Mechanisms of mesothelial cell response to viral infections: HDAC1-3 inhibition blocks poly(I:C)-induced type I interferon response and modulates the mesenchymal/inflammatory phenotype.

Infectious peritonitis is a leading cause of peritoneal functional impairment and a primary factor for therapy discontinuation in peritoneal dialysis (PD) patients. Although bacterial infections are a common cause of peritonitis episodes, emerging evidence suggests a role for viral pathogens. Toll-like receptors (TLRs) specifically recognize conserved pathogen-associated molecular patterns (PAMPs) from bacteria, viruses, and fungi, thereby orchestrating the ensuing inflammatory/immune responses. Among TLRs, TLR3 recognizes viral dsRNA and triggers antiviral response cascades upon activation. Epigenetic regulation, mediated by histone deacetylase (HDAC), has been demonstrated to control several cellular functions in response to various extracellular stimuli. Employing epigenetic target modulators, such as epidrugs, is a current therapeutic option in several cancers and holds promise in treating viral diseases. This study aims to elucidate the impact of TLR3 stimulation on the plasticity of human mesothelial cells (MCs) in PD patients and to investigate the effects of HDAC1-3 inhibition. Treatment of MCs from PD patients with the TLR3 agonist polyinosinic:polycytidylic acid (Poly(I:C)), led to the acquisition of a bona fide mesothelial-to-mesenchymal transition (MMT) characterized by the upregulation of mesenchymal genes and loss of epithelial-like features. Moreover, Poly(I:C) modulated the expression of several inflammatory cytokines and chemokines. A quantitative proteomic analysis of MCs treated with MS-275, an HDAC1-3 inhibitor, unveiled altered expression of several proteins, including inflammatory cytokines/chemokines and interferon-stimulated genes (ISGs). Treatment with MS-275 facilitated MMT reversal and inhibited the interferon signature, which was associated with reduced STAT1 phosphorylation. However, the modulation of inflammatory cytokine/chemokine production was not univocal, as IL-6 and CXCL8 were augmented while TNF-α and CXCL10 were decreased. Collectively, our findings underline the significance of viral infections in acquiring a mesenchymal-like phenotype by MCs and the potential consequences of virus-associated peritonitis episodes for PD patients. The observed promotion of MMT reversal and interferon response inhibition by an HDAC1-3 inhibitor, albeit without a general impact on inflammatory cytokine production, has translational implications deserving further analysis.

BET Protein Inhibitor JQ1 Ameliorates Experimental Peritoneal Damage by Inhibition of Inflammation and Oxidative Stress.

Peritoneal dialysis (PD) is a current replacement therapy for end-stage kidney diseases (ESKDs). However, long-term exposure to PD fluids may lead to damage of the peritoneal membrane (PM) through mechanisms involving the activation of the inflammatory response and mesothelial-to-mesenchymal transition (MMT), leading to filtration failure. Peritoneal damage depends on a complex interaction among external stimuli, intrinsic properties of the PM, and subsequent activities of the local innate-adaptive immune system. Epigenetic drugs targeting bromodomain and extra-terminal domain (BET) proteins have shown beneficial effects on different experimental preclinical diseases, mainly by inhibiting proliferative and inflammatory responses. However the effect of BET inhibition on peritoneal damage has not been studied. To this aim, we have evaluated the effects of treatment with the BET inhibitor JQ1 in a mouse model of peritoneal damage induced by chlorhexidine gluconate (CHX). We found that JQ1 ameliorated the CHX-induced PM thickness and inflammatory cell infiltration. Moreover, JQ1 decreased gene overexpression of proinflammatory and profibrotic markers, together with an inhibition of the nuclear factor-κB (NF-κB) pathway. Additionally, JQ1 blocked the activation of nuclear factor erythroid 2-related factor 2 (NRF2) and restored changes in the mRNA expression levels of NADPH oxidases (NOX1 and NOX4) and NRF2/target antioxidant response genes. To corroborate the in vivo findings, we evaluated the effects of the BET inhibitor JQ1 on PD patients' effluent-derived primary mesothelial cells and on the MeT-5A cell line. JQ1 inhibited tumor necrosis factor-α (TNF-α)-induced proinflammatory gene upregulation and restored MMT phenotype changes, together with the downmodulation of oxidative stress. Taken together, these results suggest that BET inhibitors may be a potential therapeutic option to ameliorate peritoneal damage.

Restoration of WT1/miR-769-5p axis by HDAC1 inhibition promotes MMT reversal in mesenchymal-like mesothelial cells.

Histone acetylation/deacetylation play an essential role in modifying chromatin structure and in regulating cell plasticity in eukaryotic cells. Therefore, histone deacetylase (HDAC) pharmacological inhibitors are promising tools in the therapy of fibrotic diseases and in cancer. Peritoneal fibrosis is a pathological process characterized by many cellular and molecular alterations, including the acquisition of invasive/pro-fibrotic abilities by mesothelial cells (MCs) through induction of mesothelial to mesenchymal transition (MMT). The aim of this study was to characterize the molecular mechanism of the antifibrotic role of HDAC1 inhibition. Specifically, treatment with MS-275, an HDAC1-3 inhibitor previously known to promote MMT reversal, induced the expression of several TGFBRI mRNA-targeting miRNAs. Among them, miR-769-5p ectopic expression was sufficient to promote MMT reversal and to limit MC migration and invasion, whereas miR-769-5p silencing further enhanced mesenchymal gene expression. These results were confirmed by HDAC1 genetic silencing. Interestingly, miR-769-5p silencing maintained mesenchymal features despite HDAC1 inhibition, thus indicating that it is necessary to drive MMT reversal induced by HDAC1 inhibition. Besides TGFBRI, miR-769-5p was demonstrated to target SMAD2/3 and PAI-1 expression directly. When analyzing molecular mechanisms underlying miR-769-5p expression, we found that the transcription factor Wilms' tumor 1 (WT1), a master gene controlling MC development, binds to the miR-769-5p promoter favoring its expression. Interestingly, both WT1 expression and binding to miR-769-5p promoter were increased by HDAC1 inhibition and attenuated by TGFß1 treatment. Finally, we explored the significance of these observations in the cell-to-cell communication: we evaluated the ability of miR-769-5p to be loaded into extracellular vesicles (EVs) and to promote MMT reversal in recipient mesenchymal-like MCs. Treatment of fibrotic MCs with EVs isolated from miR-769-5p over-expressing MCs promoted the down-regulation of specific mesenchymal targets and the reacquisition of an epithelial-like morphology. In conclusion, we highlighted an HDAC1-WT1-miR-769-5p axis potentially relevant for therapies aimed at counteracting organ fibrosis.

Exposure to b-LED Light While Exerting Antimicrobial Activity on Gram-Negative and -Positive Bacteria Promotes Transient EMT-like Changes and Growth Arrest in Keratinocytes.

While blue LED (b-LED) light is increasingly being studied for its cytotoxic activity towards bacteria in therapy of skin-related infections, its effects on eukaryotic cells plasticity are less well characterized. Moreover, since different protocols are often used, comparing the effect of b-LED towards both microorganisms and epithelial surfaces may be difficult. The aim of this study was to analyze, in the same experimental setting, both the bactericidal activity and the effects on human keratinocytes. Exposure to b-LED induced an intense cytocidal activity against Gram-positive (i.e, Staphylococcus aureus) and Gram-negative (i.e., Pseudomonas aeruginosa) bacteria associated with catheter-related infections. Treatment with b-LED of a human keratinocyte cell line induced a transient cell cycle arrest. At the molecular level, exposure to b-LED induced a transient downregulation of Cyclin D1 and an upregulation of p21, but not signs of apoptosis. Interestingly, a transient induction of phosphor-histone γ-H2Ax, which is associated with genotoxic damages, was observed. At the same time, keratinocytes underwent a transient epithelial to mesenchymal transition (EMT)-like phenotype, characterized by E-cadherin downregulation and SNAIL/SLUG induction. As a functional readout of EMT induction, a scratch assay was performed. Surprisingly, b-LED treatment provoked a delay in the scratch closure. In conclusion, we demonstrated that b-LED microbicidal activity is associated with complex responses in keratinocytes that certainly deserve further analysis.

Automated Peritoneal Dialysis: Patient Perspectives and Outcomes.

A steadily increasing number of end stage kidney disease (ESKD) patients are maintained on automated peritoneal dialysis (APD) worldwide, in long-standing as well as in more recently established peritoneal dialysis (PD) programs. A better understanding of the technique, paralleled by progress in involved technology, sustained this growth to the point that APD has become the prevalent mode of PD delivery in most high-income countries. While APD is now regarded to be at least as efficient as continuous ambulatory peritoneal dialysis (CAPD) with regard to major biomedical outcomes, its impact on patient-reported outcomes has been less investigated. This paper reviews the main outcomes of APD from a clinical point of view and from the person on dialysis perspective.

Intraperitoneal Pressure in Polycystic and Non-Polycystic Kidney Disease Patients, Treated by Peritoneal Dialysis.

INTRODUCTION: Intraperitoneal volume (IPV) should be individualized and aimed to maintain an intraperitoneal pressure (IPP) lower than 17 cm H2O. IPP is very variable, given its relation with body size. However, it is not yet fully understood which anthropometric variable mostly affects IPP and the relation between IPP and organomegaly in polycystic kidney disease (PKD) patients is not known. OBJECTIVES: The aim of the present study was to analyse the relation between antropometric variables and IPP in a large cohort of peritoneal dialysis (PD) patients and to identify if a relation between nephromegaly and IPP exists in PKD patients. METHODS: IPP was measured in PD patients and data was retrospectively collected. In PKD patients, total kidney volumes were measured in CT scans, and normalized with height (hTKV). RESULTS: Seventy-seven patients were included in the study, 18% affected by PKD. Mean IPP was 14.9 ± 2.9 cm H2O and it showed significant positive correlation with body mass index (BMI; ρ = 0.42, p < 0.001). No correlation was found between IPP and absolute IPV; conversely, IPP has a significant inverse correlation with IPV normalized with BMI and body surface area (ρ -0.38, p = 0.001 and ρ -0.25, p = 0.02, -respectively). Patients with IPP >17 cm H2O have significant larger BMI and lower IPV/BMI compared to those with IPP <17 cm H2O (29 ± 3.6 vs. 26 ± 4 kg/m2, p < 0.05 and 97 ± 15.5 vs. 109 ± 22 mL/kg/m2, p < 0.05). PKD patients have a wide variability in hTKV (range 645-3,787 mL/m2) and it showed a significant correlation with IPP/IPV (ρ = 0.6, p < 0.05). CONCLUSIONS: Patients with larger BMI have greater IPP, irrespectively to IPV. In PKD patients, hTKV correlate with IPP/IPV ratio. However, given the wide range of distribution of hTKV, increased IPP cannot be presumed because of pre-existing polycystic kidney, but need to be quantified.

[Peritoneal dialysis in the Lazio region: results from 2017 regional audit].

In 2011, a first peritoneal dialysis audit was held in the Lazio region to analyze the problems hindering the spread of this method and to improve the quality of care through the sharing of best practices across Centers. A scientific board was therefore set up, representing all the Centers offering PD, in order to assess clinical effectiveness using KPIs (Key Performance Indicators) and to quantify the objectives to be achieved. The analysis made it possible to identify the main problems and take action, all the while monitoring progress through KPIs. A second audit was carried out in 2017 and the collected data was analyzed and compared with the findings of the previous study. Overall, data showed an increase in prevalence, although the incidence showed a slight decrease. Indicators on the change of dialysis treatment, the dropout from domiciliary treatment and the incidence of late referral appeared stable over time. A slight improvement was observed in clinical data on peritonitis and on the length of hospitalization. All participants in the audit declared that sharing and discussing clinical practices had been really useful. In addition, through the drafting of practical documents (guides for patients, guidance on informed consent, protocols of clinical follow-up), a number of tools have been provided to ensure a uniformly high level of care across the different regional Centers.

HDAC1 inhibition by MS-275 in mesothelial cells limits cellular invasion and promotes MMT reversal.

Peritoneal fibrosis is a pathological alteration of the peritoneal membrane occurring in a variety of conditions including peritoneal dialysis (PD), post-surgery adhesions and peritoneal metastases. The acquisition of invasive and pro-fibrotic abilities by mesothelial cells (MCs) through induction of MMT, a cell-specific form of EMT, plays a main role in this process. Aim of this study was to evaluate possible effects of histone deacetylase (HDAC) inhibitors, key components of the epigenetic machinery, in counteracting MMT observed in MCs isolated from effluent of PD patients. HDAC inhibitors with different class/isoform selectivity have been used for pharmacological inhibition. While the effect of other inhibitors was limited to a partial E-cadherin re-expression, MS-275, a HDAC1-3 inhibitor, promoted: (i) downregulation of mesenchymal markers (MMP2, Col1A1, PAI-1, TGFß1, TGFßRI) (ii) upregulation of epithelial markers (E-cadherin, Occludin), (iii) reacquisition of an epithelial-like morphology and (iv) marked reduction of cellular invasiveness. Results were confirmed by HDAC1 genetic silencing. Mechanistically, MS-275 causes: (i) increase of nuclear histone H3 acetylation (ii) rescue of the acetylation profile on E-cadherin promoter, (iii) Snail functional impairment. Overall, our study, pinpointing a role for HDAC1, revealed a new player in the regulation of peritoneal fibrosis, providing the rationale for future therapeutic opportunities.

[Best practices on peritoneal dialysis: the audit of Lazio Region]. / Best practices in dialisi peritoneale: l'audit della regione Lazio.

The diffusion of peritoneal methodology can not be something out of the real organizational context and the regional directive can not be the only means to encourage the diffusion. There is the need to provide effective and sustainable levels of assistance through a clinical scientific support and sharing of best-practises. On one side, the aim is to provide an aid by the centers with great expertise in the methodology, recognized as reference points; on the other side, to establish the shared K.P.I.s (Key Performance Index), to asses the clinical effectiveness and measure the objectives to be achieved, through a modality of valuation to establish the real applicability. For this purpose, a scientific board was founded, composed by the heads of UU.OO, that provide the peritoneal dialysis, to determine which aspects to investigate and identify factors of supply improvement. The selected method was the clinical audit. The analysis of the 2011 data has allowed us to capture the situation of the peritoneal dialysis in the Lazio Region. The formative procedure has enabled the centers to share and standardize protocols and therapeutic procedures, identify the strengths of peritoneal dialysis in the Lazio Region and define the KPIs through whose compare and monitor the centers over time. The conclusive analysis of the audit has enabled to identify a series of activities to be undertaken together in order to improve the situation of the peritoneal dialysis in the Lazio Region. In the following years, surveys will be carried out to verify the KPIs trend.

Cross-Over Efficiency Comparison of Different Tidal Automated Peritoneal Dialysis Schedules.

This study compares 5 different tidal automated peritoneal dialysis (APD) prescriptions. Six low-average and 6 high-average transporters performed 3 separate sessions with 5 different schedules: (A) 50% tidal with initial fill volume (FV) 2 liters, (B) 50% tidal with 2.2 liters initial FV, (C) 2.2 liters initial FV with 70% tidal, (D) as in B, with one complete renewal of the initial FV at midsession, (E) 2.2 liters FV with breakpoint modality. Urea, creatinine and phosphate peritoneal clearances, sodium removal and ultrafiltration (UF) were compared using analysis of variance. Compared to treatment A, all the tested alternative schedules were associated with 10% significantly higher urea clearance; B, D and E, but not C, were associated with 10% higher creatinine clearance. Phosphate clearance was significantly higher with D, while sodium removal was larger with both C and D. UF was lower with A and E in high average transporters. Manipulation of the main prescriptive parameters of tidal APD has significant impact on its efficiency.

Relation between right and left ventricular function in patients undergoing chronic dialysis.

AIMS: Occurrence of heart failure during dialysis treatment is associated with high mortality. However, mechanisms underlying left ventricular dysfunction (LVD) in these patients are still elusive. In patients undergoing haemodialysis, arteriovenous fistula (AVF) is associated with right ventricular dysfunction (RVD) and a further impairment is observed when AVF is brachial rather than radial. However, it is not known whether AVF-induced RVD is associated with an impaired left ventricular function. We studied the relation between right and left ventricular function in 120 patients undergoing either haemodialysis or peritoneal dialysis and 100 healthy age-matched controls. METHODS: Echocardiography including tissue Doppler imaging (TDI) was performed for both ventricles. Average myocardial performance index (MPI) of the right ventricle (RV MPI) was obtained with a multisegmental approach by using TDI. RESULTS: RVD was higher in haemodialysis than peritoneal dialysis patients and a further increase was observed in haemodialysis patients with brachial access. Interestingly, RV MPI inversely correlated with indices of both left ventricular contraction and relaxation and the association was even stronger in haemodialysis patients, particularly in those with brachial AVF. Of note, dialysis patients in the upper tertile of RV MPI showed the larger impairment of left ventricular function. Regression analyses showed that RV MPI was independently associated with reduced left ventricular function. By contrast, LVD did not significantly affect right ventricular performance in this setting. CONCLUSION: AVF-induced RVD may contribute to LVD in dialysis patients. AVF plays a pivotal role in triggering LVD via right-to-left ventricular interdependence.

Determinants of sodium removal with tidal automated peritoneal dialysis.

In a comprehensive evaluation of dialysis adequacy, major attention has been recently paid to fluid and Na balance. Removal of Na has been reported to be significantly poorer with automated peritoneal dialysis (APD) than with continuous ambulatory peritoneal dialysis. Only limited data on Na removal with tidal APD have been published. We analyzed peritoneal Na mass balance in 122 separate nightly tidal APD sessions performed by 7 peritonitis-free, clinically stable, patients with negligible residual renal function (< 100 mL urine daily). Correlations with other efficiency measures [ultrafiltration (UF) and small-solute clearances], prescriptive parameters [duration of treatment, initial intraperitoneal fill volume (IPV) and its tidal percentage, and dialysate flux] and peritoneal transport status were tested in univariate and multivariate linear regression models. Removal of Na was 89 +/- 55 mmol per treatment, which correlated with UF (r = 0.29, p = 0.001) and was higher in patients with high-average transport (118 +/- 41 mmol vs. 81 +/- 56 mmol in low-average transporters, p = 0.0004), in whom a significant positive correlation was found with initial IPV and duration of treatment (r = 0.55; 95% confidence interval: 0.21 to 0.77; p = 0.0029; and r = 0.66; 95% confidence interval: 0.38 to 0.83; p = 0.0002 respectively). Removal of Na correlated weakly with UF in tidal APD and showed wide inter-patient variability. It should therefore be measured rather than roughly estimated from UF. Its magnitude exposes the anuric patient on nightly APD with a "dry" day to the risk of Na retention, unless controlled Na intake or dialytic strategies aimed at enhancing Na removal, or both, are implemented.

Incremental peritoneal dialysis favourably compares with hemodialysis as a bridge to renal transplantation.

Background. The value of incremental peritoneal dialysis (PD) as a bridge to renal transplantation (Tx) has not been specifically addressed. Methods. All consecutive Stage 5 CKD patients with at least 1 year predialysis followup, starting incremental PD or HD under our care and subsequently receiving their first renal Tx were included in this observational cohort study. Age, gender, BMI, underlying nephropathy, residual renal function (RRF) loss rate before dialysis and RRF at RRT start, comorbidity, RRT schedules and adequacy measures, dialysis-related morbidity, Tx waiting time, RRF at Tx, incidence of delayed graft function (DGF), in-hospital stay for Tx, serum creatinine at discharge and one year later were collected and compared between patients on incremental PD or HD before Tx. Results. Seventeen patients on incremental PD and 24 on HD received their first renal Tx during the study period. Age, underlying nephropathy, RRF loss rate in predialysis, RRF at the start of RRT and comorbidity did not differ significantly. While on dialysis, patients on PD had significantly lower epoetin requirements, serum phosphate, calciumxphosphate product and better RRF preservation. Delayed graft function (DGF) occurred in 12 patients (29%), 1 on incremental PD and 11 on HD. Serum creatinine at discharge and 1 year later was significantly higher in patients who had been on HD. Conclusions. In patients receiving their first renal Tx, previous incremental PD was associated with low morbidity, excellent preservation of RRF, easier attainment of adequacy targets and significantly better immediate and 1-year graft function than those observed in otherwise well-matched patients previously treated with HD.

Right ventricular dysfunction in patients with end-stage renal disease.

BACKGROUND: While chronic dialysis treatment has been suggested to increase pulmonary pressure values, right ventricular dysfunction (RVD) is a major cause of death in patients with end-stage renal disease. We investigated the impact of different dialysis treatments on right ventricular function. METHODS: We examined 220 subjects grouped as follows: healthy controls (n = 100), peritoneal dialysis (PD; n = 26), hemodialysis (HD) with radial arteriovenous fistula (AVF; n = 62), and HD with brachial AVF (n = 32). Echocardiography including tissue Doppler imaging (TDI) of the right ventricle was performed in all patients. RESULTS: Pulmonary pressure values progressively rose from controls across the 3 dialysis groups (21.7 ± 6.8, 29.7 ± 6.7, 37.9 ± 6.7 and 40.8 ± 6.6 mm Hg, respectively; p < 0.001). TDI indices of right ventricular function were more impaired in HD patients, particularly in those with brachial AVF. RVD, assessed by TDI myocardial performance index, was higher in HD patients compared with PD patients (71.3 vs. 34.6%, p < 0.001). Moreover, the prevalence of RVD further increased in patients with brachial AVF compared with the radial access (90.6 vs. 61.3%, p < 0.001). CONCLUSIONS: Compared to DP, HD increases the risk of RVD, particularly in the presence of brachial AVF. TDI may detect early functional failure of the right ventricle in HD patients.