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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 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 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 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.

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.