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Tubular atrophy/interstitial fibrosis (TA/IF) is a major cause of late allograft loss, and inflammation within areas of TA/IF is associated with adverse outcomes in kidney transplantation. However, there is currently no satisfactory method to suppress this inflammation to improve TA/IF. The present study aimed to determine the proinflammatory role of receptor‑interacting protein 3 (RIP3) in TA/IF to discover a novel therapeutic target. Reverse transcription‑quantitative PCR and western blotting were performed to detect the expression of RIP3 and inflammation‑associated factors. Lactate dehydrogenase release assay was used to determine necroptosis. Fluorescent 2,7‑dichlorodihydrofluorescein diacetate was used to detect the levels of reactive oxygen species (ROS). The results demonstrated that patients with chronic TA/IF exhibited upregulated receptor‑interacting protein 3 (RIP3) expression compared with the patients who had a favorable recovery after renal transplant. Therefore, the current study used normal renal tubular epithelial cells HK‑2 to establish a cellular model with a high expression level of RIP3 in order to investigate the effect of RIP3 on renal epithelial cells after transplantation. The western blotting results demonstrated that overexpression of RIP3 could significantly increase the phosphorylation level of the necroptosis executive molecule mixed lineage kinase domain‑like protein. Lactate dehydrogenase release, a key feature of necroptosis, was also markedly improved by RIP3 overexpression. Moreover, a higher inflammatory response was detected in HK‑2 cells with RIP3 overexpression, and this elevated inflammation could be restored by the necroptosis inhibitor necrosulfonamide. Of note, it was found that overexpression of RIP3 activated the NF‑κB signaling pathway via the excessive accumulation of ROS to induce necroptosis, which ultimately led to inflammation. Collectively, these findings indicated that overexpression of RIP3 promoted necroptosis via a ROS‑dependent NF‑κB pathway to induce chronic inflammation, suggesting that RIP3 may have the potential to be a therapeutic target against inflammation in TA/IF.

Purpose: Low free testosterone (T) level in men is independently associated with presence and severity of Non-Alcoholic Steatohepatitis (NASH). The histological and molecular effects of oral testosterone prodrug LPCN 1144 treatment on hepatic fibrosis and NASH features are unknown. A metabolic syndrome-induced NASH model in rabbits consuming high fat diet (HFD) has been previously used to assess treatment effects of injectable T on hepatic fibrosis and NASH features. Here we present results on LPCN 1144 in this HFD-induced, NASH preclinical model., Methods: Male rabbits were randomly assigned to five groups: regular diet (RD), HFD, HFD + 1144 vehicle (HFD + Veh), HFD + 1144 (1144), and HFD + 1144 + α-tocopherol (1144 + ALPHA). Rabbits were sacrificed after 12 weeks for liver histological, biochemical and genetic analyses. Histological scores were obtained through Giemsa (inflammation), Masson's trichrome (steatosis and ballooning), and Picrosirius Red (fibrosis) staining., Results: Compared to RD, HFD and HFD + Veh significantly worsened NASH features and hepatic fibrosis. Considering HFD and HFD + Veh arms, histological and biomarker features were not significantly different. Both 1144 and 1144 + ALPHA arms improved mean histological scores of NASH as compared to HFD arm. Importantly, percentage of fibrosis was improved in both 1144 (p < 0.05) and 1144 + ALPHA (p = 0.05) treatment arms vs. HFD. Both treatment arms also reduced HFD-induced inflammation and fibrosis mRNA markers. Furthermore, 1144 treatments significantly improved HFD-induced metabolic dysfunctions., Conclusions: Histological and biomarker analyses demonstrate that LPCN 1144 improved HFD-induced hepatic fibrosis and NASH biochemical, biomolecular and histochemical features. These preclinical findings support a therapeutic potential of LPCN 1144 in the treatment of NASH and of hepatic fibrosis., (© 2021. The Author(s).)

Heart failure (HF) and cardiac arrhythmias share overlapping pathological mechanisms that act cooperatively to accelerate disease pathogenesis. Cardiac fibrosis is associated with both pathological conditions. Our previous work identified a link between phytosterol accumulation and cardiac injury in a mouse model of phytosterolemia, a rare disorder characterized by elevated circulating phytosterols and increased cardiovascular disease risk. Here, we uncover a previously unknown pathological link between phytosterols and cardiac arrhythmias in the same animal model. Phytosterolemia resulted in inflammatory pathway induction, premature ventricular contractions (PVC) and ventricular tachycardia (VT). Blockade of phytosterol absorption either by therapeutic inhibition or by genetic inactivation of NPC1L1 prevented the induction of inflammation and arrhythmogenesis. Inhibition of phytosterol absorption reduced inflammation and cardiac fibrosis, improved cardiac function, reduced the incidence of arrhythmias and increased survival in a mouse model of phytosterolemia. Collectively, this work identified a pathological mechanism whereby elevated phytosterols result in inflammation and cardiac fibrosis leading to impaired cardiac function, arrhythmias and sudden death. These comorbidities provide insight into the underlying pathophysiological mechanism for phytosterolemia-associated risk of sudden cardiac death., (© 2021. The Author(s).)

Diabetes is a major risk factor for the development of cardiovascular disease with a higher incidence of myocardial infarction. This study explores the role of metformin, a first-line antihyperglycemic agent, in postinfarction fibrotic and inflammatory remodeling in mice. Three-month-old C57BI/6J mice were submitted to 30 min cardiac ischemia followed by reperfusion for 14 days. Intraperitoneal treatment with metformin (5 mg/kg) was initiated 15 min after the onset of reperfusion and maintained for 14 days. Real-time PCR was used to determine the levels of COL3A1, αSMA, CD68, TNF-α and IL-6. Increased collagen deposition and infiltration of macrophages in heart tissues are associated with upregulation of the inflammation-associated genes in mice after 14 days of reperfusion. Metformin treatment markedly reduced postinfarction fibrotic remodeling and CD68-positive cell population in mice. Moreover, metformin resulted in reduced expression of COL3A1, αSMA and CD68 after 14 days of reperfusion. Taken together, these results open new perspectives for the use of metformin as a drug that counteracts adverse myocardial fibroticand inflammatory remodeling after MI.

Intestinal fibrosis is a common complication of inflammatory bowel disease (IBD) and is defined as an excessive accumulation of scar tissue in the intestinal wall. Intestinal fibrosis occurs in both forms of IBD: ulcerative colitis and Crohn's disease. Small-molecule inhibitors targeting hypoxia-inducing factor (HIF) prolyl-hydroxylases are promising for the development of novel antifibrotic therapies in IBD. Herein, we evaluated the therapeutic efficacy of hydroxamate of betulinic acid (BHA), a hypoxia mimetic derivative of betulinic acid, against IBD in vitro and in vivo. We showed that BAH (5-20 μM) dose-dependently enhanced collagen gel contraction and activated the HIF pathway in NIH-3T3 fibroblasts; BAH treatment also prevented the loss of trans-epithelial electrical resistance induced by proinflammatory cytokines in Caco-2 cells. In two different murine models (TNBS- and DSS-induced IBD) that cause colon fibrosis, oral administration of BAH (20, 50 mg/kg·d, for 17 days) prevented colon inflammation and fibrosis, as detected using immunohistochemistry and qPCR assays. BAH-treated animals showed a significant reduction of fibrotic markers (Tnc, Col1a2, Col3a1, Timp-1, α-SMA) and inflammatory markers (F4/80 + , CD3 + , Il-1β, Ccl3) in colon tissue, as well as an improvement in epithelial barrier integrity and wound healing. BHA displayed promising oral bioavailability, no significant activity against a panel of 68 potential pharmacological targets and was devoid of genotoxicity and cardiotoxicity. Taken together, our results provide evidence that oral administration of BAH can alleviate colon inflammation and colitis-associated fibrosis, identifying the enhancement of colon barrier integrity as a possible mechanism of action, and providing a solid rationale for additional clinical studies.

Hyperuricemia contributes to chronic kidney disease development. However, it has been historically viewed with limited research interest. In this study, we mimicked the development of hyperuricemic nephropathy by using a potassium oxonate-induced hyperuricemia rat model. We found that administering vitamin C at 10 mg/kg/day effectively ameliorated hyperuricemic nephropathy. Compared to the control group, rats with hyperuricemia had significantly increased serum uric acid level, xanthine oxidase activity, and urine microalbumin level, by 5-fold, 1.5-fold, and 4-fold, respectively. At the same time, vitamin C supplementation reverted these values by 20% for serum uric acid level and xanthine oxidase activity and 50% for microalbumin level. Vitamin C also alleviated renal pathology and decreased the expression of pro-inflammatory and pro-fibrotic markers. A further mechanistic study suggested that vitamin C might attenuate hyperuricemic nephropathy in renal tubular epithelial cells induced by monosodium urate (MSU) crystal, at least in part, by directly inhibiting IL-6/JAK2/STAT3 signaling pathway. Meanwhile, in macrophages, vitamin C inhibited the expression of TGF-β, and reduced ROS level induced by MSU by about 35%. In short, our results suggest that vitamin C supplementation delay the progression of hyperuricemic nephropathy., (© 2021 Institute of Food Technologists®.)

As a cell surface tissue plasminogen activator (tPA)-plasminogen receptor, the annexin A2 (A2) complex facilitates plasmin generation on the endothelial cell surface, and is an established regulator of hemostasis. Whereas A2 is overexpressed in hemorrhagic disease such as acute promyelocytic leukemia, its underexpression or impairment may result in thrombosis, as in antiphospholipid syndrome, venous thromboembolism, or atherosclerosis. Within immune response cells, A2 orchestrates membrane repair, vesicle fusion, and cytoskeletal organization, thus playing a critical role in inflammatory response and tissue injury. Dysregulation of A2 is evident in multiple human disorders, and may contribute to the pathogenesis of various inflammatory disorders. The fibrinolytic system, moreover, is central to wound healing through its ability to remodel the provisional matrix and promote angiogenesis. A2 dysfunction may also promote tissue fibrogenesis and end-organ fibrosis.

Diabetic cardiomyopathy (DCM) is a common diabetic complication characterized by diastolic relaxation abnormalities, myocardial fibrosis and chronic heart failure. Although TGF-β/Smad3 signalling has been shown to play a critical role in chronic heart disease, the role and mechanisms of Smad3 in DCM remain unclear. We reported here the potential role of Smad3 in the development of DCM by genetically deleting the Smad3 gene from db/db mice. At the age of 32 weeks, Smad3WT-db/db mice developed moderate to severe DCM as demonstrated by a marked increase in the left ventricular (LV) mass, a significant fall in the LV ejection fraction (EF) and LV fractional shortening (FS), and progressive myocardial fibrosis and inflammation. In contrast, db/db mice lacking Smad3 (Smad3KO-db/db) were protected against the development of DCM with normal cardiac function and undetectable myocardial inflammation and fibrosis. Interestingly, db/db mice with deleting one copy of Smad3 (Smad3 ± db/db) did not show any cardioprotective effects. Mechanistically, we found that deletion of Smad3 from db/db mice largely protected cardiac Smad7 from Smurf2-mediated ubiquitin proteasome degradation, thereby inducing IBα to suppress NF-kB-driven cardiac inflammation. In addition, deletion of Smad3 also altered Smad3-dependent miRNAs by up-regulating cardiac miR-29b while suppressing miR-21 to exhibit the cardioprotective effect on Smad3KO-db/db mice. In conclusion, results from this study reveal that Smad3 is a key mediator in the pathogenesis of DCM. Targeting Smad3 may be a novel therapy for DCM., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Radiation enteropathy is one the most common clinical issue for patients receiving radiotherapy for abdominal/pelvic tumors which severely affect the quality of life of cancer patients due to dysplastic lesions (ischemia, ulcer, or fibrosis) that aggravate the radiation damage. Herein, this study demonstrated the prophylactic role of coenzyme Q10 (CoQ10), a powerful antioxidant, against radiotherapy-induced gastrointestinal injury. Male Sprague Dawley rats were divided into four groups: group 1 was defined as control, and group 2 was the irradiated group. Group 3 and 4 were CoQ10 control and radiation plus CoQ10 groups, respectively. CoQ10 (10 mg/kg) was orally administered for 10 days before 10 Gy whole-body radiation and was continued for 4 days post-irradiation. CoQ10 administration protected rats delivered a lethal dose of ϒ-radiation from changes in crypt-villus structures and promoted regeneration of the intestinal epithelium. CoQ10 attenuated radiation-induced oxidative stress by decreasing lipid peroxidation and increasing the antioxidant enzyme catalase activity and reduced glutathione level. CoQ10 also counteracts inflammatory response mediated after radiation exposure through downregulating intestinal NF-ĸB expression which subsequently decreased the level of inflammatory cytokine IL-6 and the expression of COX-2. Radiation-induced intestinal fibrosis confirmed via Masson's trichrome staining occurred through upregulating transforming growth factor (TGF)-β1 and matrix metalloproteinase (MMP)-9 expression, while CoQ10 administration significantly diminishes these effects which further confirmed the anti-fibrotic property of CoQ10. Therefore, CoQ10 is a promising radioprotector that could prevent intestinal complications and enhance the therapeutic ratio of radiotherapy in patients with pelvic tumors through suppressing the NF-kB/TGF-β1/MMP-9 signaling pathway., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Pathological cardiac remodeling, characterized by excessive deposition of extracellular matrix proteins and cardiac hypertrophy, leads to the development of heart failure. Meprin α (Mep1a), a zinc metalloprotease, previously reported to participate in the regulation of inflammatory response and fibrosis, may also contribute to cardiac remodeling, although whether and how it participates in this process remains unknown. Here, in this work, we investigated the role of Mep1a in pathological cardiac remodeling, as well as the effects of the Mep1a inhibitor actinonin on cardiac remodeling-associated phenotypes. We found that Mep1a deficiency or chemical inhibition both significantly alleviated TAC- and Ang II-induced cardiac remodeling and dysfunction. Mep1a deletion and blocking both attenuated TAC- and Ang II-induced heart enlargement and increases in the thickness of the left ventricle anterior and posterior walls, and reduced expression of pro-hypertrophic markers, including atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and myosin heavy chain beta (β-MHC). In addition, Mep1a deletion and blocking significantly inhibited TAC- and Ang II-induced cardiac fibroblast activation and production of extracellular matrix (ECM). Moreover, in Mep1a -/- mice and treatment with actinonin significantly reduced Ang II-induced infiltration of macrophages and proinflammatory cytokines. Notably, we found that in vitro, Mep1a is expressed in cardiac myocytes and fibroblasts and that Mep1a deletion or chemical inhibition both markedly suppressed Ang II-induced hypertrophy of rat or mouse cardiac myocytes and activation of rat or mouse cardiac fibroblasts. In addition, blocking Mep1a in macrophages reduced Ang II-induced expression of interleukin (IL)-6 and IL-1β, strongly suggesting that Mep1a participates in cardiac remodeling processes through regulation of inflammatory cytokine expression. Mechanism studies revealed that Mep1a mediated ERK1/2 activation in cardiac myocytes, fibroblasts and macrophages and contributed to cardiac remodeling. In light of our findings that blocking Mep1a can ameliorate cardiac remodeling via inhibition of cardiac hypertrophy, fibrosis, and inflammation, Mep1a may therefore serve as a strong potential candidate for therapeutic targeting to prevent cardiac remodeling., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Yes-associated protein (YAP) activation after acute ischemic kidney injury might be related to interstitial fibrosis and impaired renal tubular regeneration. Verteporfin (VP) is a photosensitizer used in photodynamic therapy to treat age-related macular degeneration. In cancer cells, VP inhibits TEA domain family member (TEAD)-YAP interactions without light stimulation. The protective role of VP in unilateral ureteral obstruction (UUO)-induced renal fibrosis and related mechanisms remains unclear. In this study, we investigate the protective effects of VP on UUO-induced renal tubulointerstitial inflammation and fibrosis and its regulation of the transforming growth factor-β1 (TGF-β1)/Smad signaling pathway. We find that VP decreased the UUO-induced increase in tubular injury, inflammation, and extracellular matrix deposition in mice. VP also decreased myofibroblast activation and proliferation in UUO kidneys and NRK-49F cells by modulating Smad2 and Smad3 phosphorylation. Therefore, YAP inhibition might have beneficial effects on UUO-induced tubulointerstitial inflammation and fibrosis by regulating the TGF-β1/Smad signaling pathway.

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm (MPN) that leads to progressive bone marrow (BM) fibrosis. Although the cellular mutations involved in the pathogenesis of PMF have been extensively investigated, the sequential events that drive stromal activation and fibrosis by hematopoietic-stromal cross-talk remain elusive. Using an unbiased approach and validation in patients with MPN, we determined that the differential spatial expression of the chemokine CXCL4/platelet factor-4 marks the progression of fibrosis. We show that the absence of hematopoietic CXCL4 ameliorates the MPN phenotype, reduces stromal cell activation and BM fibrosis, and decreases the activation of profibrotic pathways in megakaryocytes, inflammation in fibrosis-driving cells, and JAK/STAT activation in both megakaryocytes and stromal cells in 3 murine PMF models. Our data indicate that higher CXCL4 expression in MPN has profibrotic effects and is a mediator of the characteristic inflammation. Therefore, targeting CXCL4 might be a promising strategy to reduce inflammation in PMF., (© 2020 by The American Society of Hematology.)

Aims: Electronic cigarette (ECIG) has been used as an alternative to tobacco smoking as it lacks the majority of toxicants found in tobacco smoke. However, the effect of ECIG aerosol inhalation on cardiac health are not well studied. The present study aimed to compare the effects of ECIGs with that of combustible tobacco cigarette (T-Cigs) and waterpipe (WP) smoke on cardiac biomarkers of oxidative stress, inflammation, and fibrosis., Main Methods: Rats were randomized into control (fresh air, n = 12), ECIG aerosol (n = 12), T-Cig smoke (n = 15), or WP (n = 13) smoke conditions in which they were exposed 1 h/daily, 6 day/week for 4 weeks. Cardiac biomarkers of oxidative stress, inflammation, and remodeling were assessed., Key Findings: Relative to control, significant increase in heart to body weight ratio was observed in all exposed groups. Cardiac endothelin-1 and myeloperoxidase were increased for ECIG and T-Cig. Cardiac nitrite and TBARS were increased in all exposed groups, but activity of superoxide dismutase was increased for ECIG and T-Cig only while glutathione levels increased for ECIG only. No changes were observed for cardiac C-reactive protein and catalase activity. Cardiac fibrosis was observed in all exposed groups coupled with an increase in the transforming growth factor beta protein that was significant for ECIG only., Significance: ECIG aerosol may promote cardiac alterations in similar manner to tobacco smoke by promoting myocardial oxidative stress and inflammation leading to fibrosis. With regard to cardiac health, exposure to ECIG aerosol and combustible T-Cig smoke may lead to similar adverse outcomes., Competing Interests: Declaration of competing interest Dr. Eissenberg is a paid consultant in litigation against the tobacco industry and also the electronic cigarette industry and he and Dr. Shihadeh are named on a patent for a device that measures the puffing behavior of electronic cigarette users. The other authors declare that there is no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Purpose: To investigate the effect of Gypenosides (Gyps) on the inflammation and fibrosis in orbital fibroblasts (OFs) in Graves ophthalmopathy (GO)., Methods: Bioinformatics analyses were performed to identify the enriched genes and signaling pathways related to Gyps function. For ex vivo experiments, OFs were cultured from orbital connective tissues from patients with GO. OF proliferation was estimated by Cell Counting Kit-8 assay. Effects of Gyps treatment on interleukin (IL)-1β-induced inflammation and transforming growth factor-β1 (TGF-β1)-induced fibrosis were evaluated by real-time quantitative PCR (RT-qPCR), enzyme-linked immunosorbent assay (ELISA), and Western blotting. OFs were treated with IL-1β or TGF-β1 in the absence or presence of Gyps pretreatment, and the levels of related mRNA or proteins were evaluated by RT-qPCR or ELISA., Results: Eight inflammation-related target genes and nine fibrosis-related target genes were screened out. These genes were mainly enriched in pathways corresponding to inflammation and fibrosis, respectively. IL-1β-induced upregulation of inflammatory cytokines, and TGF-β-induced upregulation of fibrotic mediators in OFs were downregulated by Gyps. Moreover, Gyps reduced the activation of Toll like receptors 4/nuclear factor-κ B signaling and TGF-β1/SMAD2/SMAD4 signaling in GO OFs., Conclusions: Gyps could protect GO-derived OFs against IL-1β-induced inflammation and TGF-β1-induced fibrosis. Thus Gyps might have therapeutic potential on inflammation and fibrosis in GO.

Chronic kidney disease (CKD) progresses to end-stage renal failure via renal tubulointerstitial fibrosis. Malnutrition, inflammation, and arteriosclerosis interact to exacerbate the poor prognosis of CKD, and their effective management is thus essential. The traditional Japanese medicine Rikkunshito (RKT) exerts appetite-stimulating effects via ghrelin, which attenuates inflammation and fibrosis. We evaluated the therapeutic effect of RKT in unilateral ureter obstruction (UUO)-induced renal fibrosis/inflammation and body weight loss in mice. UUO and sham-operated mice were fed a standard diet or diet containing 3.0% RKT. Renal fibrosis was investigated by histopathology and macrophage infiltration was determined by immunohistochemistry. Expression levels of genes associated with fibrosis, inflammation, ghrelin, and mitochondrial function were determined by quantitative reverse transcription-polymerase chain reaction and western blot analyses. RKT treatment partially prevented UUO-induced weight loss but failed to attenuate renal fibrosis and inflammation. Renal expression of sirtuin 1, a ghrelin-downstream signalling molecule, and gene expression of peroxisome proliferator-activated receptor-γ coactivator 1α and Bcl-2/adenovirus E1B interacting protein 3 were unaffected by RKT. These results indicate that RKT inhibits weight loss but does not improve renal fibrosis or inflammation in a rapidly progressive renal fibrosis mouse model. RKT may have a protective effect on weight loss associated with CKD.

Background: Glomerulosclerosis and tubulointerstitial fibrosis are hallmarks of chronic kidney injury leading to end-stage renal disease. Inflammatory mechanisms contribute to glomerular and interstitial scarring, including chemokine-mediated recruitment of leucocytes. In particular, accumulation of C-C chemokine receptor type 2 (CCR2)-expressing macrophages promotes renal injury and fibrotic remodelling in diseases like glomerulonephritis and diabetic nephropathy. The functional role of CCR2 in the initiation and progression of primary glomerulosclerosis induced by podocyte injury remains to be characterized., Methods: We analysed glomerular expression of CCR2 and its chemokine ligand C-C motif chemokine ligand 2 (CCL2) in human focal segmental glomerulosclerosis (FSGS). Additionally, CCL2 expression was determined in stimulated murine glomeruli and glomerular cells in vitro. To explore pro-inflammatory and profibrotic functions of CCR2 we induced adriamycin nephropathy, a murine model of FSGS, in BALB/c wild-type and Ccr2-deficient mice., Results: Glomerular expression of CCR2 and CCL2 significantly increased in human FSGS. In adriamycin-induced FSGS, progressive glomerular scarring and reduced glomerular nephrin expression was paralleled by induced glomerular expression of CCL2. Adriamycin exposure stimulated secretion of CCL2 and tumour necrosis factor-α (TNF) in isolated glomeruli and mesangial cells and CCL2 in parietal epithelial cells. In addition, TNF induced CCL2 expression in all glomerular cell populations, most prominently in podocytes. In vivo, Ccr2-deficient mice with adriamycin nephropathy showed reduced injury, macrophage and fibrocyte infiltration and inflammation in glomeruli and the tubulointerstitium. Importantly, glomerulosclerosis and tubulointerstitial fibrosis were significantly ameliorated., Conclusions: Our data indicate that CCR2 is an important mediator of glomerular injury and progression of FSGS. CCR2- targeting therapies may represent a novel approach for its treatment., (© The Author(s) 2018. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.)

Fibrosis is a significant complication of intestinal disorders associated with microbial dysbiosis and pathobiont expansion, notably Crohn's disease (CD). Mechanisms that favor fibrosis are not well understood, and therapeutic strategies are limited. Here we demonstrate that colitis-susceptible Il10 -deficient mice develop inflammation-associated fibrosis when monoassociated with adherent/invasive Escherichia coli (AIEC) that harbors the yersiniabactin (Ybt) pathogenicity island. Inactivation of Ybt siderophore production in AIEC nearly abrogated fibrosis development in inflamed mice. In contrast, inactivation of Ybt import through its cognate receptor FyuA enhanced fibrosis severity. This corresponded with increased colonic expression of profibrogenic genes prior to the development of histological disease, therefore suggesting causality. fyuA -deficient AIEC also exhibited greater localization within subepithelial tissues and fibrotic lesions that was dependent on Ybt biosynthesis and corresponded with increased fibroblast activation in vitro Together, these findings suggest that Ybt establishes a profibrotic environment in the host in the absence of binding to its cognate receptor and indicate a direct link between intestinal AIEC and the induction of inflammation-associated fibrosis., (Copyright © 2019 Ellermann et al.)

Background: After bilateral kidney ischemia/reperfusion injury (IRI), monocytes infiltrate the kidney and differentiate into proinflammatory macrophages in response to the initial kidney damage, and then transition to a form that promotes kidney repair. In the setting of unilateral IRI (U-IRI), however, we have previously shown that macrophages persist beyond the time of repair and may promote fibrosis., Methods: Macrophage homing/survival signals were determined at 14 days after injury in mice subjected to U-IRI and in vitro using coculture of macrophages and tubular cells. Mice genetically engineered to lack Ccr2 and wild-type mice were treated ±CCR2 antagonist RS102895 and subjected to U-IRI to quantify macrophage accumulation, kidney fibrosis, and inflammation 14 and 30 days after the injury., Results: Failure to resolve tubular injury after U-IRI results in sustained expression of granulocyte-macrophage colony-stimulating factor by renal tubular cells, which directly stimulates expression of monocyte chemoattractant protein-1 ( Mcp-1 ) by macrophages. Analysis of CD45 + immune cells isolated from wild-type kidneys 14 days after U-IRI reveals high-level expression of the MCP-1 receptor Ccr2 . In mice lacking Ccr2 and wild-type mice treated with RS102895, the numbers of macrophages, dendritic cells, and T cell decreased following U-IRI, as did the expression of profibrotic growth factors and proimflammatory cytokines. This results in a reduction in extracellular matrix and kidney injury markers., Conclusions: GM-CSF-induced MCP-1/CCR2 signaling plays an important role in the cross-talk between injured tubular cells and infiltrating immune cells and myofibroblasts, and promotes sustained inflammation and tubular injury with progressive interstitial fibrosis in the late stages of U-IRI., (Copyright © 2019 by the American Society of Nephrology.)

Background: Renal fibrosis is a key pathological feature and final common pathway leading to end-stage kidney failure in many chronic kidney diseases. Myofibroblast is the master player in renal fibrosis. However, myofibroblasts are heterogeneous. Recent studies show that bone marrow-derived macrophages transform into myofibroblasts by transforming growth factor (TGF)-β-induced macrophage-myofibroblast transition (MMT) in renal fibrosis., Methods: TGF-β signaling was redirected by inhibition of β-catenin/T-cell factor (TCF) to increase β-catenin/Foxo in bone marrow-derived macrophages. A kidney fibrosis model of unilateral ureteral obstruction was performed in EGFP bone marrow chimera mouse. MMT was examined by flow cytometry analysis of GFP+F4/80+α-SMA+ cells from unilateral ureteral obstruction (UUO) kidney, and by immunofluorescent staining of bone marrow-derived macrophages in vitro. Inflammatory and anti-inflammatory cytokines were analysis by enzyme-linked immunosorbent assay., Results: Inhibition of β-catenin/TCF by ICG-001 combined with TGF-β1 treatment increased β-catenin/Foxo1, reduced the MMT and inflammatory cytokine production by bone marrow-derived macrophages, and thereby, reduced kidney fibrosis in the UUO model., Conclusions: Our results demonstrate that diversion of β-catenin from TCF to Foxo1-mediated transcription not only inhibits the β-catenin/TCF-mediated fibrotic effect of TGF-β, but also enhances its anti-inflammatory action, allowing therapeutic use of TGF-β to reduce both inflammation and fibrosis at least partially by changing the fate of bone marrow-derived macrophages., (© The Author(s) 2018. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.)

Background/aims: Renal dysfunction or renal failure is a common complication in left ventricular assist device (LVAD) recipients and is associated with reduced survival. To date, serum creatinine and glomerular filtration rate (GFR) are used for the evaluation of kidney function. However, serum creatinine and GFR have limitations. The objective of our study is to assess the levels of kidney biomarkers in LVAD recipients compared to heart failure patients and healthy controls and to examine their association with conventional clinical biomarkers., Methods: The biomarkers neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), plasminogen activator inhibitor-1 (PAI-1), and adiponectin were assessed in 51 participants: 19 heart failure patients, 16 LVAD recipients, and 16 healthy controls. Linear regressions were performed to assess whether demographic and clinical variables predict the levels of biomarkers that are associated with acute kidney injury and the risk of chronic kidney disease., Results: The levels of NGAL and adiponectin were higher in LVAD recipients and patients with heart failure as compared with healthy controls. The levels of PAI-1 and KIM-1 were not elevated in LVAD recipients. The results of linear regression analysis indicated that when controlling for the effect of CRP and BNP, 40.1% of the variance in NGAL levels can be explained by GFR (R2 = 0.401, F = 5.56, p = 0.005), while CRP can explain 35.3% of the variance in adiponectin levels (R2 = 0.353, F = 4.55, p = 0.01), when controlling for the effect of BNP and GFR., Conclusions: The levels of NGAL and adiponectin were augmented in LVAD recipients, suggesting that renal functions were not restored with circulatory support. Larger studies should assess the predictability of these biomarkers of renal dysfunction in LVAD recipients., (© 2019 S. Karger AG, Basel.)

Aim: To present treatment strategy for large volumes of injectable non-absorbable 'shell-less' soft tissue fillers (vaseline, synthol, silicone etc.)., Material and Methods: The authors present an experience of surgical treatment of 8 patients who underwent injections of medical vaseline (breast augmentation, n=5) and synthol (muscles enlargement, n=3) and review of the current literature devoted to this problem., Results: Injection of large amounts (over 50 ml) of non-absorbable fillers into soft tissues is unacceptable and leads to numerous complications. Oil-based 'shell-less' fillers cannot be removed by minimally invasive techniques (puncture, mini-incisions, etc.) due to multiple diffuse lesions in the form of oleogranulomas (cysts of different size) and surrounding widespread inflammation and fibrosis of tissues. Surgery is the only adequate method. However, this approach is followed by scars and often tissue contour deformation. Migration of these fillers to other anatomical areas (from the neck to the lower extremities) significantly complicates the situation, treatment and results. In case of categorical refusal of patients from surgical treatment and no complaints, they should be properly informed about possible consequences and complications and dynamic medical supervision is necessary. Intraoperative ultrasound examination is useful for the control of radical removal of pathological areas. Timely removal of non-absorbable fillers allows to avoid serious complications and to achieve good aesthetic results.

Despite advances in therapeutic strategies, heart failure-associated mortality rates remain high. Thus, understanding the pathophysiological molecular mechanisms involved in the remodeling process is essential for developing new and effective therapies. LRRs are present various prokaryotic and eukaryotic proteins and important for the innate immune system via regulating protein-protein interactions. LRRC25 is a member of leucine-rich repeat (LRR)-containing protein family. LRRC25 has been shown to negatively modulate nuclear factor κB (NF-κB) activation, a crucial factor related to cardiac hypertrophy. Our aim was to explore the effects of LRRC25 on cardiac hypertrophy. In the present study, LRRC25 levels were decreased in human and mouse hypertrophied hearts. LRRC25 knockout exacerbated cardiac hypertrophy responding to pressure overloading or angiotensin II (Ang II) stimulation. Deletion of LRRC25 accelerated cardiac dysfunction and fibrosis in mice subjected to aortic banding (AB). LRRC25 ablation induced a strong increase in the transcription of both hypertrophy (ANP, BNP, and β-MHC) and fibrosis associated molecules (col1, col3a1, α-SMA and fibronectin). In addition, the expression of transforming growth factor-β1 (TGF-β1), and its down-streaming signals of phosphorylated Smad2/3, was markedly induced by LRRC25 deficiency. LRRC25-knockout mice showed a significantly enhanced inflammation in response to AB surgery by promoting the activation of NF-κB signaling pathway. In mouse cardiomyocytes, LRRC25 deficiency markedly elevated TGF-β1 and NF-κB activation stimulated by Ang II. Treatment with a combination of TGF-β1 or NF-κB inhibitor abolished the effects of LRRC25-knockout on the promotion of cardiac hypertrophy in vitro. Together, our study identified LRRC25 as a critical molecular switch whose down-regulation resulted in cardiac hypertrophy in a TGF-β1- and NF-κB-dependent manner., (Copyright © 2018. Published by Elsevier Inc.)

Aims: Tobacco smoking is considered a global health issue, contributing to increased risk of cardiovascular disease (CVD) and diabetes (DM). We aimed to assess effects of cigarette smoking on cardiac inflammation, oxidative stress and fibrosis in rat model of streptozotocin (STZ)-induced diabetes., Main Methods: Adults Wistar rats were assigned into control (fresh air, intraperitoneal injection (i.p) of citrate buffer), cigarette smoking (1 h daily for 4 weeks, i.p citrate buffer), DM (35 STZ mg/kg single i.p, fresh air), and DM + Smoking groups for 4 weeks. Cardiac biomarkers of oxidative stress, inflammation, and fibrosis were evaluated., Key Findings: STZ-induced diabetes as documented by the persistent increase in blood glucose. Relative to control, a significant decrease in body weight was observed in diabetic groups paralleled with increased heart to body weight ratio and systolic blood pressure in all groups. Levels of total nitrite, thiobarbituric acid substances, endothelin -1, interleukin-6 and myeloperoxidase were increased in the DM, Smoking and DM + Smoking groups without changes in C-reactive protein. Cardiac levels of GSH were increased in Smoking groups whereas activities of catalase and superoxide dismutase increased in DM, Smoking and DM + Smoking groups. DM but not smoking increased cardiac fibrosis with a parallel increase in transforming growth factor beta. Cardiac levels of matrix metalloproteinase-2 were elevated in Smoking groups and decreased in DM., Significance: Exposure to cigarette smoke may increase risk of CVD in DM by increased cardiac oxidative stress and inflammation. Smoking was associated with increased oxidant enzymes and metalloproteinase-2 probably to prevent cardiac fibrosis., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Chronic graft-versus-host disease (cGVHD) is a marked complication of hematopoietic stem cell transplantation, and multiple organs can be affected by cGVHD-induced inflammation and fibrosis. In clinical settings, immunosuppressive agents have been the last resort to treat cGVHD. However, it has been only partially effective for cGVHD. Hence, efficacious treatment of cGVHD is eagerly awaited. Our previous work suggested that oxidative stress was elevated in cGVHD-disordered lacrimal glands and that epithelial-to-mesenchymal transition (EMT) was implicated in fibrosis caused by ocular cGVHD. In addition, our recent article demonstrated that thioredoxin interaction protein (TXNIP) and transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-𝛋B) were associated with the development of cGVHD. After our search for effective drugs, we chose tranilast to combat systemic cGVHD. Tranilast is known to (1) act as an inhibitor of the inflammatory molecules TXNIP and NF-κB and (2) exert anti-fibrotic, anti-EMT and anti-oxidative effects. To investigate the effectiveness of tranilast for cGVHD, we used an MHC-compatible, multiple minor histocompatibility antigen-mismatched murine model of cGVHD. Tranilast or a solvent-vehicle were orally given to the allogeneic bone marrow transplantation (allo-BMT) recipients from the day before allo-BMT (Day-1) to Day 27 after allo-BMT. Their cGVHD-vulnerable organs were collected Day 28 after allo-BMT and analyzed by using various methods such as histology, immunohistochemistry and immunoblotting. As indicated by our results, tranilast alleviated cGVHD-elicited inflammation and fibrosis by suppressing the expression and/or activation of TXNIP and NF-κB and preventing EMT. Taken together, although this strategy may not be a complete cure for cGVHD, tranilast could be a promising medication to ameliorate cGVHD-triggered disabling symptoms., Competing Interests: Y. O. has a patent in Japan (Patent No. 4966019; Name; Topical application and oral intake of tranilast for the treatment of chronic GVHD-related dye eye disease.) Any other relevant declarations relating to employment, consultancy, patents, products in development or modified products. The other authors have no conflict of interest. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Vitamin D deficiency is especially prevalent in pregnant women and children. Our recent study demonstrated that vitamin D deficiency in early life disturbed testicular development. This study investigated the effects of vitamin D deficiency in early life on prostatic hyperplasia in middle-aged mice. In control group, dams and their male pups were fed with standard-chow diets. In VDD group, dams were fed with vitamin D deficient (VDD) diets throughout pregnancy and lactation. After weaning, male pups continued to be fed with VDD diets. As expected, prostate weight was elevated and prostatic hyperplasia was observed in VDD-fed mice. The number of prostatic Ki-67-positive epithelial cells, a proliferation marker, was increased in VDD-fed mice. Further analysis found that vitamin D deficiency promoted inflammatory infiltration and stromal fibrosis in prostate of middle-aged mice. Moreover, vitamin D deficiency activated NF-κB and up-regulated Il-6 mRNA in prostate of middle-aged mice. In addition, vitamin D deficiency activated prostatic STAT3, a proliferation pathway in middle-aged mice. Of interest, VDD-induced prostatic inflammation and hyperplasia were partially reversed when VDD diets was replaced with standard diets. These results provide evidence that vitamin D deficiency in early life promotes prostatic hyperplasia in middle-aged mice through exacerbating local inflammation., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Trauma or burn injuries that affect the deep dermis often produce a hypertrophic scar, which limits patients' joint movement and generates an aesthetic problem. Inflammation is believed to be one of the main pathogenic mechanisms. We found that IL-17 was increased in scar tissues from patients with hypertrophic scar compared with normal skin. Recombinant mouse IL-17 was subcutaneously injected into mice that underwent full-thickness excision surgery to investigate the role of IL-17 in scar formation. Mice stimulated with IL-17 showed aggravated fibrogenesis, delayed wound healing, and increased inflammation. In addition, macrophage infiltration was also increased. According to the results of the Transwell assay, IL-17 promoted macrophage infiltration through an indirect mechanism. After depleting macrophages with clodronate liposomes, the effect of IL-17 disappeared. Levels of monocyte chemotactic protein (MCP) 1, MCP2, and MCP3 (together referred to as MCPs) were increased by IL-17 stimulation. Bindarit (an inhibitor of MCPs) was used to verify the role of MCPs. In addition, the Ly6C-low macrophages were responsible for wound fibrogenesis in mice. In this study, we detected the increased levels of IL-17 for the first time and revealed that IL-17 induced the infiltration of a specific subtype of macrophages to aggravate fibrosis through an MCP-dependent mechanism. Thus, our results provide a better understanding of scar formation and new strategies for scar prevention., (Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Vitamin D (VD) and its analogues play critical roles in metabolic and cardiovascular diseases. Recent studies have demonstrated that VD exerts a protective role in cardiovascular diseases. However, the beneficial effect of VD on pressure overload-induced cardiac remodeling and dysfunction and its underlying mechanisms are not fully elucidated. In this study, cardiac dysfunction and hypertrophic remodeling in mice were induced by pressure overload. Cardiac function was evaluated by echocardiography, and myocardial histology was detected by H&E and Masson's trichrome staining. Cardiomyocyte size was detected by wheat germ agglutinin staining. The protein levels of signaling mediators were examined by western blotting while mRNA expression of hypertrophic and fibrotic markers was examined by qPCR analysis. Oxidative stress was detected by dihydroethidine staining. Our results showed that administration of VD3 significantly ameliorates pressure overload-induced contractile dysfunction, cardiac hypertrophy, fibrosis and inflammation in mice. In addition, VD3 treatment also markedly inhibited cardiac oxidative stress and apoptosis. Moreover, protein levels of calcineurin A, ERK1/2, AKT, TGF-β, GRP78, cATF6, and CHOP were significantly reduced whereas SERCA2 level was upregulated in the VD3-treated hearts compared with control. These results suggest that VD3 attenuates cardiac remodeling and dysfunction induced by pressure overload, and this protective effect is associated with inhibition of multiple signaling pathways., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Alport syndrome (AS) is a hereditary, progressive nephritis caused by mutation of type IV collagen. Previous studies have shown that activation of signal transducer and activator of transcription 3 (STAT3) exacerbates other renal diseases, but whether STAT3 activation exacerbates AS pathology is still unknown. Here we aim to investigate the involvement of STAT3 in the progression of AS., Method: Phosphorylated STAT3 expression was assessed by immunoblotting analysis of kidneys and glomeruli of an AS mouse model (Col4a5 G5X mutant). To determine the effect of blocking STAT3 signaling, we treated AS mice with the STAT3 inhibitor stattic (10 mg/kg i.p., three times per week for 10 weeks; n = 10). We assessed the renal function [proteinuria, blood urea nitrogen (BUN), serum creatinine] and analyzed the glomerular injury score, fibrosis and inflammatory cell invasion by histological staining. Moreover, we analyzed the gene expression of nephritis-associated molecules., Results: Phosphorylated STAT3 was upregulated in AS kidneys and glomeruli. Treatment with stattic ameliorated the progressive renal dysfunction, such as increased levels of proteinuria, BUN and serum creatinine. Stattic also significantly suppressed the gene expression levels of renal injury markers (Lcn2, Kim-1), pro-inflammatory cytokines (Il-6, KC), pro-fibrotic genes (Tgf-β, Col1a1, α-Sma) and Mmp9. Stattic treatment decreased the renal fibrosis congruently with the decrease of transforming growth factor beta (TGF-β) protein and increase of antifibrosis-associated markers p-Smad1, 5 and 8, which are negative regulators of TGF-β signaling., Conclusion: STAT3 inhibition significantly ameliorated the renal dysfunction in AS mice. Our finding identifies STAT3 as an important regulator in AS progression and provides a promising therapeutic target for AS., (© The Author 2017. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.)

Hypoxic acute kidney injury (AKI) is often incompletely repaired and leads to chronic kidney disease (CKD), which is characterized by tubulointerstitial inflammation and fibrosis. The Slit2 family of secreted glycoproteins is expressed in the kidney, it has been shown to exert an anti-inflammatory activity and prevent ischemic renal injury in vivo. However, whether Slit2 reduces renal fibrosis and inflammation after hypoxic and inflammatory epithelial cells injury in vitro remains unknown. In this study, we aimed to evaluate whether Slit2 ameliorated fibrosis and inflammation in two renal epithelial cells line challenged with hypoxia and lipopolysaccharide (LPS). Renal epithelial cells were treated with hypoxia and LPS to induce cell injury. Hoechst staining and Western blot analysis was conducted to examine epithelial cells injury. Immunofluorescence staining and Western blot analysis was performed to evaluate tubulointerstitial fibrosis. Real-time polymerase chain reaction (PCR) tested the inflammatory factor interleukin (IL)-1β and tumor necrosis factor (TNF)-α, and Western blot analysis determined the hypoxia-inducible factor (HIF)-1α, Toll-like receptor 4 (TLR4) and nuclear factor (NF)-κB. Results revealed that hypoxia induced epithelial cells apoptosis, inflammatory factor IL-1β and TNF-α release and tubulointerstitial fibrosis. LPS could exacerbate hypoxia -induced epithelial cells apoptosis, IL-1β and TNF-α release and fibrosis. Slit2 reduced the expression of fibronectin, the rate of epithelial cell apoptosis, and the expression of inflammatory factor. Slit2 could also inhibit the expression of TLR4 and NF-κB, but not the expression of HIF-1α. Therefore, Slit2 attenuated inflammation and fibrosis after LPS- and hypoxia-induced epithelial cells injury via the TLR4/NF-κB signaling pathway, but not depending on the HIF-1α signaling pathway., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Renal interstitial fibrosis is characterized by excessive accumulation of extracellular matrix, which leads to end-stage renal failure., Purpose: The aim of this study was to explore the effect of Elsholtzia ciliata (Thunb.) Hylander ethanol extract (ECE) on renal interstitial fibrosis induced by unilateral ureteral obstruction (UUO)., Study Design: After quantitative analysis of ECE using the high performance liquid chromatography-photodiode array (HPLC-PDA) method, an in vitro study was performed to assess the anti-inflammatory and anti-fibrotic effects of ECE, using lipopolysaccharide (LPS) and transforming growth factor-ß (TGF-ß), respectively., Methods: For in vivo study, all male Sprague Dawley (SD) rats (n=10/group), except for those in the control group, underwent UUO. The rats were orally treated with water (control), captopril (positive control, 200 mg/kg), and ECE (300 and 500 mg/kg) for 14 days., Results: In ECE, luteolin and rosmarinic acid were relatively abundant among the other flavonoids and phenolic acids. ECE treatment ameliorated LPS-induced overexpression of nuclear factor-κB, tumor necrosis factor (TNF-α), and interleukin-6 and improved oxidative stress in RAW 264.7 cells. Furthermore, ECE treatment suppressed TGF-ß-induced α-smooth muscle actin and matrix metalloproteinase 9 expression in human renal mesangial cells. In the UUO model, 14 consecutive days of ECE treatment improved UUO-induced renal damage and attenuated histopathological alterations and interstitial fibrosis. Moreover, the renal expression of TNF-α, TGF-ß, and Smad 3 were inhibited by ECE treatment., Conclusion: Taken together, the effects of ECE may be mediated by blocking the activation of TGF-ß and inflammatory cytokines, leading subsequently to degradation of the ECM accumulation pathway. Based on these findings, ECE might serve as an improved treatment strategy for renal fibrotic disease., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Context: A substantial number of obese individuals are relatively insulin sensitive and the etiology for this variation remains unknown., Objective: The primary objective was to detect factors in adipose tissue differentiating obese insulin-sensitive (OBIS) from obese insulin-resistant (OBIR) individuals and investigate whether adipose tissue hypoxia is a contributing factor in the pathogenesis of insulin resistance., Design and Setting: This was a cross-sectional study in the general community., Participants: Subjects consisted of nondiabetic OBIS and OBIR subjects with similar body mass index, age, and total body fat but different insulin sensitivity index as well as lean insulin-sensitive subjects., Interventions(s): There were no interventions., Main Outcome Measure(s): We examined adipocytokines and the expression of candidate genes regulating hypoxia, inflammation, and lipogenesis in adipose tissue and adipose tissue oxygenation., Results: OBIS subjects had increased plasma adiponectin but similar plasma TNFα and leptin levels as compared with OBIR subjects. Genes regulating inflammation (CD68, MCP1, scavenger receptor A, and oxidized LDL receptor 1) were increased by 40%–60% (P < .05) in OBIR vs OBIS cohorts. In addition, genes involved in extracellular matrix formation such as collagen VI and MMP7 were up-regulated by 43% and 78% (P < .05), respectively, in OBIR vs OBIS. The expression of HIF1α and VEGF gene expression was increased by 37% and 52%, respectively, in OBIR vs OBIS (P < .01). Despite the differential expression in hypoxia-related genes, adipose tissue oxygenation measured by a Licox oxygen probe was not different between OBIS and OBIR subjects, but it was higher in lean subjects as compared with obese subjects., Conclusions: We confirmed that adipose tissue inflammation and fibrosis play an important role in the pathogenesis of insulin resistance independent of obesity in humans. Whether hypoxia is simply a consequence of adipose tissue expansion or is related to the pathogenesis of obesity-induced insulin resistance is yet to be understood.

Background: Intestinal fibrosis is mainly associated with Crohn's disease and is defined as a progressive and excessive deposition of extracellular matrix components. No specific antifibrotic therapies are available. In this study, we evaluate the antifibrotic effect of a novel 5-ASA analog able to activate the peroxisome proliferator-activated receptor γ, named GED-0507-34 Levo., Methods: Colonic fibrosis was induced in 110 C57BL/6 mice by 3 cycles of 2.5% (wt/vol) dextran sulfate sodium administration for 6 weeks. The preventive effects of oral daily GED (30 mg · kg(-1) · d(-1)) administration were evaluated using a macroscopic and histological score and also through biological endpoints. Expression of main markers of myofibroblasts activation was determined in transforming growth factor (TGF-β)-stimulated intestinal fibroblasts and epithelial cells., Results: GED improved macroscopic and microscopic intestinal lesions in dextran sulfate sodium-treated animals and reduced the profibrotic gene expression of Acta2, COL1a1, and Fn1 by 1.48-folds (P < 0.05), 1.93-folds (P < 0.005), and 1.03-fold (P < 0.05), respectively. It reduced protein levels of main markers of fibrosis (α-SMA and Collagen I-II) and the main TGF-β/Smad pathway components. GED also decreased the interleukin-13 and connective tissue growth factor expression by 1.89-folds (P < 0.05) and 2.2-folds (P < 0.005), respectively. GED inhibited TGF-β-induced activation of both fibroblast and intestinal epithelial cell lines, by regulating mRNA expression of α-SMA and fibronectin, and restoring the TGF-β-induced loss of intestinal epithelial cell markers. GED treatment also reduced the TGF-β and ACTA1 expression in primary human intestinal fibroblasts from ulcerative colitis patients., Conclusions: GED ameliorates intestinal fibrosis in dextran sulfate sodium-induced chronic colitis in mice and regulates major profibrotic cellular and molecular mechanisms.

The purpose of this study was to compare the biocompatibility of two types of calcium hydroxide paste in subcutaneous tissue in rat. Twenty-two Wistar rats were divided into 4 experimental (n=5 each) and one control (n=2) group. A polyethylene tube filled with either Dentsply or Sure-Paste was implanted in each rat in the experimental groups, while an empty polyethylene tube was used in the control group. After 15 or 60 days, the animals were sacrificed and histopathological examination carried out. Tissue reaction was assessed by inflammatory cell infiltration using a 4-point scoring system, ranging from 0 to 3. Data were analyzed with the Kruskal-Wallis, Wilcoxon, and McNemar tests. Both types of paste induced an inflammatory response at each time point, although the intensity varied. A significant reduction in the number of inflammatory cells was observed at 60 days. Dentsply appeared to induce a more marked inflammatory response at both time points, although the difference was not significant. These results suggest that both types of paste are biocompatible with subcutaneous tissue in rat.

Methionine sulfoxide reductase A (MsrA), which stereospecifically catalyzes the reduction of methionine-S-sulfoxide, is an important reactive oxygen species (ROS) scavenger. Tissue fibrosis is a maladaptive repair process following injury, associated with oxidative stress. In this study, we investigated the role of MsrA in unilateral ureteral obstruction (UUO)-induced kidney fibrosis and its underlying mechanisms by using MsrA gene-deleted mice (MsrA(-/-)). MsrA deletion increased collagen deposition in the interstitium and the expression of collagen III and α-smooth muscle actin in the UUO kidneys, indicating that MsrA deficiency exacerbated the progression of UUO-induced kidney fibrosis. UUO reduced the kidney expression of MsrA, MsrB1, and MsrB2, thereby decreasing MsrA and MsrB activity. UUO increased hydrogen peroxide and lipid peroxidation levels and the ratio of oxidized glutathione (GSSG) to total glutathione (GSH) in the kidneys. The UUO-induced elevations in the levels of these oxidative stress markers and leukocyte markers were much higher in the MsrA(-/-) than in the MsrA(+/+) kidneys, the latter suggesting that the exacerbated kidney fibrosis in MsrA(-/-) mice was associated with enhanced inflammatory responses. Collectively, our data suggest that MsrA plays a protective role in the progression of UUO-induced kidney fibrosis via suppression of fibrotic responses caused by oxidative stress and inflammation., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The authors describe 3 children who presented with progressively enlarging skin-covered solid masses over the shunt catheter in the neck/clavicular region. The authors reviewed the clinical, laboratory, pathological, radiographic, and follow-up data for all 3 patients and reviewed the literature on the subject. The patients had no clinical evidence of an infectious process. Surgical exploration revealed that masses were surrounding and encasing the shunt tubing to which they were strongly attached. Pathological studies of the tissues demonstrated varying degrees of exuberant chronically inflamed granulation tissues, interstitial fibrosis, and dystrophic calcification. One patient had associated thinning of the skin overlying the mass and subsequently developed ulceration. No infectious organisms were observed. The cerebrospinal fluid aspirates from the shunts did not yield any organisms. There has been no recurrence of the masses. The presence of a growing mass over the shunt tube in the neck or the chest region without clinical evidence of infection does not indicate that the mass should be treated with antibiotics and complete shunt removal. Rather, the mass can be cured by extirpation and with "bypass" new shunt tubing locally.

Diabetes affects cardiac structure and function, and it has been suggested that diabetes leads to cardiomyopathy. Arachidonate 12/15-lipoxygenase (LOX) has been suggested to play an important role in atherogenesis and heart failure. However, the role of 12/15-LOX in diabetic cardiomyopathy has not been examined. In this study, we investigated the effects of cardiac 12/15-LOX on diabetic cardiomyopathy. We created streptozotocin (STZ)-induced diabetic mice and compared them with Alox15-deficient mice. Expression of 12/15-LOX and inflammatory cytokines such as tumor necrosis factor (TNF)-α and nuclear factor (NF)-κB were upregulated in STZ-induced diabetic hearts. Disruption of 12/15-LOX significantly improved STZ-induced cardiac dysfunction and fibrosis. Moreover, deletion of 12/15-LOX inhibited the increases of TNF-α and NF-κB as well as the production of STZ-induced reactive oxygen species in the heart. Administration of N-acetylcysteine in diabetic mice prevented STZ-induced cardiac fibrosis. Neonatal cultured cardiomyocytes exposed to high glucose conditions induced the expression of 12/15-LOX as well as TNF-α, NF-κB, and collagen markers. These increases were inhibited by treatment of the 12/15-LOX inhibitor. Our results suggest that cardiac 12/15-LOX-induced inflammation and oxidative stress are involved in the development of diabetic cardiomyopathy and that inhibition of 12/15-LOX could be a novel treatment for this condition., (© 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

Purpose of Review: To provide a perspective by investigating the potential cross-talk between the adipose tissue and the kidney during obesity., Recent Findings: It is well established that excessive caloric intake contributes to organ injury. The associated increased adiposity initiates a cascade of cellular events that leads to progressive obesity-associated diseases such as kidney disease. Recent evidence has indicated that adipose tissue produces bioactive substances that contribute to obesity-related kidney disease, altering the renal function and structure. In parallel, proinflammatory processes within the adipose tissue can also lead to pathophysiological changes in the kidney during the obese state., Summary: Despite considerable efforts to better characterize the pathophysiology of obesity-related metabolic disease, there are still a lack of efficient therapeutic strategies. New strategies focused on regulating adipose function with respect to AMP-activated protein kinase activation, NADPH oxidase function, and TGF-β may contribute to reducing adipose inflammation that may also provide renoprotection.

Background: Radiotherapy for lung cancer may induce pneumonitis. However, histological effects of radiotherapy on normal lung tissue are unknown. Transbronchial biopsy (TBB) is safe and accurate in monitoring parenchymal lesions in lung-transplanted patients. The aim of this prospective study was to evaluate whether histological changes of the healthy lung parenchyma after radiotherapy are present on TBB biopsies., Patients and Methods: Twelve patients with lung cancer necessitating radiation therapy participated in the study. Serial TBBs were obtained from lung parenchyma contra-lateral to the tumor before, just after radiotherapy, and at six months post-irradiation. Evaluation of each specimen was based on the presence of congestion, inflammation, hemorrhage and fibrosis., Results: A significant increase of interstitial fibrosis (thickness) and congestion was observed between the point prior to radiotherapy and after completion of radiotherapy (p=0.047), as well as between the pre-radiotherapy point and at six months after radiotherapy (p=0.014). Six patients (50%) showed intra-alveolar fibroblastic growth after radiotherapy. No patient showed clinical or radiographic findings of radiation pneumonitis., Conclusion: Even in the absence of clinical or radiographic findings, the lung parenchyma contra-lateral to the tumor suffers early histological lesions after radiation therapy, as monitored by serial TBBs., (Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Background: The purpose of this study was to evaluate the associations between patient characteristics or surgical site classifications and the histologic remodeling scores of synthetic meshes biopsied from their abdominal wall repair sites in the first attempt to generate a multivariable risk prediction model of non-constructive remodeling., Methods: Biopsies of the synthetic meshes were obtained from the abdominal wall repair sites of 51 patients during a subsequent abdominal re-exploration. Biopsies were stained with hematoxylin and eosin, and evaluated according to a semi-quantitative scoring system for remodeling characteristics (cell infiltration, cell types, extracellular matrix deposition, inflammation, fibrous encapsulation, and neovascularization) and a mean composite score (CR). Biopsies were also stained with Sirius Red and Fast Green, and analyzed to determine the collagen I:III ratio. Based on univariate analyses between subject clinical characteristics or surgical site classification and the histologic remodeling scores, cohort variables were selected for multivariable regression models using a threshold p value of ≤0.200., Results: The model selection process for the extracellular matrix score yielded two variables: subject age at time of mesh implantation, and mesh classification (c-statistic = 0.842). For CR score, the model selection process yielded two variables: subject age at time of mesh implantation and mesh classification (r (2) = 0.464). The model selection process for the collagen III area yielded a model with two variables: subject body mass index at time of mesh explantation and pack-year history (r (2) = 0.244)., Conclusion: Host characteristics and surgical site assessments may predict degree of remodeling for synthetic meshes used to reinforce abdominal wall repair sites. These preliminary results constitute the first steps in generating a risk prediction model that predicts the patients and clinical circumstances for which non-constructive remodeling of an abdominal wall repair site with synthetic mesh reinforcement is most likely to occur.

Background: Newer biomarkers, reflective of biological processes, such as inflammation and fibrosis, cardiac stretch or damage and vascular health may be useful in understanding clinical events in chronic kidney disease (CKD). We assessed whether these newer biomarkers, alone or as a panel, improve risk prediction for renal replacement therapy or death, over and above conventional clinical, demographic and laboratory variables., Methods: We conducted a prospective observational Canadian cohort study in 2544 CKD patients with estimated glomerular filtration rate (eGFR) of 15-45 mL/min/1.73 m(2), under nephrology care, in urban and rural centers. We measured traditional clinical and laboratory risk factors, as well as newer biomarkers: cystatin C, high sensitivity c-reactive protein (hsCRP), interleukin 6 (IL6), transforming growth factor β1 (TGFβ1), fibroblast growth factor 23 (FGF23), N-terminal probrain natriuretic peptide (NT-proBNP), troponin I and asymmetric dimethylarginine (ADMA). Key outcomes were renal replacement therapy (RRT, dialysis or transplantation) and death, during the first year follow-up after enrollment: a time point important for clinical decision-making for patients and providers., Results: Newer biomarkers do not improve the prediction of RRT, when added to conventional risk factors such as eGFR, urine albumin to creatinine ratio, hemoglobin, phosphate and albumin. However, in predicting death within 1 year, cystatin C, NT-proBNP, hsCRP and FGF23 values significantly improved model discrimination and reclassification: c statistic increased by absolute 4.3% and Net Reclassification Improvement for categories of low, intermediate and high risk at 11.2%., Conclusions: Our findings suggest that the addition of newer biomarkers may be useful in predicting death in patients with established CKD within a 1-year timeframe. This information may be useful in informing prognosis and redirect resources to serve patients at higher risk to improve outcomes and sustainability of the nephrology care system.

Decreasing organ quality is prompting research toward new methods to alleviate ischemia reperfusion injury (IRI). Oxidative stress and nuclear factor kappa beta (NF-κB) activation are well-described elements of IRI. We added cyclodextrin-complexed curcumin (CDC), a potent antioxidant and NF-κB inhibitor, to University of Wisconsin (UW) solution (Belzer's Solution, Viaspan), one of the most effective clinically approved preservative solutions. The effects of CDC were evaluated on pig endothelial cells and in an autologous donation after circulatory death (DCD) kidney transplantation model in large white pigs. CDC allowed rapid and lasting uptake of curcumin into cells. In vitro, CDC decreased mitochondrial loss of function, improved viability and lowered endothelial activation. In vivo, CDC improved function recovery, lowered histological injury and doubled animal survival (83.3% vs. 41.7%). At 3 months, immunohistochemical staining for epithelial-to-mesenchymal transition (EMT) and fibrosis markers was intense in UW grafts while it remained limited in the UW + CDC group. Transcriptional analysis showed that CDC treatment protected against up-regulation of several pathophysiological pathways leading to inflammation, EMT and fibrosis. Thus, use of CDC in a preclinical transplantation model with stringent IRI rescued kidney grafts from an unfavorable prognosis. As curcumin has proved well tolerated and nontoxic, this strategy shows promise for translation to the clinic., (© Copyright 2014 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Effects of mineralocorticoids are not restricted to regulation of epithelial salt transport, extracellular volume and blood pressure; mineralocorticoids also influence a wide variety of seemingly unrelated functions such as inflammation and fibrosis. The present brief review addresses the role of mineralocorticoids in the orchestration of these latter processes. Mineralocorticoids foster inflammation as well as vascular, cardiac, renal and peritoneal fibrosis. Mechanisms involved in mineralocorticoid-sensitive inflammation and fibrosis include the serum- and glucocorticoid-inducible kinase 1 (SGK1), which is genomically upregulated by mineralocorticoids and transforming growth factor β (TGF-β), and stimulated by mineralocorticoid-sensitive phosphatidylinositide 3-kinase. SGK1 upregulates the inflammatory transcription factor nuclear factor-κB, which in turn stimulates the expression of diverse inflammatory mediators including connective tissue growth factor. Moreover, SGK1 inhibits the degradation of the TGF-β-dependent transcription factors Smad2/3. Mineralocorticoids foster the development of TH17 cells, which is compromised following SGK1 deletion. Excessive SGK1 expression is observed in a wide variety of fibrosing diseases including lung fibrosis, diabetic nephropathy, glomerulonephritis, obstructive kidney disease, experimental nephrotic syndrome, obstructive nephropathy, liver cirrhosis, fibrosing pancreatitis, peritoneal fibrosis, Crohn's disease and celiac disease. The untoward inflammatory and fibrosing effects of mineralocorticoids could be blunted or even reversed by mineralocorticoid receptor blockers, which may thus be considered in the treatment of inflammatory and/or fibrosing disease., (© 2014 S. Karger AG, Basel.)

Background: Glucose-sensor-induced tissue reactions (e.g., inflammation and wound healing) are known to negatively impact sensor function in vivo. The roles of cytokine networks in controlling these tissue reactions (i.e., sensor biofouling) is not understood. In the present study, we investigated the role of interleukin-1 receptor antagonist (IL-1Ra), a key anti-inflammatory antagonist of the proinflammatory interleukin-1 cytokines [i.e. interleukin-1 (IL-1) alpha and IL-1 beta] in controlling continuous glucose monitoring (CGM)., Methods: To investigate the role of IL-1Ra in long-term CGM in vivo, we compared CGM in transgenic mice that overexpress IL-1Ra [interleukin-1 receptor antagonist overexpresser (IL-1Ra~OE), B6.Cg-Tg(IL1rn)1Dih/J] or are deficient in IL-1Ra [interleukin-1 receptor antagonist knockout (IL-1Ra~KO), B6.129S-IL1rn(tm1Dih)/J] with mice that have normal levels of IL-1Ra (C57BL/6) over a 28-day time period., Results: Mean absolute relative difference (MARD) analysis of CGM results among the mice of varying IL-1Ra levels demonstrated that during the first 21 days, IL-1~KO mice had the greatest tissue inflammation and the poorest sensor performance (i.e., higher MARD values) when compared with normal or IL-1Ra~OE mice. By 28 days post-sensor implantation, the inflammatory reactions had subsided and were replaced by varying degrees of fibrosis., Conclusions: These data support our hypothesis on the importance of the IL-1 family of agonists and antagonists in controlling tissue reactions and sensor function in vivo. These data also suggest that local delivery of IL-1Ra genes or recombinant proteins (anakinra) or other IL-1 antagonists such as antibodies or soluble IL-1 receptors would suppress sensor-induced tissue reactions and likely enhance glucose sensor function by inhibiting inflammation and wound healing at sensor implantation sites., (© 2013 Diabetes Technology Society.)

Introduction: Chronically relapsing inflammation, tissue remodeling and fibrosis are hallmarks of inflammatory bowel diseases. The aim of this study was to investigate changes in connective tissue in a chronic murine model resulting from repeated cycles of dextran sodium sulphate (DSS) ingestion, to mimic the relapsing nature of the human disease., Materials and Methods: C57BL/6 mice were exposed to DSS in drinking water for 1 week, followed by a recovery phase of 2 weeks. This cycle of exposure was repeated for up to 3 times (9 weeks in total). Colonic inflammation, fibrosis, extracellular matrix proteins and colonic gene expression were studied. In vivo MRI T 2 relaxometry was studied as a potential non-invasive imaging tool to evaluate bowel wall inflammation and fibrosis., Results: Repeated cycles of DSS resulted in a relapsing and remitting disease course, which induced a chronic segmental, transmural colitis after 2 and 3 cycles of DSS with clear induction of fibrosis and remodeling of the muscular layer. Tenascin expression mirrored its expression in Crohn's colitis. Microarray data identified a gene expression profile different in chronic colitis from that in acute colitis. Additional recovery was associated with upregulation of unique genes, in particular keratins, pointing to activation of molecular pathways for healing and repair. In vivo MRI T2 relaxometry of the colon showed a clear shift towards higher T2 values in the acute stage and a gradual regression of T2 values with increasing cycles of DSS., Conclusions: Repeated cycles of DSS exposure induce fibrosis and connective tissue changes with typical features, as occurring in Crohn's disease. Colonic gene expression analysis revealed unique expression profiles in chronic colitis compared to acute colitis and after additional recovery, pointing to potential new targets to intervene with the induction of fibrosis. In vivo T2 relaxometry is a promising non-invasive assessment of inflammation and fibrosis.

The incidence of nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) has increased in parallel with the incidence of obesity. While both NAFLD and NASH are characterized by hepatosteatosis, NASH is characterized by hepatic damage, inflammation, oxidative stress, and fibrosis. We previously reported that feeding Ldlr(-/-) mice a high-fat, high-cholesterol diet containing menhaden oil attenuated several markers of NASH, including hepatosteatosis, inflammation, and fibrosis. Herein, we test the hypothesis that DHA [22:6 (n-3)] is more effective than EPA [20:5 (n-3)] at preventing Western diet (WD)-induced NASH in Ldlr(-/-) mice. Mice were fed the WD supplemented with either olive oil (OO), EPA, DHA, or EPA + DHA for 16 wk. WD + OO feeding induced a severe NASH phenotype, characterized by robust hepatosteatosis, inflammation, oxidative stress, and fibrosis. Whereas none of the C20-22 (n-3) fatty acid treatments prevented WD-induced hepatosteatosis, all 3 (n-3) PUFA-containing diets significantly attenuated WD-induced inflammation, fibrosis, and hepatic damage. The capacity of dietary DHA to suppress hepatic markers of inflammation (Clec4F, F4/80, Trl4, Trl9, CD14, Myd88), fibrosis (Procol1α1, Tgfβ1), and oxidative stress (NADPH oxidase subunits Nox2, p22phox, p40phox, p47phox, p67phox) was significantly greater than dietary EPA. The effects of DHA on these markers paralleled DHA-mediated suppression of hepatic Fads1 mRNA abundance and hepatic arachidonic acid content. Because DHA suppression of NASH markers does not require a reduction in hepatosteatosis, dietary DHA may be useful in combating NASH in obese humans.

We previously reported that kidney transplants with early acute injury express transcripts indicating injury repair--the acute kidney injury signal. This study investigated the significance of this signal in transplants with other conditions, including rejection and recurrent disease. The injury signal was elevated in biopsies in many different conditions, including T cell-mediated rejection and potentially progressive diseases such as antibody-mediated rejection and glomerulonephritis. A high injury signal correlated with poor function and with inflammation in areas of fibrosis, but not with fibrosis without inflammation. In multivariate survival analysis, the injury signal in late kidney transplant biopsies strongly predicted future graft loss, similar to a published molecular risk score derived in late kidneys. Indeed, the injury signal shared many individual transcripts with the risk score, e.g. ITGB6, VCAN, NNMT. The injury signal was a better predictor of future graft loss than fibrosis, inflammation or expression of collagen genes. Thus the acute injury signal, first defined in early reversible injury, is present in many diseases as a reflection of parenchymal distress, where its significance is dictated by the inducing insult, i.e. treatable/self-limited versus untreatable and sustained. Progression in troubled transplants is primarily a function of ongoing parenchymal injury by disease, not fibrogenesis., (© Copyright 2013 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Fibrosis is a serious condition complicating chronic inflammatory processes affecting the intestinal tract. Advances in this field that rely on human studies have been slow and seriously restricted by practical and logistic reasons. As a consequence, well-characterized animal models of intestinal fibrosis have emerged as logical and essential systems to better define and understand the pathophysiology of fibrosis. In point of fact, animal models allow the execution of mechanistic studies as well as the implementation of clinical trials with novel, pathophysiology-based therapeutic approaches. This review provides an overview of the currently available animal models of intestinal fibrosis, taking into consideration the methods of induction, key characteristics of each model, and underlying mechanisms. Currently available models will be classified into seven categories: spontaneous, gene-targeted, chemical-, immune-, bacteria-, and radiation-induced as well as postoperative fibrosis. Each model will be discussed in regard to its potential to create research opportunities to gain insights into the mechanisms of intestinal fibrosis and stricture formation and assist in the development of effective and specific antifibrotic therapies.

The lymphotoxin system (LT) regulates interactions between lymphocytes and stromal cells to maintain lymphoid microenvironmental homeostasis. Soluble LT beta-receptor-Ig (LTβRIg) blocks lymphocyte LTα1β2-stromal cell LTβR signaling. In a murine cardiac allograft model, LTbRIg treatment reversed the tolerance induced by anti-CD40L antibody leading to graft inflammation and fibrosis. LTβRIg treatment decreased PD-L1 expression by blood endothelial cells, and decreased VCAM-1 while increasing CXCL1, CXCL2, CXCL12, CCL5, CCL21 and IL-6 expression in fibroblastic reticular cells. In secondary lymphoid organs these effects caused T- and B cell zone disruption, loss of CD35(+) follicular dendritic cells and abnormal recruitment of CD11b(+) Ly6G(+) neutrophils. These disruptions correlated with increased numbers of CD8(+) T cells and CD11b(+) Ly6G(+) neutrophils, and decreased numbers of CD4(+) T cells and Foxp3(+) regulatory T cells in the grafts. Depleting neutrophils or blocking neutrophil-attracting chemokines restored normal histology in lymph node, spleen and grafts. Taken together, LTβRIg treatment altered stromal subset, particularly fibroblastic reticular cell, production of cytokines and chemokines, resulting in changes in neutrophil recruitment in spleen, lymph node and grafts, and inflammation and fibrosis associated with decreased Foxp3(+) regulatory T cells and increased CD8(+) T cell infiltration of grafts., (© Copyright 2012 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Background: Crohn's disease causes intestinal inflammation leading to intestinal fibrosis. Spironolactone is an antifibrotic medication commonly used in heart failure to reduce mortality. We examined whether spironolactone is antifibrotic in the context of intestinal inflammation., Methods: In vitro, spironolactone repressed fibrogenesis in transforming growth factor beta (TGF-β)-stimulated human colonic myofibroblasts. However, spironolactone therapy significantly increased mortality in two rodent models of inflammation-induced intestinal fibrosis, suggesting spironolactone could be harmful during intestinal inflammation. Since inflammatory bowel disease (IBD) patients rarely receive spironolactone therapy, we examined whether spironolactone use was associated with mortality in a common cause of inflammatory colitis, Clostridium difficile infection (CDI)., Results: Spironolactone use during CDI infection was associated with increased mortality in a retrospective cohort of 4008 inpatients (15.9% vs. 9.1%, n = 390 deaths, P < 0.0001). In patients without liver disease, the adjusted odds ratio (OR) for inpatient mortality associated with 80 mg spironolactone was 1.99 (95% confidence interval [CI]: 1.51-2.63) In contrast to the main effect of spironolactone mortality, multivariate modeling revealed a protective interaction between liver disease and spironolactone dose. The adjusted OR for mortality after CDI was 1.96 (95% CI: 1.50-2.55) for patients without liver disease on spironolactone vs. 1.28 (95% CI: 0.82-2.00) for patients with liver disease on spironolactone when compared to a reference group without liver disease or spironolactone use., Conclusions: We propose that discontinuation of spironolactone in patients without liver disease during CDI could reduce hospital mortality by 2-fold, potentially reducing mortality from CDI by 35,000 patients annually across Europe and the U.S., (Copyright © 2011 Crohn's & Colitis Foundation of America, Inc.)