Calprotectin blockade inhibits long-term vascular pathology following peritoneal dialysis-associated bacterial infection.

Bacterial infections and the concurrent inflammation have been associated with increased long-term cardiovascular (CV) risk. In patients receiving peritoneal dialysis (PD), bacterial peritonitis is a common occurrence, and each episode further increases late CV mortality risk. However, the underlying mechanism(s) remains to be elucidated before safe and efficient anti-inflammatory interventions can be developed. Damage-Associated Molecular Patterns (DAMPs) have been shown to contribute to the acute inflammatory response to infections, but a potential role for DAMPs in mediating long-term vascular inflammation and CV risk following infection resolution in PD, has not been investigated. We found that bacterial peritonitis in mice that resolved within 24h led to CV disease-promoting systemic and vascular immune-mediated inflammatory responses that were maintained up to 28 days. These included higher blood proportions of inflammatory leukocytes displaying increased adhesion molecule expression, higher plasma cytokines levels, and increased aortic inflammatory and atherosclerosis-associated gene expression. These effects were also observed in infected nephropathic mice and amplified in mice routinely exposed to PD fluids. A peritonitis episode resulted in elevated plasma levels of the DAMP Calprotectin, both in PD patients and mice, here the increase was maintained up to 28 days. In vitro, the ability of culture supernatants from infected cells to promote key inflammatory and atherosclerosis-associated cellular responses, such as monocyte chemotaxis, and foam cell formation, was Calprotectin-dependent. In vivo, Calprotectin blockade robustly inhibited the short and long-term peripheral and vascular consequences of peritonitis, thereby demonstrating that targeting of the DAMP Calprotectin is a promising therapeutic strategy to reduce the long-lasting vascular inflammatory aftermath of an infection, notably PD-associated peritonitis, ultimately lowering CV risk.

Targeting carcinoma-associated mesothelial cells with antibody-drug conjugates in ovarian carcinomatosis.

Ovarian carcinomatosis is characterized by the accumulation of carcinoma-associated mesothelial cells (CAMs) in the peritoneal stroma and mainly originates through a mesothelial-to-mesenchymal transition (MMT) process. MMT has been proposed as a therapeutic target for peritoneal metastasis. Most ovarian cancer (OC) patients present at diagnosis with peritoneal seeding, which makes tumor progression control difficult by MMT modulation. An alternative approach is to use antibody-drug conjugates (ADCs) targeted directly to attack CAMs. This strategy could represent the cornerstone of precision-based medicine for peritoneal carcinomatosis. Here, we performed complete transcriptome analyses of ascitic fluid-isolated CAMs in advanced OC patients with primary-, high-, and low-grade, serous subtypes and following neoadjuvant chemotherapy. Our findings suggest that both cancer biological aggressiveness and chemotherapy-induced tumor mass reduction reflect the MMT-associated changes that take place in the tumor surrounding microenvironment. Accordingly, MMT-related genes, including fibroblast activation protein (FAP), mannose receptor C type 2 (MRC2), interleukin-11 receptor alpha (IL11RA), myristoylated alanine-rich C-kinase substrate (MARCKS), and sulfatase-1 (SULF1), were identified as specific actionable targets in CAMs of OC patients, which is a crucial step in the de novo design of ADCs. These cell surface target receptors were also validated in peritoneal CAMs of colorectal cancer peritoneal implants, indicating that ADC-based treatment could extend to other abdominal tumors that show peritoneal colonization. As proof of concept, a FAP-targeted ADC reduced tumor growth in an OC xenograft mouse model with peritoneal metastasis-associated fibroblasts. In summary, we propose MMT as a potential source of ADC-based therapeutic targets for peritoneal carcinomatosis. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Novel Aspects of the Immune Response Involved in the Peritoneal Damage in Chronic Kidney Disease Patients under Dialysis.

Chronic kidney disease (CKD) incidence is growing worldwide, with a significant percentage of CKD patients reaching end-stage renal disease (ESRD) and requiring kidney replacement therapies (KRT). Peritoneal dialysis (PD) is a convenient KRT presenting benefices as home therapy. In PD patients, the peritoneum is chronically exposed to PD fluids containing supraphysiologic concentrations of glucose or other osmotic agents, leading to the activation of cellular and molecular processes of damage, including inflammation and fibrosis. Importantly, peritonitis episodes enhance peritoneum inflammation status and accelerate peritoneal injury. Here, we review the role of immune cells in the damage of the peritoneal membrane (PM) by repeated exposure to PD fluids during KRT as well as by bacterial or viral infections. We also discuss the anti-inflammatory properties of current clinical treatments of CKD patients in KRT and their potential effect on preserving PM integrity. Finally, given the current importance of coronavirus disease 2019 (COVID-19) disease, we also analyze here the implications of this disease in CKD and KRT.

Potential Therapeutic Effects of Natural Plant Compounds in Kidney Disease.

BACKGROUND: The blockade of the progression or onset of pathological events is essential for the homeostasis of an organism. Some common pathological mechanisms involving a wide range of diseases are the uncontrolled inflammatory reactions that promote fibrosis, oxidative reactions, and other alterations. Natural plant compounds (NPCs) are bioactive elements obtained from natural sources that can regulate physiological processes. Inflammation is recognized as an important factor in the development and evolution of chronic renal damage. Consequently, any compound able to modulate inflammation or inflammation-related processes can be thought of as a renal protective agent and/or a potential treatment tool for controlling renal damage. The objective of this research was to review the beneficial effects of bioactive natural compounds on kidney damage to reveal their efficacy as demonstrated in clinical studies. METHODS: This systematic review is based on relevant studies focused on the impact of NPCs with therapeutic potential for kidney disease treatment in humans. RESULTS: Clinical studies have evaluated NPCs as a different way to treat or prevent renal damage and appear to show some benefits in improving OS, inflammation, and antioxidant capacity, therefore making them promising therapeutic tools to reduce or prevent the onset and progression of KD pathogenesis. CONCLUSIONS: This review shows the promising clinical properties of NPC in KD therapy. However, more robust clinical trials are needed to establish their safety and therapeutic effects in the area of renal damage.

Alanyl-Glutamine Restores Tight Junction Organization after Disruption by a Conventional Peritoneal Dialysis Fluid.

Understanding and targeting the molecular basis of peritoneal solute and protein transport is essential to improve peritoneal dialysis (PD) efficacy and patient outcome. Supplementation of PD fluids (PDF) with alanyl-glutamine (AlaGln) increased small solute transport and reduced peritoneal protein loss in a recent clinical trial. Transepithelial resistance and 10 kDa and 70 kDa dextran transport were measured in primary human endothelial cells (HUVEC) exposed to conventional acidic, glucose degradation products (GDP) containing PDF (CPDF) and to low GDP containing PDF (LPDF) with and without AlaGln. Zonula occludens-1 (ZO-1) and claudin-5 were quantified by Western blot and immunofluorescence and in mice exposed to saline and CPDF for 7 weeks by digital imaging analyses. Spatial clustering of ZO-1 molecules was assessed by single molecule localization microscopy. AlaGln increased transepithelial resistance, and in CPDF exposed HUVEC decreased dextran transport rates and preserved claudin-5 and ZO-1 abundance. Endothelial clustering of membrane bound ZO-1 was higher in CPDF supplemented with AlaGln. In mice, arteriolar endothelial claudin-5 was reduced in CPDF, but restored with AlaGln, while mesothelial claudin-5 abundance was unchanged. AlaGln supplementation seals the peritoneal endothelial barrier, and when supplemented to conventional PD fluid increases claudin-5 and ZO-1 abundance and clustering of ZO-1 in the endothelial cell membrane.

Targeting Toll-like receptors with soluble Toll-like receptor 2 prevents peritoneal dialysis solution-induced fibrosis.

Peritoneal membrane failure due to fibrosis limits the use of peritoneal dialysis (PD). Peritoneal fibrosis may potentially be induced by sterile inflammation caused by ongoing cellular stress due to prolonged exposure to PD solutions (PDS). Effective therapies to prevent this process remain to be developed. Toll-like receptors (TLRs) mediate sterile inflammation by recognizing damage-associated molecular patterns (DAMPs) released by cellular stress. We evaluated the involvement of TLRs and DAMPs in PDS-induced fibrosis models and the therapeutic potential of TLR-DAMP targeting for preventing fibrosis. A range of PDS elicited pro-inflammatory and fibrotic responses from PD patient peritoneal leukocytes, mesothelial cells and mouse peritoneal leukocytes. TLR2/4 blockade of human peritoneal cells or TLR2/4 knockouts inhibited these effects. PDS did not induce rapid ERK phosphorylation or IκB-α degradation, suggesting that they do not contain components capable of direct TLR activation. However, PDS increased the release of Hsp70 and hyaluronan, both TLR2/4 DAMP ligands, by human and mouse peritoneal cells, and their blockade decreased PDS-driven inflammation. Soluble TLR2, a TLR inhibitor, reduced PDS-induced pro-inflammatory and fibrotic cytokine release ex vivo. Daily catheter infusion of PDS in mice caused peritoneal fibrosis, but co-administration of soluble TLR2 prevented fibrosis, suppressed pro-fibrotic gene expression and pro-inflammatory cytokine production, reduced leukocyte/neutrophil recruitment, recovered Treg cell levels and increased the Treg:Th17 ratio. Thus, TLR2/4, Hsp70 and hyaluronan showed major roles in PDS-induced peritoneal inflammation and fibrosis. The study demonstrates the therapeutic potential of a TLR-DAMP targeting strategy to prevent PDS-induced fibrosis.

Genomic reprograming analysis of the Mesothelial to Mesenchymal Transition identifies biomarkers in peritoneal dialysis patients.

Peritoneal dialysis (PD) is an effective renal replacement therapy, but a significant proportion of patients suffer PD-related complications, which limit the treatment duration. Mesothelial-to-mesenchymal transition (MMT) contributes to the PD-related peritoneal dysfunction. We analyzed the genetic reprograming of MMT to identify new biomarkers that may be tested in PD-patients. Microarray analysis revealed a partial overlapping between MMT induced in vitro and ex vivo in effluent-derived mesothelial cells, and that MMT is mainly a repression process being higher the number of genes that are down-regulated than those that are induced. Cellular morphology and number of altered genes showed that MMT ex vivo could be subdivided into two stages: early/epithelioid and advanced/non-epithelioid. RT-PCR array analysis demonstrated that a number of genes differentially expressed in effluent-derived non-epithelioid cells also showed significant differential expression when comparing standard versus low-GDP PD fluids. Thrombospondin-1 (TSP1), collagen-13 (COL13), vascular endothelial growth factor A (VEGFA), and gremlin-1 (GREM1) were measured in PD effluents, and except GREM1, showed significant differences between early and advanced stages of MMT, and their expression was associated with a high peritoneal transport status. The results establish a proof of concept about the feasibility of measuring MMT-associated secreted protein levels as potential biomarkers in PD.

UNR/CSDE1 Drives a Post-transcriptional Program to Promote Melanoma Invasion and Metastasis.

RNA binding proteins (RBPs) modulate cancer progression through poorly understood mechanisms. Here we show that the RBP UNR/CSDE1 is overexpressed in melanoma tumors and promotes invasion and metastasis. iCLIP sequencing, RNA sequencing, and ribosome profiling combined with in silico studies unveiled sets of pro-metastatic factors coordinately regulated by UNR as part of RNA regulons. In addition to RNA steady-state levels, UNR was found to control many of its targets at the level of translation elongation/termination. Key pro-oncogenic targets of UNR included VIM and RAC1, as validated by loss- and gain-of-function studies. Our results identify UNR as an oncogenic modulator of melanoma progression, unravel the underlying molecular mechanisms, and identify potential targets for this therapeutically challenging malignancy.

Metastatic risk and resistance to BRAF inhibitors in melanoma defined by selective allelic loss of ATG5.

Melanoma is a paradigm of aggressive tumors with a complex and heterogeneous genetic background. Still, melanoma cells frequently retain developmental traits that trace back to lineage specification programs. In particular, lysosome-associated vesicular trafficking is emerging as a melanoma-enriched lineage dependency. However, the contribution of other lysosomal functions such as autophagy to melanoma progression is unclear, particularly in the context of metastasis and resistance to targeted therapy. Here we mined a broad spectrum of cancers for a meta-analysis of mRNA expression, copy number variation and prognostic value of 13 core autophagy genes. This strategy identified heterozygous loss of ATG5 at chromosome band 6q21 as a distinctive feature of advanced melanomas. Importantly, partial ATG5 loss predicted poor overall patient survival in a manner not shared by other autophagy factors and not recapitulated in other tumor types. This prognostic relevance of ATG5 copy number was not evident for other 6q21 neighboring genes. Melanocyte-specific mouse models confirmed that heterozygous (but not homozygous) deletion of Atg5 enhanced melanoma metastasis and compromised the response to targeted therapy (exemplified by dabrafenib, a BRAF inhibitor in clinical use). Collectively, our results support ATG5 as a therapeutically relevant dose-dependent rheostat of melanoma progression. Moreover, these data have important translational implications in drug design, as partial blockade of autophagy genes may worsen (instead of counteracting) the malignant behavior of metastatic melanomas.

Paricalcitol reduces peritoneal fibrosis in mice through the activation of regulatory T cells and reduction in IL-17 production.

Fibrosis is a significant health problem associated with a chronic inflammatory reaction. The precise mechanisms involved in the fibrotic process are still poorly understood. However, given that inflammation is a major causative factor, immunomodulation is a possible therapeutic approach to reduce fibrosis. The vitamin D receptor (VDR) that is present in all hematopoietic cells has been associated with immunomodulation. We investigated whether the intraperitoneal administration of paricalcitol, a specific activator of the VDR, modulates peritoneal dialysis fluid (PDF)-induced peritoneal fibrosis. We characterized the inflammatory process in the peritoneal cavity of mice treated or not treated with paricalcitol and analyzed the ensuing fibrosis. The treatment reduced peritoneal IL-17 levels, which strongly correlated with a significantly lower peritoneal fibrotic response. In vitro studies demonstrate that both CD4+ and CD8+ regulatory T cells appear to impact the regulation of IL-17. Paricalcitol treatment resulted in a significantly increased frequency of CD8+ T cells showing a regulatory phenotype. The frequency of CD4+ Tregs tends to be increased, but it did not achieve statistical significance. However, paricalcitol treatment increased the number of CD4+ and CD8+ Treg cells in vivo. In conclusion, the activation of immunological regulatory mechanisms by VDR signaling could prevent or reduce fibrosis, as shown in peritoneal fibrosis induced by PDF exposure in mice.

A genome-wide search for quantitative trait loci affecting the cortical surface area and thickness of Heschl's gyrus.

Heschl's gyrus (HG) is a core region of the auditory cortex whose morphology is highly variable across individuals. This variability has been linked to sound perception ability in both speech and music domains. Previous studies show that variations in morphological features of HG, such as cortical surface area and thickness, are heritable. To identify genetic variants that affect HG morphology, we conducted a genome-wide association scan (GWAS) meta-analysis in 3054 healthy individuals using HG surface area and thickness as quantitative traits. None of the single nucleotide polymorphisms (SNPs) showed association P values that would survive correction for multiple testing over the genome. The most significant association was found between right HG area and SNP rs72932726 close to gene DCBLD2 (3q12.1; P=2.77 × 10(-7) ). This SNP was also associated with other regions involved in speech processing. The SNP rs333332 within gene KALRN (3q21.2; P=2.27 × 10(-6) ) and rs143000161 near gene COBLL1 (2q24.3; P=2.40 × 10(-6) ) were associated with the area and thickness of left HG, respectively. Both genes are involved in the development of the nervous system. The SNP rs7062395 close to the X-linked deafness gene POU3F4 was associated with right HG thickness (Xq21.1; P=2.38 × 10(-6) ). This is the first molecular genetic analysis of variability in HG morphology.

Effect of lixiviated sediments affected with treated water on Selenastrum capricornutum, Printz and Origanum vulgare L.

Xochimilco is an area of Mexico City fulfilling important ecological functions. However, the water of the canal network in the lacustrine zone of Xochimilco is supplied by the water treatment plants of the city, implying a risk of accumulated contaminants in the sediments. This study reports the effect of lixiviates obtained from sediments collected in the canals of Xochimilco on the growth of the alga Selenastrum capricornutum and the angiosperm Origanum vulgare. Three factors were tested: (a) water source in terms of the effluent from the two water treatment plants (urban waste-water, located at Cerro de la Estrella (CE) and urban-rural waters at San Luis Tlaxialtemalco (SLT); (b) sampling season (January, dry season; May and September, rainy season; and (c) distances from the water discharge point in the Xochimilco's main canal (5200 and 1000 m for CE, and 0, 200 m for SLT). The chemical water properties analyzed were: pH, electrical conductivity, N-NO(3), N-NH(3), N(Total), P-PO(4) and P(Total). The alga was more sensitive to the contaminants than O. vulgare, showing growth inhibition of 93-100%. The effect of sampling season on the inhibition of algal growth was ordered as follows: September > May > January. Lixiviates obtained from sediment samples 200 and 1000 m from the main point of water discharge caused a higher algal growth inhibition than the samples obtained at the source point. Lixiviate promoted the growth of seedlings of O. vulgare.

Characterization of epithelial-to-mesenchymal transition of mesothelial cells in a mouse model of chronic peritoneal exposure to high glucose dialysate.

Animal models of peritoneal dialysis fluid (PDF) exposure are key tools in the study of mechanisms involved in alterations of the peritoneal membrane and in the design of therapies. We recently developed a mouse model of chronic peritoneal exposure to high glucose dialysate. Herein, we make a sequential analysis of the effects of glucose-based PDF on mouse peritoneal membrane and on mesothelium. We demonstrate that chronic exposure to PDF induces thickness and fibrosis of the peritoneal membrane in a time-dependent manner. We also show that mesothelial cells progressively detach and lose cytokeratin expression. In addition, we demonstrate that some mesothelial cells invade the submesothelial space, where they appear as cytokeratin- and alpha-smooth muscle actin-positive cells. These findings demonstrate that epithelial-to-mesenchymal transition (EMT) of mesothelial cells takes place in mouse peritoneum exposed to PDF, validating this model for the study of effects of drugs on the EMT process as a therapy for peritoneal deterioration.

Fixed versus removable microdialysis probes for in vivo neurochemical analysis: implications for behavioral studies.

The levels of several neurochemicals, i.e., uric acid (UA), dopamine (DA), dihydroxyphenylacetic acid, and 5-hydroxyindoleacetic acid, collected daily from the rat striatum with either fixed or removable microdialysis probes for 7 days after surgery were compared. The implantation of the fixed cannula was followed by a 10-fold increase in the UA content in the dialysates collected from the first day after surgery onward and by a steady decrease in dihydroxyphenylacetic acid levels, whereas those of DA remained fairly stable. With the removable cannula system, only a smaller, transient increase in UA during the first 3 days after surgery was observed, with no change in DA or monoamine metabolites. The glial reaction around the cannula tracks was assessed by both quantitative histological techniques and measuring the glutamine levels in the dialysates collected at the time of surgery and 7 days later. Both the glial cell number and nuclear size, as well as the glutamine outflow, were considerably larger in the animals implanted with the fixed probes. It is, therefore, likely that the UA levels in the dialysate reflect the glial reaction to the probe. The suitability of the removable probe system for behavioral experiments involving repeated microdialysis sampling was illustrated in an experiment showing that the DA release in the nucleus accumbens of male rats assessed daily at postsurgery days 5-10 was virtually identical in three alternating sessions of sexual behavior as was the smaller release of this neurotransmitter detected during intervening nonsexual social interactions.

Voltammetric monitoring of brain extracellular levels of serotonin, 5-hydroxyindoleacetic acid and uric acid as assessed by simultaneous microdialysis.

We have previously developed a microcomputer-assisted curve-fitting method for measuring the components of the mixed electrochemical signals recorded by differential normal pulse voltammetry in the living brain. It was initially used for resolution of the dopamine and dihydroxyphenylacetic acid components of the catechol signal (peak 2). This report shows how it can be applied to analysis of the indoleamine/uric acid (UA) components of the more complex peak 3. The voltammogram is modeled as a mixture of 3 normal curves of known parameters corresponding to the oxidation of UA, 5-hydroxyindoleacetic acid and serotonin, which is solved by non-linear iterative procedures. Performance was assessed by treatments with drugs having well-known effects on the substances monitored, pargyline and allopurinol, and by the chromatographic analysis of microdialysates collected simultaneously from the contralateral side.

Mathematical resolution of mixed in vivo voltammetry signals. Models, equipment, assessment by simultaneous microdialysis sampling.

A microcomputer-assisted curve-fitting procedure was developed for the quantitative estimation of the components of the mixed "catechol peak" recorded with differential normal pulse voltammetry (DNPV) at electrochemically pretreated carbon fiber microelectrodes in the living brain. The contribution of each of the relevant electroactive species is fitted by a normal probability function, the parameters of which are previously determined in vitro for each electrode and substance. The voltammogram is thus modeled as a mixture of normal curves corresponding to the individual oxidizable substances plus a low order polynomial approximating the baseline. In a former approach the function was solved by linear least squares techniques. As a further improvement, we now propose a non-linear model of the voltammogram and a Gauss-Newton iterative algorithm with stepwise regression for parameter estimation. This report shows the application of the method for the resolution of the dopamine (DA) and dihydroxyphenylacetic acid (DOPAC) components of the DNPV signal recorded from the striatum of freely moving animals in response to amphetamine and pargyline. The method was validated by the chemical assay of contralateral microdialysates collected simultaneously. The changes detected by both methodologies were closely parallel, with highly significant correlation coefficients (0.87 and 0.99 for DA and DOPAC, respectively, P less than 0.001). This study further illustrates that the in vivo voltammetry methodology can be improved substantially by incorporating a suitable mathematical treatment of the electrochemical signals.

Sex-related olfactory stimuli induce a selective increase in dopamine release in the nucleus accumbens of male rats. A voltammetric study.

Changes in the dopaminergic (DA) transmission in the nucleus accumbens were investigated in male rats exposed to sociosexual olfactory stimuli from different conspecifics: receptive female, non-receptive female and intact male. DAergic transmission was assessed by measurement of extracellular levels of DA and dihydroxyphenylacetic acid (DOPAC). Both compounds were recorded by using differential normal pulse voltammetry (DNPV) with electrochemically pretreated carbon fiber electrodes and numerical analysis of the catechol peak. Exposition to receptive female odors induced a marked and selective increase in DA release compared to control values. Exposition to non-receptive female odors and male odors induced an increase in DA release not significantly different from that following the change of environment. In conclusion, mesencephalic DAergic neurons reaching the nucleus accumbens appear to be involved in the perception of behaviorally significant olfactory cues.

In vivo voltammetry study of the modulatory action of prolactin on the mesolimbic dopaminergic system.

The effects of an acute subcutaneous injection of ovine prolactin (PRL) or its vehicle on total motor activity and the extracellular levels of dopamine (DA), DOPAC and ascorbic acid (AA) in the nucleus accumbens were monitored simultaneously in freely behaving male rats. The neurochemical data were obtained by Differential Normal Pulse Voltammetry using electrochemically pretreated carbon fiber microelectrodes and numerical waveform analysis of the catechol peak. PRL treatment increased the extracellular levels of DA and DOPAC in the nucleus accumbens, which is consistent with previous reports using other methodologies. Furthermore, there was a concomitant decrease in the AA signals and motor activity. These findings suggest an antagonistic action of PRL on dopaminergic transmission in the mesolimbic system.