediated by transforming growth factor-beta (TGF-ß). As TGF-ß has also been implicated in senescence of HPMCs, we have performed an exploratory study to examine if senescent HPMCs can undergo EMT. METHODS: Omentum-derived HPMCs were rendered senescent by repeated passages in culture. Features of EMT were assessed by immunostaining and quantitative polymerase chain reaction (qPCR) at various stages of the HPMC lifespan and after treatment with or without TGF-ß. The motility of HPMCs was assessed in a scratch wound migration assay. RESULTS: Replicative senescence of HPMCs was associated with a gradual increase in the constitutive expression of EMT markers, including increased production of extracellular matrix proteins. However, senescent HPMCs also retained epithelial cell features such as cytokeratin, calretinin, and E-cadherin and showed decreased, rather than increased, motility. In contrast, exposure to TGF-ß resulted in an up-regulation of mesenchymal markers and down-regulation of epithelial markers. Such effects of TGF-ß occurred both in young and senescent cells, although they were less pronounced in senescence. CONCLUSIONS: Senescence of HPMCs is associated with spontaneous development of several EMT features. At the same time, senescent HPMCs preserve epithelial cell-like characteristics and are less prone to develop a full EMT phenotype in response to TGF-ß. These observations may support the concept of cellular senescence being antagonistically pleiotropic with regard to EMT.

Thy-1+/- fibroblast subsets in the human peritoneum.

Fibrotic thickening of the peritoneum develops in patients receiving peritoneal dialysis (PD) for renal failure. For unknown reasons, however, in some patients it progresses to extensive fibrosis that compromises dialysis capacity of the peritoneum. It is increasingly clear that fibroblasts display large heterogeneity not only between but also within tissues. Differential surface expression of thymocyte differentiation antigen 1 (Thy-1) has been shown to identify functionally distinct fibroblast subsets in several organs. Here, we isolated Thy-1+/- subsets of human peritoneal fibroblasts (HPFB) and analyzed them in terms of profibrotic myofibroblast features. In healthy individuals, Thy-1+ cells constituted ~45% of the HPFB population found in the greater omentum but were not detected in the parietal peritoneum. When propagated in culture and compared with Thy-1- cells, omentum-derived Thy-1+ HPFB consistently displayed an increased expression of α-smooth muscle actin, collagen I, and transforming growth factor-ß1. They also showed greater proliferation capacity and enhanced contractile properties. The number of Thy-1+ HPFB increased significantly in PD patients and made up more than 70 and 95% of all HPFB found in the omentum and parietal peritoneum, respectively. These data indicate that the expansion of Thy-1+ fibroblasts may contribute to fibrotic thickening of the peritoneal membrane during PD.

Telomere length profiles in primary human peritoneal mesothelial cells are consistent with senescence.

Mesothelial cell (MC) senescence contributes to malignancy and tissue fibrosis. The role of telomere erosion in MC senescence remains controversial, with evidence for both telomere-dependent and telomere-independent mechanisms reported. Single telomere length analysis revealed considerable telomere length heterogeneity in freshly isolated human peritoneal MCs, reflecting a heterogeneous proliferative history and providing high-resolution evidence for telomere-dependent senescence. By contrast the attenuated replicative lifespan, lack of telomere erosion and induction of p16 expression in in vitro-aged cells was consistent with stress-induced senescence. Given the potential pathophysiological impact of senescence in mesothelial tissues, high-resolution MC telomere length analysis may provide clinically useful information.

IL-6 Trans-Signaling Links Inflammation with Angiogenesis in the Peritoneal Membrane.

Vascular endothelial growth factor (VEGF) is implicated in the peritoneal membrane remodeling that limits ultrafiltration in patients on peritoneal dialysis (PD). Although the exact mechanism of VEGF induction in PD is unclear, VEGF concentrations in drained dialysate correlate with IL-6 levels, suggesting a link between these cytokines. Human peritoneal mesothelial cells (HPMCs), the main source of IL-6 and VEGF in the peritoneum, do not bear the cognate IL-6 receptor and are thus unable to respond to classic IL-6 receptor signaling. Here, we investigated whether VEGF release by HPMCs is controlled by IL-6 in combination with its soluble receptor (IL-6 trans-signaling). Although treatment with either IL-6 or soluble IL-6 receptor (sIL-6R) alone had no effect on VEGF production, stimulation of HPMCs with IL-6 in combination with sIL-6R promoted VEGF expression and secretion through a transcriptional mechanism involving STAT3 and SP4. Conditioned medium from HPMCs cultured with IL-6 and sIL-6R promoted angiogenic endothelial tube formation, which could be blocked by silencing SP4. In vivo, induction of peritoneal inflammation in wild-type and IL-6-deficient mice showed IL-6 involvement in the control of Sp4 and Vegf expression and new vessel formation, confirming the role of IL-6 trans-signaling in these processes. Taken together, these findings identify a novel mechanism linking IL-6 trans-signaling and angiogenesis in the peritoneal membrane.

Increased storage and secretion of phosphatidylcholines by senescent human peritoneal mesothelial cells.

BACKGROUND/AIMS: Human peritoneal mesothelial cells (HPMC) secrete phosphatidylcholines (PC) which form a lipid bilayer lining the peritoneum. They prevent frictions and adhesions and act as a barrier to the transport of water-soluble solutes while permitting water flux. PC may play an essential role in peritoneal integrity and function, the role of PD induced HPMC senescence on PC homeostasis, however, is unknown. METHODS: HPMC cell lines were isolated from four non-uremic patients. Expression of the three PC synthesis genes (rt-PCR), and cellular storage and secretion of PC (ESI-mass-spectrometry) were analyzed in young and senescent HPMC (>Hayflick-limit). RESULTS: Senescent cells displayed significantly altered morphology; flow cytometry demonstrated extensive staining for senescence-associated beta galactosidase. Nine different PC were detected in HPMC with palmitoyl-myristoyl phosphatidylcholine (PMPC) being most abundant. In senescent HPMC mRNA expression of the three key PC synthesis genes was 1.5-, 2.4- and 6-fold increased as compared to young HPMC, with the latter, phosphatidylcholine cytidylyltransferase, being rate limiting. Intracellular storage of the nine PC was 75-450 % higher in senescent vs. young HPMC, PC secretion rates were 100-300 % higher. Intracellular PC concentrations were not correlated with the PC secretion rates. Electron microscopy demonstrated lamellar bodies, the primary storage site of PC, in senescent but not in young cells. CONCLUSION: Senescent HPMC store and secrete substantially more PC than young cells. Our findings indicate a novel protective mechanism, which should counteract peritoneal damage induced by chronic exposure to PD fluids.

Senescence-Associated Changes in Proteome and O-GlcNAcylation Pattern in Human Peritoneal Mesothelial Cells.

INTRODUCTION: Senescence of peritoneal mesothelial cells represents a biological program defined by arrested cell growth and altered cell secretory phenotype with potential impact in peritoneal dialysis. This study aims to characterize cellular senescence at the level of global protein expression profiles and modification of proteins with O-linked N-acetylglucosamine (O-GlcNAcylation). METHODS: A comparative proteomics analysis between young and senescent human peritoneal mesothelial cells (HPMC) was performed using two-dimensional gel electrophoresis. O-GlcNAc status was assessed by Western blot under normal conditions and after modulation with 6-diazo-5-oxo-L-norleucine (DON) to decrease O-GlcNAcylation or O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino N-phenyl carbamate (PUGNAc) to increase O-GlcNAcylation. RESULTS: Comparison of protein pattern of senescent and young HPMC revealed 29 differentially abundant protein spots, 11 of which were identified to be actin (cytoplasmic 1 and 2), cytokeratin-7, cofilin-2, transgelin-2, Hsp60, Hsc70, proteasome ß-subunits (type-2 and type-3), nucleoside diphosphate kinase A, and cytosolic 5'(3')-deoxyribonucleotidase. Although the global level of O-GlcNAcylation was comparable, senescent cells were not sensitive to modulation by PUGNAc. DISCUSSION: This study identified changes of the proteome and altered dynamics of O-GlcNAc regulation in senescent mesothelial cells. Whereas changes in cytoskeleton-associated proteins likely reflect altered cell morphology, changes in chaperoning and housekeeping proteins may have functional impact on cellular stress response in peritoneal dialysis.

New developments in peritoneal fibroblast biology: implications for inflammation and fibrosis in peritoneal dialysis.

Uraemia and long-term peritoneal dialysis (PD) can lead to fibrotic thickening of the peritoneal membrane, which may limit its dialytic function. Peritoneal fibrosis is associated with the appearance of myofibroblasts and expansion of extracellular matrix. The extent of contribution of resident peritoneal fibroblasts to these changes is a matter of debate. Recent studies point to a significant heterogeneity and complexity of the peritoneal fibroblast population. Here, we review recent developments in peritoneal fibroblast biology and summarize the current knowledge on the involvement of peritoneal fibroblasts in peritoneal inflammation and fibrosis.