Profibrotic Markers Research Articles

Increased pro-remodeling and pro-thrombotic responses in the left compared to the right atrial appendage: role of low-grade inflammation and the AT1R/NADPH oxidases/SGLT2 pro-oxidant pathway

Abstract Introduction In atrial fibrillation (AF), there is a complex interplay between arrhythmia burden and cardiovascular risk factors leading to left atrial remodeling, an increased risk of stroke and heart failure. Most thrombi get formed in the atrial appendage. Several studies suggested that sodium-glucose cotransporter 2 inhibitors (SGLT2i), besides showing major benefits on heart failure, might lower the risk of incident AF. Therefore, we examined the expression level of SGLT2 in human right and left atrial appendages (RAA, LAA) and determined its role in pro-oxidant, pro-fibrotic and pro-thrombotic responses. Methods Human RAA and LAA were collected from patients undergoing cardiac surgery at a university hospital. The LAA was cut into a proximal part (low stasis, high shear) and a distal part (high stasis, low shear). The level of reactive oxygen species (ROS) was determined using dihydroethidium, mRNA and protein expression levels by RT-qPCR, and Western blot analysis and immunofluorescence, respectively, and the level of fibrosis by Sirius red staining. Results The distal LAA had higher mRNA levels of ICAM-1 and p53, and protein levels of ICAM-1, AT1R, p53, p21 and tissue factor than the proximal LAA. LAA displayed higher mRNA and protein levels of SGLT2 and SGLT1, pro-adhesive, pro-thrombotic, pro-remodeling, pro-fibrotic and senescence markers, and components of the angiotensin system, in addition to IL-1 mRNA levels and p-p65 NF-kB protein levels compared to RAA. These responses were associated with higher levels of oxidative stress, nitrotyrosine and fibrosis. SGLT2 immunofluorescence signals in distal LAA were colocalized with those of CD31, CD68, TNF-α and troponin T. Oxidative stress levels in distal and proximal LAA and RAA were reduced by inhibitors of NADPH oxidases, ACE1 and SGLT2, an AT1R antagonist, and by a TNF-α neutralizing antibody whereas NG-nitro-L-Arginine methyl ester, the non-selective NO synthase inhibitor, inhibited ROS in distal and proximal LAA without affecting RAA. In addition, the level of TNF-α in LAA was positively correlated to the indexed LA volume indicating a link between inflammation and remodeling. Conclusions The distal part of the LAA of patients undergoing heart surgery showed greater impaired endothelial function and senescence associated with more pronounced pro­-oxidant, pro­-remodeling and pro-­inflammatory responses than the proximal part and the RAA. These responses were associated with an increased expression level of SGLT 2 in endothelial cells, cardiomyocytes, and macrophages in areas showing an inflammatory response. Moreover, the pro-oxidant signal was sensitive to inhibitors of the local angiotensin system, NO synthase, SGLT2 and TNF-α. Thus, the AT1R/NADPH oxidases/SGLT2 pathway appears as an interesting target to blunt the stimulatory pro-oxidant signal in cardiac tissues affected by low-grade inflammation that leads to remodeling and fibrosis.

Antifibrotic effect of apremilast in systemic sclerosis dermal fibroblasts and bleomycin-induced mouse model

Phosphodiesterase (PDE) 4 inhibitors have been reported to suppress the progression of dermal fibrosis in patients with systemic sclerosis (SSc); however, the precise mechanisms remain to be elucidated. Therefore, we conducted experiments focusing on the antifibrotic and anti-inflammatory effects of apremilast using dermal fibroblasts derived from patients with SSc and an SSc mouse model. Dermal fibroblasts derived from healthy controls and patients with SSc were incubated with apremilast in the presence or absence of 10 ng/ml transforming growth factor (TGF)-β1 for the measurement of intracellular cAMP levels and evaluation of mRNA and protein expression. A bleomycin-induced dermal fibrosis mouse model was used to evaluate the inhibitory effects of apremilast on the progression of dermal fibrosis. Intracellular cAMP levels were significantly reduced in dermal fibroblasts derived from patients with SSc compared with those derived from healthy controls. Apremilast reduced the mRNA expression of profibrotic markers and the protein expression of type I collagen and Cellular Communication Network Factor 2 (CCN2) in dermal fibroblasts. Additionally, apremilast inhibited the progression of dermal fibrosis in mice, partly by acting on T cells. These results suggest that apremilast may be a potential candidate for treating dermal fibrosis in SSc.

Hypoxia enhances anti-fibrotic properties of extracellular vesicles derived from hiPSCs via the miR302b-3p/TGFβ/SMAD2 axis

BackgroundCardiac fibrosis is one of the top killers among fibrotic diseases and continues to be a global unaddressed health problem. The lack of effective treatment combined with the considerable socioeconomic burden highlights the urgent need for innovative therapeutic options. Here, we evaluated the anti-fibrotic properties of extracellular vesicles (EVs) derived from human induced pluripotent stem cells (hiPSCs) that were cultured under various oxygen concentrations.MethodsEVs were isolated from three hiPSC lines cultured under normoxia (21% O2; EV-N) or reduced oxygen concentration (hypoxia): 3% O2 (EV-H3) or 5% O2 (EV-H5). The anti-fibrotic activity of EVs was tested in an in vitro model of cardiac fibrosis, followed by a detailed investigation of the underlying molecular mechanisms. Sequencing of EV miRNAs combined with bioinformatics analysis was conducted and a selected miRNA was validated using a miRNA mimic and inhibitor. Finally, EVs were tested in a mouse model of angiotensin II-induced cardiac fibrosis.ResultsWe provide evidence that an oxygen concentration of 5% enhances the anti-fibrotic effects of hiPS-EVs. These EVs were more effective in reducing pro-fibrotic markers in activated human cardiac fibroblasts, when compared to EV-N or EV-H3. We show that EV-H5 act through the canonical TGFβ/SMAD pathway, primarily via miR-302b-3p, which is the most abundant miRNA in EV-H5. Our results show that EV-H5 not only target transcripts of several profibrotic genes, including SMAD2 and TGFBR2, but also reduce the stiffness of activated fibroblasts. In a mouse model of heart fibrosis, EV-H5 outperformed EV-N in suppressing the inflammatory response in the host and by attenuating collagen deposition and reducing pro-fibrotic markers in cardiac tissue.ConclusionsIn this work, we provide evidence of superior anti-fibrotic properties of EV-H5 over EV-N or EV-H3. Our study uncovers that fine regulation of oxygen concentration in the cellular environment may enhance the anti-fibrotic effects of hiPS-EVs, which has great potential to be applied for heart regeneration.Graphical

ASH2L-mediated H3K4me3 drives diabetic nephropathy through HIPK2 and Notch1 pathway

Diabetic nephropathy (DN) is one of the complications of diabetes. Long-term hyperglycemia in the kidney results in renal insufficiency, and eventually leads to end-stage renal disease. Epigenetic factor ASH2L has long been identified as a transcriptional activator, and we previously indicated that ASH2L aggravated fibrosis and inflammation in high glucose-induced glomerular mesangial cells, but the pathophysiological relevance and the mechanism of ASH2L-mediated H3K4me3 in DN is not well understood. Here we demonstrated that ASH2L is upregulated in glomeruli isolated from db/db mice. Loss of ASH2L protected glomerular injury caused by hyperglycemia, as evidenced by reduced albuminuria, preserved structure, decreased glomerular extracellular matrix deposition, and lowered renal glomerular expression of proinflammatory and profibrotic markers in db/db mice. Furthermore, we demonstrated that enrichment of ASH2L-mediated H3K4me3 on the promoter regions of ADAM17 and HIPK2 triggered their transcription, leading to aberrant activation of Notch1 signaling pathway, thereby contributing to fibrosis and inflammation in DN. The findings of this study provide compelling evidence for targeting ASH2L as a potential therapeutic strategy to prevent or slow down the progression of DN.

Blocking Mitochondrial Pyruvate Transport Alters Corneal Myofibroblast Phenotype: A New Target for Treating Fibrosis.

The purpose of this study was to critically test the hypothesis that mitochondrial pyruvate carrier (MPC) function is essential for maintenance of the corneal myofibroblast phenotype in vitro and in vivo. Protein and mRNA for canonical profibrotic markers were assessed in cultured cat corneal myofibroblasts generated via transforming growth factor (TGF)-β1 stimulation and treated with either the thiazolidinedione (TZD) troglitazone or the MPC inhibitor alpha-cyano-beta-(1-phenylindol-3-yl) acrylate (UK-5099). RNA sequencing was used to gain insight into signaling modules related to instructive, permissive, or corollary changes in gene expression following treatment. A feline photorefractive keratectomy (PRK) model of corneal wounding was used to test the efficacy of topical troglitazone at reducing α-smooth muscle actin (SMA)-positive staining when applied 2 to 4 weeks postoperatively, during peak fibrosis. Troglitazone caused cultured myofibroblasts to adopt a fibroblast-like phenotype through a noncanonical, peroxisome proliferator-activated receptor (PPAR)-γ-independent mechanism. Direct MPC inhibition using UK-5099 recapitulated this effect, but classic inhibitors of oxidative phosphorylation (OXPHOS) did not. Gene Set Enrichment Analysis (GSEA) of RNA sequencing data converged on energy substrate utilization and the Mitochondrial Permeability Transition pore as key players in myofibroblast maintenance. Finally, troglitazone applied onto an established zone of active fibrosis post-PRK significantly reduced stromal α-SMA expression. Our results provide empirical evidence that metabolic remodeling in myofibroblasts creates selective vulnerabilities beyond simply mitochondrial energy production, and that these are critical for maintenance of the myofibroblast phenotype. For the first time, we provide proof-of-concept data showing that this remodeling can be exploited to treat existing corneal fibrosis via inhibition of the MPC.

A spatial sequencing atlas of age-induced changes in the lung during influenza infection

Influenza virus infection causes increased morbidity and mortality in the elderly. Aging impairs the immune response to influenza, both intrinsically and because of altered interactions with endothelial and pulmonary epithelial cells. To characterize these changes, we performed single-cell RNA sequencing (scRNA-seq), spatial transcriptomics, and bulk RNA sequencing (bulk RNA-seq) on lung tissue from young and aged female mice at days 0, 3, and 9 post-influenza infection. Our analyses identified dozens of key genes differentially expressed in kinetic, age-dependent, and cell type-specific manners. Aged immune cells exhibited altered inflammatory, memory, and chemotactic profiles. Aged endothelial cells demonstrated characteristics of reduced vascular wound healing and a prothrombotic state. Spatial transcriptomics identified novel profibrotic and antifibrotic markers expressed by epithelial and non-epithelial cells, highlighting the complex networks that promote fibrosis in aged lungs. Bulk RNA-seq generated a timeline of global transcriptional activity, showing increased expression of genes involved in inflammation and coagulation in aged lungs. Our work provides an atlas of high-throughput sequencing methodologies that can be used to investigate age-related changes in the response to influenza virus, identify novel cell-cell interactions for further study, and ultimately uncover potential therapeutic targets to improve health outcomes in the elderly following influenza infection.

Type 1 diabetes contributes to combined pulmonary fibrosis and emphysema in male alpha 1 antitrypsin deficient mice.

Type 1 diabetes (T1D) is a metabolic disease characterized by hyperglycemia and can affect multiple organs, leading to life-threatening complications. Increased prevalence of pulmonary disease is observed in T1D patients, and diabetes is a leading cause of comorbidity in several lung pathologies. A deficiency of alpha-1 antitrypsin (AAT) can lead to the development of emphysema. Decreased AAT plasma concentrations and anti-protease activity are documented in T1D patients. The objective of this study was to determine whether T1D exacerbates the progression of lung damage in AAT deficiency. First, pulmonary function testing (PFT) and histopathological changes in the lungs of C57BL/6J streptozotocin (STZ)-induced T1D mice were investigated 3 and 6 months after the onset of hyperglycemia. PFT demonstrated a restrictive pulmonary pattern in the lungs of STZ-injected mice, along with upregulation of mRNA expression of pro-fibrotic markers Acta2, Ccn2, and Fn1. Increased collagen deposition was observed 6 months after the onset of hyperglycemia. To study the effect of T1D on the progression of lung damage in AAT deficiency background, C57BL/6J AAT knockout (KO) mice were used. Control and STZ-challenged AAT KO mice did not show significant changes in lung function 3 months after the onset of hyperglycemia. However, histological examination of the lung demonstrated increased collagen accumulation and alveolar space enlargement in STZ-induced AAT KO mice. AAT pretreatment on TGF-β-stimulated primary lung fibroblasts reduced mRNA expression of pro-fibrotic markers ACTA2, CCN2, and FN1. Induction of T1D in AAT deficiency leads to a combined pulmonary fibrosis and emphysema (CPFE) phenotype in male mice.

OR35-02 Novel Androgen Receptor PROTAC Degrader ARV-110 Ameliorates Androgen Mediated Renal Injury in a Mouse Model of Polycystic Ovary Syndrome

Abstract Disclosure: J. Basnet: None. S. Rezq: None. A.M. Huffman: None. T.E. Asala: None. L.L. Yanes Cardozo: None. D.G. Romero: None. Background: Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in reproductive-age women. PCOS women often have increased central adiposity, insulin resistance, and renal injury. Hyperandrogenism is prevalent in PCOS women and is associated with renal injury and dysfunction. Androgens bind to the androgen receptor (AR) to impart downstream transcriptional activity. Targeting the AR may alleviate PCOS complications; however, the use of AR blockers is limited by their severe adverse effects in females; thus, safer alternative approaches are highly needed. The novel AR Proteolysis Targeting Chimera (PROTAC) degrader ARV-110 has shown high efficacy to prevent AR signaling in prostate cancer; however, its efficacy in PCOS is unknown. Using a well-established hyperandrogenemic mouse model of PCOS, we aim to test the hypothesis that decreasing AR protein levels with ARV-110 ameliorates androgen-mediated derangements in PCOS. Methods: Three-week old female mice (C57BL/6N) were implanted with Silastic tubes filled with the androgen dihydrotestosterone (DHT, 8mg, s.c., 90 days) or vehicle (n=6/grp). Four weeks post-DHT administration, mice were treated with ARV-110 (1 mg/kg/day, s.c.) for an additional 8 weeks. Body composition (EchoMRI) and oral glucose tolerance test (OGTT) were assessed. Kidney weight was measured by gravimetry. AR, extracellular matrix (ECM) remodeling marker MMP9, pro-fibrotic marker TGF-β, renal injury markers (urinary albumin to creatinine ration (UACR), KIM1, and NGAL) were assessed by Western-blot or ELISA. Results: DHT mice showed significant (p<0.05) increases in body weight (27.3±0.6 vs 23.9±0.26 g), fat mass (4.2±0.6 vs 2.1±0.1 g), lean mass (21.6±0.2 vs 19.8±0.2 g), as well as kidney weight (491.3±7.69 vs 303.1±5.5 mg) compared to controls. DHT increased fasting glucose (183.2±25.2 vs 141.5±9.9 mg/dL, p<0.05) and impaired OGTT (AUC:34557±6255 vs 23459±1896 mg.min/dL, p<0.05). ARV-110 treatment ameliorated all aforementioned DHT-mediated changes. At the molecular level, DHT-treated mice had higher renal AR (2.5-fold) and MMP9 (1.5-fold) protein expression. DHT upregulated renal TGF-β levels (2-fold, p<0.05), UACR (966.8±160.9 vs 381.5±41.2 ng albumin/mg creatinine), and urinary KIM1 (3.5-fold) and NGAL (6-fold). ARV-110 successfully decreased renal AR protein levels by 70% in DHT mice compared to vehicle-treated DHT. Excitingly, while ARV-110 did not modulate renal TGF-β levels, it completely abolished DHT-mediated increase in renal MMP9 while significantly lowering the levels of urinary renal injury markers UACR, KIM1 and NGAL. Conclusion and significance: Our findings suggest that DHT-mediated kidney hypertrophy, associated ECM remodeling and injury are ameliorated by ARV-110 in DHT-treated mice. AR PROTACs could be a novel therapeutic approach to treat renal dysfunction in PCOS. Supported by NIH P20GM121334. Presentation Date: Sunday, June 18, 2023

Disruption of IL-18 signaling via engineered IL-18BP biologics alleviates experimental cholestatic liver disease

Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease characterized by progressive inflammation and fibrosis around intrahepatic and extrahepatic bile ducts leading to severe hepatic cirrhosis and high mortality. Although there is an urgent clinical unmet need for PSC, no effective medical therapy has been developed to delay the disease progression until today. IL-18 binding protein (IL-18BP) is well-known to be a natural negative feedback regulator for IL-18, and we have developed a recombinant long-acting IL-18BP referred to as APB-R3 as a therapeutic agent to treat IL-18-related inflammatory diseases. Here, we aimed to study whether disrupted IL-18 signaling by APB-R3 treatment can inhibit PSC injuries in the experimental DDC diet-induced PSC rodent model. First, we found that the amounts of free IL-18 are augmented under PSC condition with increased expression of biliary IL-18 receptors. Administration of APB-R3 effectively attenuated key diagnostic parameters of PSC such as plasma ALP and GGT levels as well as bile acids levels. We also observed that blockade of IL-18 suppressed ductular reactive and proliferative phenotypes of cholangiocytes. Additionally, APB-R3 significantly ameliorated DDC diet-induced periductal fibrosis and transcriptional expressions of pro-fibrotic marker genes. Enhanced senescence associated secretory phenotype (SASP) markers in cholestatic liver disease were diminished by APB-R3 treatment. Our findings clearly demonstrate that the administration of IL-18BP biologics, APB-R3, effectively alleviates DDC diet-induced biliary injuries in rodent PSC model, implying APB-R3 can be a promising therapeutic reagent which warrants clinical human trials as new therapeutic options.

A novel BRD4 degrader, ARV-825, attenuates lung fibrosis through senolysis and antifibrotic effect

BackgroundRecent studies suggest that cellular senescence is related to the pathogenesis of idiopathic pulmonary fibrosis. However, cellular senescence has yet to be targeted therapeutically in clinical practice. ARV825, a recently developed BRD4 degrader, has been reported as a novel senolytic drug. Conversely, it has also been reported that BRD4 regulates the pro-fibrotic gene expression of fibroblasts. Therefore, this study focuses on the senolytic and anti-fibrotic effects of ARV825 and evaluated these effects on lung fibrosis. MethodsLung fibroblasts were induced to senescence through serial passage. The expression of senescence markers and pro-fibrotic markers were determined through quantitative PCR or immunoblot analysis. Lung fibrosis was induced in mice through intratracheal administration of bleomycin. Mice treated with ARV825 underwent histological analysis of lung fibrosis using the Ashcroft score. Total lung collagen was quantified through a hydroxyproline assay. Respiratory mechanics analysis was performed using the flexiVent system. ResultsFor senescent cells, ARV825 induced the expression of an apoptosis marker while reducing the expression of BRD4 and senescence markers. On the other hand, for early passage pre-senescent cells, ARV825 reduced the expression of collagen type 1 and α-smooth muscle actin. In an experimental mouse model of lung fibrosis, ARV825 attenuated lung fibrosis and improved lung function. Immunohistochemical staining revealed a significant decrease in the number of senescent alveolar type 2 cells in lung tissue due to ARV825 treatment. ConclusionsThese results suggest that ARV825 may impact the progressive and irreversible course of fibrotic lung diseases.

P2X7 Receptor Regulates Collagen Expression in Human Intestinal Fibroblasts: Relevance in Intestinal Fibrosis.

Intestinal fibrosis is a common complication that affects more than 50% of Crohn´s Disease (CD) patients. There is no pharmacological treatment against this complication, with surgery being the only option. Due to the unknown role of P2X7 in intestinal fibrosis, we aim to analyze the relevance of this receptor in CD complications. Surgical resections from CD and non-Inflammatory Bowel Disease (IBD) patients were obtained. Intestinal fibrosis was induced with two different murine models: heterotopic transplant model and chronic-DSS colitis in wild-type and P2X7-/- mice. Human small intestine fibroblasts (HSIFs) were transfected with an siRNA against P2X7 and treated with TGF-β. A gene and protein expression of P2X7 receptor was significantly increased in CD compared to non-IBD patients. The lack of P2X7 in mice provoked an enhanced collagen deposition and increased expression of several profibrotic markers in both murine models of intestinal fibrosis. Furthermore, P2X7-/- mice exhibited a higher expression of proinflammatory cytokines and a lower expression of M2 macrophage markers. Moreover, the transient silencing of the P2X7 receptor in HSIFs significantly induced the expression of Col1a1 and potentiated the expression of Col4 and Col5a1 after TGF-β treatment. P2X7 regulates collagen expression in human intestinal fibroblasts, while the lack of this receptor aggravates intestinal fibrosis.

Abstract P2099: Small Proline-rich Protein 1a Is A Key Downstream Effector Of Microrna-150 During Both Maladaptive Cardiac Remodeling In Mice And Human Cardiac Fibroblast Activation

Background & Objectives: MicroRNA-150 (miR-150) is conserved between rodents and humans, is significantly downregulated during heart failure (HF), and correlates with patient outcomes. We reported that miR-150 is protective during myocardial infarction (MI) in part by decreasing cardiomyocyte (CM) apoptosis and that proapoptotic small proline-rich protein 1a ( Sprr1a ) is a direct CM target of miR-150. We also showed that Sprr1a knockdown in mice improves cardiac dysfunction and fibrosis post-MI and that Sprr1a is upregulated in pathological mouse cardiac fibroblasts (CFs) from ischemic myocardium. However, the direct functional relationship between miR-150 and SPRR1A during both post-MI remodeling in mice and human CF (HCF) activation was not established. Methods & Results: Here, using a novel miR-150 knockout (KO); Sprr1a -hypomorphic ( Sprr1a hypo/hypo ) mouse model, we demonstrate that Sprr1a knockdown blunts adverse post-MI effects caused by miR-150 loss. MiR-150 KO; Sprr1a hypo/hypo mice also exhibit decreased profibrotic markers in the infarct areas of hearts as compared to miR-150 KO mice. Moreover, primary adult HCF studies reveal that SPRR1A is upregulated in hypoxia/reoxygenation-treated HCFs and is downregulated in HCFs exposed to the cardioprotective β-blocker carvedilol, which is inversely associated with miR-150 expression. Significantly, we show that the protective roles of miR-150 in HCFs are directly mediated by functional repression of profibrotic SPRR1A (See Figure). Conclusions: These findings delineate a pivotal functional interaction between miR-150 and SPRR1A as a novel regulatory mechanism pertinent to CF activation and ischemic HF.

Coadministration of 3′5-dimaleamylbenzoic acid and quercetin decrease pulmonary fibrosis in a systemic sclerosis model

Systemic sclerosis (SSc) is an autoimmune disease characterized by microvascular compromise and fibrosis. Pulmonary fibrosis, a prominent pulmonary complication in SSc, results in impaired lung function due to excessive accumulation of extracellular matrix components. This study aimed to investigate the effects of coadministration of 3′5-dimaleamylbenzoic acid (AD) and quercetin (Q) on key events in the development and maintenance of pulmonary fibrosis in a bleomycin (BLM)-induced SSc mouse model. The model was induced in CD1 mice through BLM administration using osmotic mini pumps. Subsequently, mice were treated with AD (6 mg/kg) plus Q (10 mg/kg) and sacrificed at 21 and 28 days post BLM administration. Histopathological analysis was performed by hematoxylin and eosin staining and Masson's trichrome staining. Immunohistochemistry was used to determine the expression of proliferation, proinflammatory, profibrotic and oxidative stress markers. The coadministration of AD and Q during the fibrotic phase of the BLM-induced SSc model led to attenuated histological alterations and pulmonary fibrosis, reflected in the recovery of alveolar spaces (30 %, p < 0.01) and decreased collagen deposits (50 %, p < 0.001). This effect was achieved by decreasing the expression of the proliferative markers cyclin D1 (87 %, p < 0.0001) and PCNA (43 %, p < 0.0001), inflammatory markers COX-2 (71 %, p < 0.0001) and iNOS (84 %, p < 0.0001), profibrotic markers α-SMA (80 %, p < 0.0001) and TGF-β (81 %, p < 0.0001) and the lipid peroxidation marker 4-HNE (43 %, p < 0.01). The antifibrotic effect of this combined therapy is associated with the regulation of proliferation, inflammation and oxidative stress, mechanisms involved in the development and progression of the fibrotic process. Our novel therapeutic strategy is the first approach to propose the use of the combination of prooxidant and antioxidant compounds as a potential strategy for SSc-associated pulmonary fibrosis.

Effect of E-Vaping on Kidney Health in Mice Consuming a High-Fat Diet.

High-fat diet (HFD) consumption and tobacco smoking are risk factors for chronic kidney disease. E-cigarettes have gained significant popularity among younger populations worldwide, especially among overweight individuals. It is unclear whether vaping interacts with HFD consumption to impact renal health. In this study, Balb/c mice (male, 7 weeks old) were fed a pellet HFD (43% fat, 20 kJ/g) for 16 weeks when exposed to nicotine or nicotine-free e-vapour from weeks 11 to 16. While HFD alone increased collagen Ia and IV depositions, it did not cause significant oxidative stress and inflammatory responses in the kidney itself. On the other hand, e-vapour exposure alone increased oxidative stress and damaged DNA and mitochondrial oxidative phosphorylation complexes without significant impact on fibrotic markers. However, the combination of nicotine e-vapour and HFD increased inflammatory responses, oxidative stress-induced DNA injury, and pro-fibrotic markers, suggesting accelerated development of renal pathology. Nicotine-free e-vapour exposure and HFD consumption suppressed the production of mitochondrial OXPHOS complexes and extracellular matrix protein deposition, which may cause structural instability that can interrupt normal kidney function in the future. In conclusion, our study demonstrated that a HFD combined with e-cigarette vapour exposure, especially when containing nicotine, can increase susceptibility to kidney disease.

Isoliquiritigenin attenuates high glucose-induced proliferation, inflammation, and extracellular matrix deposition in glomerular mesangial cells by suppressing JAK2/STAT3 pathway.

To investigate the effect of isoliquiritigenin (ISL) on high glucose (HG)-induced glomerular mesangial cells (GMCs) proliferation, extracellular matrix (ECM) deposition and inflammation, and the underlying mechanisms. Mouse GMCs (SV40-MES-13) were cultured in HG medium, with or without ISL. The proliferation of GMCs was determined by MTT assay. The production of proinflammatory cytokines was detected by qRT-PCR and ELISA. The expression of connective tissue growth factor (CTGF), TGF-β1, collagen IV, and fibronectin was measured by qRT-PCR and western blot. The phosphorylation of JAK2 and STAT3 was examined by western blot. Next, JAK2 inhibitor AG490 was applied to HG-exposed GMCs. The levels of JAK2/STAT3 phosphorylation and pro-fibrotic markers were analyzed by western blot, and the secretion of TNF-α and IL-1β was evaluated by ELISA. GMCs were treated with HG, HG plus ISL or HG plus ISL, and recombinant IL-6 (rIL-6) which is a JAK2 activator. The levels of JAK2/STAT3 activation, ECM formation, and proinflammatory cytokines secretion were determined by western blot and ELISA, respectively. In mouse GMCs, ISL successfully repressed HG-induced hyperproliferation; production of TNF-α and IL-1β; expression of CTGF, TGF-β1, collagen IV, and fibronectin; and activation of JAK2/STAT3. Similar to ISL, AG490 was able to reverse the inflammation and ECM generation caused by HG. Moreover, rIL-6 impeded the amelioration of ISL on HG-induced adverse effects. Our study demonstrated that ISL displayed preventive effects on HG-exposed GMCs through inhibiting JAK2/STAT3 pathway and provided an insight into the application of ISL for diabetic nephropathy (DN) treatment.

Radiation induced changes in profibrotic markers in the submental muscles and their correlation with tongue movement.

Swallowing impairment is a major complication of radiation treatment for oropharyngeal cancers. Developing targeted therapies that improve swallowing outcomes relies on an understanding of the mechanisms that influence motor function after radiation treatment. The purpose of this study was to determine whether there is a correlation between radiation induced changes in tongue movement and structural changes in irradiated submental muscles, as well as assess other possible causes for dysfunction. We hypothesized that a clinically relevant total radiation dose to the submental muscles would result in: a) quantifiable changes in tongue strength and displacement during drinking two months post treatment; and b) a profibrotic response and/or fiber type transition in the irradiated tissue. Sprague-Dawley adult male rats received radiation to the submental muscles at total dose-volumes known to provoke dysphagia in humans. A clinical linear accelerator administered 8 fractions of 8Gy for a total of 64Gy. Comparisons were made to sham-treated rats that received anesthesia only. Swallowing function was assessed using videofluoroscopy and tongue strength was analyzed via force lickometer. TGFβ1 expression was analyzed via ELISA. The amount of total collagen was analyzed by picrosirius red staining. Immunofluorescence was used to assess fiber type composition and size. Significant changes in licking function during drinking were observed at two months post treatment, including a slower lick rate and reduced tongue protrusion during licking. In the mylohyoid muscle, significant increases in TGFβ1 protein expression were found post radiation. Significant increases in the percentage of collagen content were observed in the irradiated geniohyoid muscle. No changes in fiber type expression were observed. Results indicate a profibrotic transition within the irradiated swallowing muscles that contributes to tongue dysfunction post-radiation treatment.

#4151 POLYCYSTINS ARE REQUIRED FOR RENAL TUBULOINTERSTITIAL FIBROSIS

Abstract Background and Aims Renal fibrosis is the common pathway of various chronic kidney diseases progressing to end stage renal failure. Polycystin-1 (encoded by PKD1 gene) and polycystin-2 (encoded by PKD2 gene) form a transmembrane complex and function as a stress sensor, which is located in the primary cilia. Polycystins are involved in the disease condition of different organs. Mutation of PKD genes causes autosomal dominant polycystic kidney disease and deletion of polycystin attenuates heart injury induced cardiac fibrosis. The role of polycystins in renal tubulointerstitial fibrosis is currently unclear. Method Unilateral ureteral obstruction (UUO), unilateral ischemia-reperfusion injury (UIRI), aristolochic acid or folic acid induced mouse models of renal fibrosis were established for this study. Results Here we showed that polycystin-2 is up-regulated in these three mouse models of renal fibrosis and tightly correlated with the expression of collagen-I in a time dependent manner. Treatment with triptolide inhibited the expression of polycystin-2 and pro-fibrotic markers in UUO and UIRI models. Moreover, triptolide or PKD2 siRNA inhibited the expression of polycystin-2 and pro-fibrotic markers in vitro. Using Pkd2 conditional knockout mice, we showed that genetic deletion of Pkd2 reduced the expression of pro-fibrotic markers in UUO kidneys. Polycystin-1 was also up-regulated in renal fibrotic models and conditional deletion of Pkd1 reduced the expression of pro-fibrotic markers in UUO or folic acid induced fibrotic kidneys. Furthermore, the expression of the methyltransferase EZH2 is positively correlated with the expression of polycystins in fibrotic kidneys. Conditional knockout of EZH2 attenuated the anti-fibrotic responses induced by Pkd1 deletion in UUO kidneys. Conclusion In conclusion, polycystins are up-regulated in fibrotic kidneys and promote renal tubulointerstitial fibrosis through up-regulation of EZH2, suggesting that primary cilia are required for renal tubulointerstitial fibrosis.

#5645 SHORT-CHAIN FATTY ACIDS, PROPIONATE AND BUTYRATE, RESEMBLE THE BENEFICIAL EFFECTS OF VITAMIN D DURING ACUTE KIDNEY DAMAGE

Abstract Background and Aims The vitamin D receptor (VDR) is a nuclear receptor that acts as a ligand-induced transcription factor regulating the renal expression of numerous genes with anti-inflammatory and anti-fibrotic effects, among others. For that, VDR requires the binding of its ligand, 1,25 (OH)2D3, to further heterodimerize with its co-activator, RXR, and translocate into the nucleus. Vitamin D deficiency in patients with renal disease leads to a decrease in VDR-mediated signaling and, consequently, of its beneficial functions. Additionally, these patients have intestinal dysbiosis, which leads to alterations in the production of microbial metabolites. Short-chain fatty acids (SCFAs) are metabolites with a clear anti-inflammatory and immunoregulatory role. Until now, some studies have studied their role as regulators of the inflammation and oxidative stress during the renal damage.1,2 However, their role in the VDR-mediated signaling in the kidney and the potential consequences to prevent the progression of the disease have not yet been explored in detail. Method The tubular epithelial cell line, HK2, was used to analyze the effect of the SCFAs, Propionate (Prop,1-15mM) and Butyrate (But, 0.5-3 mM), in the VDR expression and their target-genes. In vitro treatment with histone deacetylases (HDACs) inhibitors and specific HDAC1 and HDAC3 siRNAs were used to determine the role of both SCFAs as epigenetic remodelers. The binding of VDR to HDAC1/3 and RXR was determined by ChIP and co-immunoprecipitation assays. The in vivo effect of these SCFAs was evaluated in an acute kidney injury mice model induced by folic acid (250 mg/kg) and administration of Prop (200mg/kg) or But (500mg/kg) at different time points. The expression of VDR and its target genes, inflammatory cell infiltration, renal damage markers and renal function parameters were evaluated at shorter (24h) and longer times (40 days). Results Treatment of tubular cells with Prop and But induces, in a dose-dependent manner, the VDR gene transcription. This effect is similar to the one obtained by using specific inhibitors and siRNA treatments against HDAC1/3. We determine using ChIP assay, that HDAC1/3 are recruited to the promoter regions of the VDR gene, blocking its expression. In presence of Prop and But, these HDACs are displaced, increasing the acetylation levels and VDR transcription. Moreover, Prop and But prevent the degradation of VDR by the proteasome, increasing its stability and enhancing VDR protein levels. In the presence of both SCFAs, VDR dimerizes with RXR initiating its translocation to the nucleus and allowing the transcription of its dependent genes, such as Cyp24a1 and E-cadherin. Of note, VDR activation by Prop and But is additive to the effect achieved by the vitamin D alone. In vivo studies reported that administration of Prop or But prevents the loss of VDR expression 24h after induction of the damage, leading to its activation and to the expression of its target genes. Additionally, a decrease in the recruitment of neutrophils to the kidney was observed associated to a reduced expression of IL-6. These changes are accompanied by a decrease of the kidney damage markers (KIM-1 and NGAL) and a significant reduction of the creatinine and blood urea nitrogen serum levels. In a second model of AKI-to CKD transition, administration of both SCFAs shows a decrease in the inflammatory (Ccl20, Ccl2, Lif, Ltb, Csf2SF2, Il18, Ccl5, Tnf-α) and pro-fibrotic markers (Fsp1, α-Sma, Col1a1, Fn1) and a partial recovery of the glomerular filtration rate at long term. Conclusion Propionate and Butyrate, not only induce the VDR gene transcription in renal tubular cells but are also able to stabilize and activate the VDR protein. Accordingly, both metabolites are able to restore the loosed expression and activation of VDR due to induced renal damage, reduce the infiltration of immune cells, and partially recover the renal function. Thus, strategies aimed to increase the propionate and butyrate levels with postbiotics could be useful to ameliorate the AKI renal damage and CKD transition.

#2999 APABETALONE REDUCES CARDIAC EVENTS IN CKD PATIENTS BY DOWNREGULATING FIBROTIC AND INFLAMMATORY PROCESSES

Abstract Background and Aims Major adverse cardiac events (MACE) are a leading cause of mortality in patients with chronic kidney disease (CKD). Apabetalone is an orally available inhibitor of bromodomain &amp; extraterminal (BET) proteins – epigenetic readers that modulate gene expression involved in fibrosis &amp; inflammation. In the phase 3 BETonMACE trial in patients with CVD &amp; diabetes mellitus (NCT02586155), apabetalone reduced MACE by 50% in the subpopulation with CKD (eGFR &amp;lt; 60 mL/min/1.73 m2; HR 0.50 95% CI 0.26,0.96 p = 0.04) indicating favorable response along the kidney-heart axis. This study examines effects of apabetalone in human renal mesangial cells (HRMCs) on fibrotic &amp; inflammatory pathways. In the clinic, we assess plasma levels of fibrotic factors in CKD patients &amp; matched controls after receiving apabetalone. Method TGF-β1 is a pro-fibrotic cytokine that activates HRMCs to over-produce extracellular matrix (ECM). HRMCs were stimulated with TGF-β1 or LPS ± 1-25 µM apabetalone, 0.15-0.5 µM JQ1 or 0.1 µM MZ1 - BET inhibitors (BETi) with chemical scaffolds different than apabetalone. Gene expression was measured by RNA-seq &amp; real-time PCR. RNA-seq was evaluated by Gene Ontology (GO) &amp; Ingenuity Pathway Analysis (IPA). Proteins were measured by immunofluorescence microscopy or ELISA. Collagen gel contraction was assessed in 3D culture. In human subjects, proteomic analysis of plasma was performed via SOMAscan 1.3k 12 hours after 100 mg of apabetalone in CKD patients (n = 8 eGFR &amp;lt; 30 stage 4 or 5) and matched controls (n = 8 eGFR &amp;gt; 60). Results GO analysis of RNA-seq from TGF-β1 stimulated HRMCs showed multiple gene sets associated with ECM production &amp; remodeling in the top 20 affected by BETi. IPA predicted NfkB-RelA and NFkB (complex) were inhibited by apabetalone, consistent with suppressed inflammation. IPA also predicted apabetalone activated pathways of glucose utilization &amp; tolerance of ROS production, including Oxidative Phosphorylation (z-score 5.7, p = 2.95 × 10−3 at 25 µM) &amp; NRF2-Mediated Oxidative Stress Response (z score 2.3, p = 7.9 × 10−12 at 25 µM; z-score 1.6, p = 1.5 × 10−5 at 5 µM). Predicted changes may allow tolerance of elevated glucose. Mechanistically, apabetalone suppressed TGF-β1 induced α-smooth muscle actin (α-SMA) gene expression, a marker of activation, up to 89% (p = 2.8 × 10−6 at 25 µM), and abolished α-SMA filament formation. Consequently, apabetalone opposed TGF-β1 induced collagen gel contraction up to 24% (p = 0.031 at 25 µM). Further, apabetalone countered TGF-β1 induced expression &amp; protein production of key drivers of fibrosis including (a) thrombospondin, an activator of latent TGF-β1 (b) fibronectin, a key ECM component (c) periostin, a promoter of ECM production (d) osteonectin, a regulator of TGF-β1 expression and (e) IL6, a pro-inflammatory cytokine (Table 1). In addition, apabetalone dose dependently opposed LPS stimulated expression of inflammatory genes: IL6 up to 94%, IL1B up to 95% and PTGS2 up to 94% (p&amp;lt;0.001 for each). In all studies, JQ1 or MZ1 had similar activity as apabetalone, confirming on-target BETi effects. In humans, plasma levels of profibrotic and inflammatory markers were reduced 12 hours after receiving apabetalone specifically in those with CKD. Only periostin was reduced in subjects without renal impairment (Table 1). Conclusion Through epigenetic regulation of transcription in HRMCs, apabetalone reduces production of fibrotic &amp; inflammatory factors known to signal along the kidney-heart axis &amp; exacerbate kidney dysfunction. Predicted changes in energy metabolism suggest apabetalone facilitates adaptation to high glucose in kidneys. In a clinical trial, plasma levels of fibrotic &amp; inflammatory factors were reduced specifically in CKD patients receiving apabetalone. Our results provide mechanistic insights into reduced MACE in CKD patients receiving apabetalone in the phase 3 BETonMACE trial &amp; will be further evaluated in an upcoming Phase 3 trial.

Total sesquiterpenoids from Eupatorium lindleyanum DC. attenuate bleomycin-induced lung fibrosis by suppressing myofibroblast transition

Eupatorium lindleyanum DC. has been used as a functional food in China for a long time. However, the antifibrotic activity of total sesquiterpenoids from Eupatorium lindleyanum DC. (TS-EL) is still unknown. In this study, we discovered that TS-EL reduced the increase in α-smooth muscle actin (α-SMA), type I collagen and fibronectin content, the formation of cell filaments and collagen gel contraction in transforming growth factor-β1-stimulated human lung fibroblasts. Intriguingly, TS-EL did not change the phosphorylation of Smad2/3 and Erk1/2. TS-EL decreased the levels of serum response factor (SRF), a critical transcription factor of α-SMA, and SRF knockdown alleviated the transition of lung myofibroblasts. Furthermore, TS-EL significantly attenuated bleomycin (BLM)-induced lung pathology and collagen deposition and reduced the levels of two profibrotic markers, total lung hydroxyproline and α-SMA. TS-EL also decreased the levels of SRF protein expression in BLM-induced mice. These results suggested that TS-EL attenuates pulmonary fibrosis by inhibiting myofibroblast transition via the downregulation of SRF.