High-Dose Everolimus May Induce Pro-Inflammatory/Fibrotic Transcriptomic Changes In Bronchial Epithelial Cells From Cystic Fibrosis Patients.

Background: Solid organ transplantation is an available therapeutic option for cystic fibrosis (CF) patients without lung transplantation. However, the use of immunosuppressive agents may cause severe adverse events. In particular, patients treated with mTOR-inhibitors (mTOR-I) may aggravate pulmonary complications. It has been recently described that these drugs may induce epithelial to mesenchymal transition (EMT) of airway cells. Objective: The purpose of this study was to evaluate the effects of mTOR-I on primary bronchial epithelial cells carrying F508del. Materials and Methods: Human bronchial epithelial cells homozygous for F508del were treated with 5 and 100nM EVE for 24 hours and their RNA was extracted and hybridized to the Human HT-12 v3 Expression BeadChip (Illumina). Microarray results were validated by Real-Time PCR. Transepithelial resistance was measured by Millicell-ERS ohmmeter. Results: High dosage EVE induced a significant up-regulation of 48 genes and a down-regulation of 14 genes. After pathway analysis by GSEA, we found that most of them were implicated in the inflammatory and pro-fibrotic pathways. Real-Time PCR confirmed that 100nM EVE was able to up-regulate some identified genes (IL-1 α IL-8, Pim-1) as well as pro-fibrotic elements ( α -SMA, connective tissue growth factor and metalloproteinase-12). Additionally, high dosage of EVE was also able to reduce the transepithelial resistance. In contrast, a lower level of EVE did not produce similar effects. Conclusion: Although performed in vitro, our study suggested that in solid organ transplant recipients with CF without a lung transplant, mTOR-I should be used at a low dosage to reduce its contribution to pulmonary inflammation and fibrosis.

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.

Circulating Ouabain Modulates Expression of Claudins in Rat Intestine and Cerebral Blood Vessels

The ability of exogenous low ouabain concentrations to affect claudin expression and therefore epithelial barrier properties was demonstrated previously in cultured cell studies. We hypothesized that chronic elevation of circulating ouabain in vivo can affect the expression of claudins and tight junction permeability in different tissues. We tested this hypothesis in rats intraperitoneally injected with ouabain (1 μg/kg) for 4 days. Rat jejunum, colon and brain frontal lobes, which are variable in the expressed claudins and tight junction permeability, were examined. Moreover, the porcine jejunum cell line IPEC-J2 was studied. In IPEC-J2-cells, ouabain (10 nM, 19 days of incubation) stimulated epithelial barrier formation, increased transepithelial resistance and the level of cSrc-kinase activation by phosphorylation, accompanied with an increased expression of claudin-1, -5 and down-regulation of claudin-12; the expression of claudin-3, -4, -8 and tricellulin was not changed. In the jejunum, chronic ouabain increased the expression of claudin-1, -3 and -5 without an effect on claudin-2 and -4 expression. In the colon, only down-regulation of claudin-3 was observed. Chronic ouabain protected the intestine transepithelial resistance against functional injury induced by lipopolysaccharide treatment or by modeled acute microgravity; this regulation was most pronounced in the jejunum. Claudin-1 was also up-regulated in cerebral blood vessels. This was associated with reduction of claudin-3 expression while the expression of claudin-5 and occludin was not affected. Altogether, our results confirm that circulating ouabain can functionally and tissue-specifically affect barrier properties of epithelial and endothelial tissues via Na,K-ATPase-mediated modulation of claudins expression.

A new role for heat shock factor 27 in the pathophysiology of Clostridium difficile toxin B

Clostridium difficile (CD) is a common pathogen that causes severe gastrointestinal inflammatory diarrhea in patients undergoing antibiotic therapy. Its virulence derives from two toxins, toxin CD, A and B (TcdA and TcdB) (Borriello et al. Rev Infect Dis 12, Suppl 2: S185-191, 1990). Among the prime candidates for CD colonization are patients with cystic fibrosis (CF), who are routinely treated with antibiotics and frequently hospitalized. Indeed, ~50% of patients with CF are colonized with virulent forms of CD but do not exhibit diarrhea (Bauer et al. Clin Microbiol Infect 20: O446–O449, 2014; Binkovitz et al. Am J Roentgenol 172: 517–521, 199; Zemljic et al. Anaerobe 16: 527–532, 2010). We found that TcdB has global effects on colonic cells, including reducing the steady-state levels of sodium-proton exchange regulatory factors, reducing the levels of heat shock protein (Hsp) 27, and increasing the fraction of total Hsp27 bound to the cystic fibrosis transmembrane conductance regulator (CFTR). Also, since some mutations in CFTR seem to be protective, we asked whether CFTR is a target of TcdB. We show here that TcdB increases the maturation of CFTR and transiently increases its function. These combined effects promote increased surface expression of CFTR, resulting in a transient increase in Cl− secretion. This increase is followed by a precipitous decline in both CFTR-dependent Cl− secretion and transepithelial resistance (TER), suggesting a breakdown in the epithelial cells’ tight junctions. We also found that overexpressing Hsp27 reverses some of the deleterious effects of TcdB, in particular preserving TER and therefore likely the maintenance of barrier function. Thus, our data suggest that Hsp27 plays a role in the diarrhea generated by CD infection and is a potential therapeutic target for treating this diarrhea. NEW & NOTEWORTHY Clostridium difficile (CD) is a common pathogen that causes severe gastrointestinal inflammatory diarrhea in patients undergoing antibiotic therapy. We provide new evidence that heat shock protein (Hsp) 27 is one of the key players in CD pathology and that increasing Hsp27 can prevent the decrease in transepithelial resistance induced by toxin CD B, pointing the way for pharmacologic therapies for patients with chronic CD infection that can increase Hsp27 as a means to mitigate the effects of CD on gastrointestinal pathology.

Claudin-19 Is Regulated by Extracellular Osmolality in Rat Kidney Inner Medullary Collecting Duct Cells

The inner medullary collecting duct (IMCD) is subject to severe changes in ambient osmolality and must either allow water transport or be able to seal the lumen against a very high osmotic pressure. We postulate that the tight junction protein claudin-19 is expressed in IMCD and that it takes part in epithelial adaptation to changing osmolality at different functional states. Presence of claudin-19 in rat IMCD was investigated by Western blotting and immunofluorescence. Primary cell culture of rat IMCD cells on permeable filter supports was performed under different osmotic culture conditions and after stimulation by antidiuretic hormone (AVP). Electrogenic transepithelial transport properties were measured in Ussing chambers. IMCD cells cultivated at 300 mosm/kg showed high transepithelial resistance, a cation selective paracellular pathway and claudin-19 was mainly located in the tight junction. Treatment by AVP increased cation selectivity but did not alter transepithelial resistance or claudin-19 subcellular localization. In contrast, IMCD cells cultivated at 900 mosm/kg had low transepithelial resistance, anion selectivity, and claudin-19 was relocated from the tight junctions to intracellular vesicles. The data shows osmolality-dependent transformation of IMCD epithelium from tight and sodium-transporting to leaky, with claudin-19 expression in the tight junction associated to tightness and cation selectivity under low osmolality.

Diurnal heat stress reduces pig intestinal integrity and increases endotoxin translocation

Heat stress negatively affects performance and intestinal integrity of pigs. The objective of this study was to characterize the effects of diurnal heat stress (dHS) on nursery-grower pig performance, intestinal integrity, and lipopolysaccharide (LPS) translocation. Forty-eight nursery-grower gilts, individually penned, were randomly assigned to two treatments. Twenty-four pigs were then exposed to dHS for 3 d, 6 h at 38°C and 18 h at 32°C, at 40–60% humidity. The remaining pigs were maintained under thermal neutral (TN) conditions. Changes in pig rectal temperatures (Tr), respiration rates (RR), performance, and blood parameters were evaluated. Additionally, ex vivo ileum integrity was assessed with the Ussing chamber by measuring transepithelial resistance (TER), and 4 kDa fluorescein isothiocyanate (FITC)–dextran (FD4) and FITC–LPS mucosal to serosal flux. As expected, dHS increased pig Tr and RR (P < 0.05) and reduced pig performance (P < 0.05) on the 3-d period. Compared with TN, ileum TER (P = 0.04), FITC–LPS (P < 0.001), and FD4 (P = 0.011) permeability were significantly increased due to dHS. Compared with TN pigs, dHS increased serum endotoxin by 150% (P = 0.031). Altogether, 3-d dHS significantly reduced pig performance and intestinal integrity and increased blood endotoxin concentrations.

GRHL2 Is Required for Collecting Duct Epithelial Barrier Function and Renal Osmoregulation

Collecting ducts make up the distal-most tubular segments of the kidney, extending from the cortex, where they connect to the nephron proper, into the medulla, where they release urine into the renal pelvis. During water deprivation, body water preservation is ensured by the selective transepithelial reabsorption of water into the hypertonic medullary interstitium mediated by collecting ducts. The collecting duct epithelium forms tight junctions composed of barrier-enforcing claudins and exhibits a higher transepithelial resistance than other segments of the renal tubule exhibit. However, the functional relevance of this strong collecting duct epithelial barrier is unresolved. Here, we report that collecting duct–specific deletion of an epithelial transcription factor, grainyhead-like 2 (GRHL2), in mice led to reduced expression of tight junction–associated barrier components, reduced collecting duct transepithelial resistance, and defective renal medullary accumulation of sodium and other osmolytes. In vitro, Grhl2-deficient collecting duct cells displayed increased paracellular flux of sodium, chloride, and urea. Consistent with these effects, Grhl2-deficient mice had diabetes insipidus, produced dilute urine, and failed to adequately concentrate their urine after water restriction, resulting in susceptibility to prerenal azotemia. These data indicate a direct functional link between collecting duct epithelial barrier characteristics, which appear to prevent leakage of interstitial osmolytes into urine, and body water homeostasis.