Role of CDKL1-SOX11 signaling axis in Acute Kidney Injury.

The biology of CDKL (Cyclin-Dependent Kinase-Like) kinase family remains enigmatic. Contrary to their nomenclature, CDKLs do not rely on cyclins for activation and are not involved in cell cycle regulation. Instead, they share structural similarities with MAPKs (Mitogen-Activated Protein Kinases) and GSK3 (glycogen synthase kinase 3), though their specific functions and associated signaling pathways are still unknown. Previous studies have shown that the activation of CDKL5 kinase contributes to the development of acute kidney injury (AKI) by suppressing the protective SOX9-dependent transcriptional program in tubular epithelial cells. In the current study, we measured the functional activity of all the five CDKL kinases and discovered that, in addition to CDKL5, CDKL1 is also activated in tubular epithelial cells during AKI. To explore the role of CDKL1, we generated a germline knockout mouse which exhibited no abnormalities under normal conditions. Notably, when these mice were challenged with bilateral ischemia reperfusion and rhabdomyolysis, they were found to be protected from AKI. Further mechanistic investigations revealed that CDKL1 phosphorylates and destabilizes SOX11, contributing to tubular dysfunction. In summary, these studies have unveiled a previously unknown CDKL1-SOX11 axis that drives tubular dysfunction during AKI.

Network pharmacology analysis of the regulatory effects and mechanisms of ALAE on sow reproduction in vivo and in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Reproductive ability of sows is a primary element influencing the development of pig farming. Herbal extracts of Angelica sinensis (Oliv.) Diels, Astragalus mongholicus Bunge, Eucommia ulmoides Oliv., and Polypodium glycyrrhiza D.C.Eaton showed effects on improvement of reproduction in sows. AIMS OF THE STUDY: To investigate the mechanism of the treatment effects by a compound of these four Chinese herbs in a 1:1:1:1 ratio (ALAE) on endometriosis, endometritis, uterine adhesion, intrauterine growth retardation, pre-eclampsia, and its enhancement of reproductive efficiency in sows. MATERIALS AND METHODS: Active components of ALAE were identified by using ultra-performance liquid chromatography-mass spectrometry analysis and network pharmacology. Then we used the results to construct a visualization network. Key targets and pathways of ALAE involved in sow reproduction improvement were validated in sow animals and porcine endometrial epithelial cells (PEECs). RESULTS: A total of 62 active compounds were found in ALAE (41 in Polypodium glycyrrhiza D.C.Eaton, 5 in Astragalus mongholicus Bunge, 11 in Eucommia ulmoides Oliv., 5 in Angelica sinensis (Oliv.) Diels) with 563 disease-related targets (e.g. caspase-3, EGFR, IL-6) involved in EGFR tyrosine kinase inhibitor resistance, PI3K-AKT, and other signaling pathways. Molecular docking results indicated GC41 (glabridin), GC18 (medicarpin), EGFR and CCND1 are possible key components and target proteins related to reproductive improvement in sows. In PEECs, EGFR expression decreased at the mRNA and protein levels by three doses (160, 320, and 640 µg/mL) of ALAE. The phosphorylation of downstream pathway PI3K-AKT1was enhanced. The expression of inflammatory factors (IL-6, IL-1ß), ESR1 and caspase-3 decreased through multiple pathways. Additionally, the expression levels of an anti-inflammatory factor (IL-10), angiogenesis-related factors (MMP9, PIGF, PPARγ, IgG), and placental junction-related factors (CTNNB1, occludin, and claudin1) increased. Furthermore, the total born number of piglets, the number of live and healthy litters were significantly increased. The number of stillbirths decreased by ALAE treatment in sow animals. CONCLUSIONS: Dministration of ALAE significantly increased the total number of piglets born, the numbers of live and healthy litters and decreased the number of stillbirths through improving placental structure, attenuating inflammatory response, modulating placental angiogenesis and growth factor receptors in sows. The improvement of reproductive ability may be related to activation of the EGFR-PI3K-AKT1 pathway in PEECs. Moreover, ALAE maybe involved in modulation of estrogen receptors, apoptotic factors, and cell cycle proteins.

Primary nasal influenza infection rewires tissue-scale memory response dynamics.

The nasal mucosa is often the initial site of respiratory viral infection, replication, and transmission. Understanding how infection shapes tissue-scale primary and memory responses is critical for designing mucosal therapeutics and vaccines. We generated a single-cell RNA-sequencing atlas of the murine nasal mucosa, sampling three regions during primary influenza infection and rechallenge. Compositional analysis revealed restricted infection to the respiratory mucosa with stepwise changes in immune and epithelial cell subsets and states. We identified and characterized a rare subset of Krt13+ nasal immune-interacting floor epithelial (KNIIFE) cells, which concurrently increased with tissue-resident memory T (TRM)-like cells. Proportionality analysis, cell-cell communication inference, and microscopy underscored the CXCL16-CXCR6 axis between KNIIFE and TRM cells. Secondary influenza challenge induced accelerated and coordinated myeloid and lymphoid responses without epithelial proliferation. Together, this atlas serves as a reference for viral infection in the upper respiratory tract and highlights the efficacy of local coordinated memory responses.

Extracellular glucose triggers metabolic reprogramming of cultured human bronchial epithelial cells and indirect fibroblast activation.

Glucose is essential for energy metabolism, and its usage can determine other cellular functions, depending on the cell type. In some pathological conditions, cells are exposed to high concentrations of glucose for extended periods. In this study, we investigated metabolic, oxidative stress, and cellular senescence pathways in human bronchial epithelial cells (HBECs) cultured in media with physiologically low (5 mm) and high (12.5 mm) glucose concentrations. HBECs exposed to 12.5 mm glucose showed increased glucose routing toward the pentose phosphate pathway, lactate synthesis, and glycogen, but not triglyceride synthesis. These metabolic shifts were not associated with changes in cell proliferation rates, oxidative stress, or cellular senescence pathways. Since hyperglycemia is associated with fibrosis in the lung, we asked whether HBECS could activate fibroblasts. Primary human lung fibroblasts cultured in media conditioned by 12.5 mm glucose-exposed HBECs showed a 1.3-fold increase in the gene expression of COL1A1 and COL1A2, along with twofold increased protein levels of smooth muscle cell actin and 2.4-fold of COL1A1. Consistently, HBECs cultured with 12.5 mm glucose secreted proteins associated with inflammation and fibrosis, such as interleukins IL-1ß, IL-10, and IL-13, CC chemokine ligands CCL2 and CCL24, and with extracellular matrix remodeling, such as metalloproteinases (MMP)-1, MMP-3, MMP-9, and MMP-13 and tissue inhibitors of MMPs (TIMP)-1 and -2. This study shows that HBECs undergo metabolic reprogramming and increase the secretion of profibrotic mediators following exposure to high concentrations of glucose, and it contributes to the understanding of the metabolic crosstalk of neighboring cells in diabetes-associated pulmonary fibrosis.

Expression of ISG60 is induced by TLR3 signaling in BEAS­2B bronchial epithelial cells: Possible involvement in CXCL10 expression.

Viral infections in the respiratory tract are common, and, in recent years, severe acute respiratory syndrome coronavirus 2 outbreaks have highlighted the effect of viral infections on antiviral innate immune and inflammatory reactions. Specific treatments for numerous viral respiratory infections have not yet been established and they are mainly treated symptomatically. Therefore, understanding the details of the innate immune system underlying the airway epithelium is crucial for the development of new therapies. The present study aimed to investigate the function and expression of interferon (IFN)­stimulated gene (ISG)60 in non­cancerous bronchial epithelial BEAS­2B cells exposed to a Toll­like receptor 3 agonist. BEAS­2B cells were treated with a synthetic TLR3 ligand, polyinosinic­polycytidylic acid (poly IC). The mRNA and protein expression levels of ISG60 were analyzed using reverse transcription­quantitative PCR and western blotting, respectively. The levels of C­X­C motif chemokine ligand 10 (CXCL10) were examined using an enzyme­linked immunosorbent assay, and the effects of knockdown of IFN­ß, ISG60 and ISG56 were examined using specific small interfering RNAs. Notably, ISG60 expression was increased in proportion to poly IC concentration, and recombinant human IFN­ß also induced ISG60 expression. By contrast, knockdown of IFN­ß and ISG56 decreased ISG60 expression, and ISG60 knockdown reduced CXCL10 and ISG56 expression. These findings suggested that ISG60 is partly implicated in CXCL10 expression and that ISG60 may serve a role in the innate immune response of bronchial epithelial cells. The present study highlights ISG60 as a potential target for new therapeutic strategies against viral infections in the airway.

BMSCs promote alveolar epithelial cell autophagy to reduce pulmonary fibrosis by inhibiting core fucosylation modifications.

BACKGROUND: Idiopathic pulmonary fibrosis is a chronic progressive interstitial lung disease characterized by alveolar epithelial cell (AEC) injury and fibroblast activation. Inadequate autophagy in AECs may result from the activation of several signaling pathways following AEC injury, with glycoproteins serving as key receptor proteins. The core fucosylation (CF) modification in glycoproteins is crucial. Mesenchymal stem cells derived from bone marrow (BMSCs) have the ability to regenerate damaged tissue and treat pulmonary fibrosis (PF). This study aimed to elucidate the relationship and mechanism of interaction between BMSCs, CF modification, and autophagy in PF. METHODS: C57BL/6 male mice, alveolar epithelial cell-specific FUT8 conditional knockout (CKO) mice, and MLE12 cells were administered bleomycin (BLM), FUT8 siRNA, and mouse BMSCs, respectively. Experimental techniques including tissue staining, western blotting, immunofluorescence, autophagic flux detection, and flow cytometry were utilized in this study. RESULTS: First, we found that autophagy was inhibited while FUT8 expression was elevated in PF mice and BLM-induced AEC injury models. Subsequently, CKO mice and MLE12 cells transfected with FUT8 siRNA were employed to demonstrate that inhibition of CF modification induces autophagy in AECs and mitigates PF. Finally, mouse BMSCs were utilized to demonstrate that they alleviate the detrimental autophagy of AECs by inhibiting CF modification and decreasing PF. CONCLUSIONS: Suppression of CF modification enhanced the suppression of AEC autophagy and reduced PF in mice. Additionally, through the prevention of CF modification, BMSCs can assist AECs deficient in autophagy and partially alleviate PF.

Different localization of P2X4 and P2X7 receptors in native mouse lung - lack of evidence for a direct P2X4-P2X7 receptor interaction.

Introduction: P2X receptors are a family of homo- and heterotrimeric cation channels gated by extracellular ATP. The P2X4 and P2X7 subunits show overlapping expression patterns and have been involved in similar physiological processes, such as pain and inflammation as well as various immune cell functions. While formation of P2X2/P2X3 heterotrimers produces a distinct pharmacological phenotype and has been well established, functional identification of a P2X4/P2X7 heteromer has been difficult and evidence for and against a physical association has been found. Most of this evidence stems, however, from in vitro model systems. Methods: Here, we used a P2X7-EGFP BAC transgenic mouse model as well as P2X4 and P2X7 knock-out mice to re-investigate a P2X4-P2X7 interaction in mouse lung by biochemical and immunohistochemical experiments as well as quantitative expression analysis. Results: No detectable amounts of P2X4 could be co-purified from mouse lung via P2X7-EGFP. In agreement with these findings, immuno-histochemical analysis using a P2X7-specific nanobody revealed only limited overlap in the cellular and subcellular localizations of P2X4 and P2X7 in both the native lung tissue and primary cells. Comparison of P2X4 and P2X7 transcript and protein levels in the respective gene-deficient and wild type mice showed no mutual interrelation between their expression levels in whole lungs. However, a significantly reduced P2rx7 expression was found in alveolar macrophages of P2rx4 -/- mice. Discussion: In summary, our detailed analysis of the cellular and subcellular P2X4 and P2X7 localization and expression does not support a physiologically relevant direct association of P2X4 and P2X7 subunits or receptors in vivo.

Mechanisms of Bleomycin-induced Lung Fibrosis: A Review of Therapeutic Targets and Approaches.

Pulmonary toxicity is a serious side effect of some specific anticancer drugs. Bleomycin is a well-known anticancer drug that triggers severe reactions in the lungs. It is an approved drug that may be prescribed for the treatment of testicular cancers, Hodgkin's and non-Hodgkin's lymphomas, ovarian cancer, head and neck cancers, and cervical cancer. A large number of experimental studies and clinical findings show that bleomycin can concentrate in lung tissue, leading to massive oxidative stress, alveolar epithelial cell death, the proliferation of fibroblasts, and finally the infiltration of immune cells. Chronic release of pro-inflammatory and pro-fibrotic molecules by immune cells and fibroblasts leads to pneumonitis and fibrosis. Both fibrosis and pneumonitis are serious concerns for patients who receive bleomycin and may lead to death. Therefore, the management of lung toxicity following cancer therapy with bleomycin is a critical issue. This review explains the cellular and molecular mechanisms of pulmonary injury following treatment with bleomycin. Furthermore, we review therapeutic targets and possible promising strategies for ameliorating bleomycin-induced lung injury.

Arsenic activated GLUT1-mTORC1/HIF-1α-PKM2 positive feedback networks promote proliferation and migration of bladder epithelial cells.

Arsenic (As) is recognized as a potent environmental contaminant associated with bladder carcinogenesis. However, its molecular mechanism remains unclear. Metabolic reprogramming is one of the hallmarks of cancer and is as a central feature of malignancy. Here, we performed the study of cross-talk between the mammalian target of rapamycin complex 1 (mTORC1)/ Hypoxia-inducible factor 1 alpha (HIF-1α) pathway and aerobic glycolysis in promoting the proliferation and migration of bladder epithelial cells treated by arsenic in vivo and in vitro. We demonstrated that arsenite promoted N-methyl-N-nitrosourea (MNU)-induced tumor formation in the bladder of rats and the malignant behavior of human ureteral epithelial (SV-HUC-1) cell. We found that arsenite positively regulated the mTORC1/HIF-1α pathway through glucose transporter protein 1 (GLUT1), which involved in the malignant progression of bladder epithelial cells relying on glycolysis. In addition, pyruvate kinase M2 (PKM2) increased by arsenite reduced the protein expressions of succinate dehydrogenase (SDH) and fumarate hydratase (FH), leading to the accumulation of tumor metabolites of succinate and fumarate. Moreover, heat shock protein (HSP)90, functioning as a chaperone protein, stabilized PKM2 and thereby regulated the proliferation and aerobic glycolysis in arsenite treated SV-HUC-1 cells. Taken together, these results provide new insights into mTORC1/HIF-1α and PKM2 networks as critical molecular targets that contribute to the arsenic-induced malignant progression of bladder epithelial cells.

BRD4 plays an antiaging role in the senescence of renal tubular epithelial cells.

Background: Age-related kidney failure is often induced by a decrease in the bioavailability of tubular epithelial cells in elderly chronic kidney disease (CKD) patients. BRD4, an epigenetic regulator and a member of the bromodomain and extraterminal (BET) protein family, acts as a super-enhancer (SE) organizing and regulating genes expression during embryogenesis and cancer development. But the physiological function of BRD4 in normal cells has been less studied. This study aimed to research certain biological roles of BRD4 in the process of normal cell aging and discuss the potential mechanisms. Methods: In this study, we investigated the biological functions of BRD4 proteins in the aging of renal tubular cells. At first, we used a D-galactose (D-gal) and BRD4 inhibitor (Abbv-075) to replicate kidney senescence in vivo. D-gal and Abbv-075 were then used to measure the aging-related changes, such as changes in cell cycle, ß-galactosidase activity, cell migration, and p16 protein expression in vitro. At last, we knocked down and over-expressed BRD4 to investigate the aging-related physiological phenomena in renal tubular cells. Results: In vitro, D-gal treatment induced noticeable aging-related changes such as inducing cell apoptosis and cell cycle arrest, increasing ß-galactosidase activity as well as up-regulating p16 protein expression in primary human tubular epithelial cells. In the aging mice model, D-gal significantly induced renal function impairment and attenuated BRD4 protein expression. At the same time, the BRD4 inhibitor (Abbv-075) was able to mimic D-gal-induced cell senescence. In vivo, Abbv-075 also decreased kidney function and up-regulated p21 protein expression. When we knocked down the expression of BRD4, the senescence-associated ß-galactosidase (SA-ß-gal) activity increased dramatically, cell migration was inhibited, and the proportion of cells in the G0/G1 phase increased. Additionally, the knockdown also promoted the expression of the senescence-related proteins p16. When the renal tubular cells were overexpressed with BRD4, cell aging-related indicators were reversed in the D-gal-induced cell aging model. Conclusions: BRD4 appears to have an active role in the aging of renal tubular cells in vivo and in vitro. The findings also suggest that BRD4 inhibitors have potential nephrotoxic effects for oncology treatment. BRD4 may be a potential therapeutic biomarker and drug target for aging-related kidney diseases, which warrants additional studies.

The mammosphere-derived epithelial cell secretome modulates neutrophil functions in the bovine model.

Background: Innovative therapies against bacterial infections are needed. One approach is to focus on host-directed immunotherapy (HDT), with treatments that exploit natural processes of the host immune system. The goals of this type of therapy are to stimulate protective immunity while minimizing inflammation-induced tissue damage. We use non-traditional large animal models to explore the potential of the mammosphere-derived epithelial cell (MDEC) secretome, consisting of all bioactive factors released by the cells, to modulate host immune functions. MDEC cultures are enriched for mammary stem and progenitor cells and can be generated from virtually any mammal. We previously demonstrated that the bovine MDEC secretome, collected and delivered as conditioned medium (CM), inhibits the growth of bacteria in vitro and stimulates functions related to tissue repair in cultured endothelial and epithelial cells. Methods: The immunomodulatory effects of the bovine MDEC secretome on bovine neutrophils, an innate immune cell type critical for resolving bacterial infections, were determined in vitro using functional assays. The effects of MDEC CM on neutrophil molecular pathways were explored by evaluating the production of specific cytokines by neutrophils and examining global gene expression patterns in MDEC CM-treated neutrophils. Enzyme linked immunosorbent assays were used to determine the concentrations of select proteins in MDEC CM and siRNAs were used to reduce the expression of specific MDEC-secreted proteins, allowing for the identification of bioactive factors modulating neutrophil functions. Results: Neutrophils exposed to MDEC secretome exhibited increased chemotaxis and phagocytosis and decreased intracellular reactive oxygen species and extracellular trap formation, when compared to neutrophils exposed to control medium. C-X-C motif chemokine 6, superoxide dismutase, peroxiredoxin-2, and catalase, each present in the bovine MDEC secretome, were found to modulate neutrophil functions. Conclusion: The MDEC secretome administered to treat bacterial infections may increase neutrophil recruitment to the site of infection, stimulate pathogen phagocytosis by neutrophils, and reduce neutrophil-produced ROS accumulation. As a result, pathogen clearance might be improved and local inflammation and tissue damage reduced.

Spatio-Temporal Regulation of Notch Activation in Asymmetrically Dividing Sensory Organ Precursor Cells in Drosophila melanogaster Epithelium.

The Notch communication pathway, discovered in Drosophila over 100 years ago, regulates a wide range of intra-lineage decisions in metazoans. The division of the Drosophila mechanosensory organ precursor is the archetype of asymmetric cell division in which differential Notch activation takes place at cytokinesis. Here, we review the molecular mechanisms by which epithelial cell polarity, cell cycle and intracellular trafficking participate in controlling the directionality, subcellular localization and temporality of mechanosensitive Notch receptor activation in cytokinesis.

Cryptosporidium impacts epithelial turnover and is resistant to induced death of the host cell.

Infection with the apicomplexan parasite Cryptosporidium is a leading cause of diarrheal disease. Cryptosporidiosis is of particular importance in infants and shows a strong association with malnutrition, both as a risk factor and as a consequence. Cryptosporidium invades and replicates within the small intestine epithelial cells. This is a highly dynamic tissue that is developmentally stratified along the villus axis. New cells emerge from a stem cell niche in the crypt and differentiate into mature epithelial cells while moving toward the villus tip, where they are ultimately shed. Here, we studied the impact of Cryptosporidium infection on this dynamic architecture. Tracing DNA synthesis in pulse-chase experiments in vivo, we quantified the genesis and migration of epithelial cells along the villus. We found proliferation and epithelial migration to be elevated in response to Cryptosporidium infection. Infection also resulted in significant cell loss documented by imaging and molecular assays. Consistent with these observations, single-cell RNA sequencing of infected intestines showed a gain of young and a loss of mature cells. Interestingly, enhanced epithelial cell loss was not a function of enhanced apoptosis of infected cells. To the contrary, Cryptosporidium-infected cells were less likely to be apoptotic than bystanders, and experiments in tissue culture demonstrated that infection provided enhanced resistance to chemically induced apoptosis to the host but not bystander cells. Overall, this study suggests that Cryptosporidium may modulate cell apoptosis and documents pronounced changes in tissue homeostasis due to parasite infection, which may contribute to its long-term impact on the developmental and nutritional state of children. IMPORTANCE: The intestine must balance its roles in digestion and nutrient absorption with the maintenance of an effective barrier to colonization and breach by numerous potential pathogens. An important component of this balance is its constant turnover, which is modulated by a gain of cells due to proliferation and loss due to death or extrusion. Here, we report that Cryptosporidium infection changes the dynamics of this process increasing both gain and loss of enterocytes speeding up the villus elevator. This leads to a much more immature epithelium and a reduction of the number of those cells typically found toward the villus apex best equipped to take up key nutrients including carbohydrates and lipids. These changes in the cellular architecture and physiology of the small intestine may be linked to the profound association between cryptosporidiosis and malnutrition.

Single-Cell RNA Sequencing Reveals Transcriptional Signatures and Cell-Cell Communication in Diabetic Retinopathy.

BACKGROUND: Diabetic retinopathy (DR) is a major cause of vision loss in workingage individuals worldwide. Cell-to-cell communication between retinal cells and retinal pigment epithelial cells (RPEs) in DR is still unclear, so this study aimed to generate a single-cell atlas and identify receptor‒ligand communication between retinal cells and RPEs. METHODS: A mouse single-cell RNA sequencing (scRNA-seq) dataset was retrieved from the GEO database (GSE178121) and was further analyzed with the R package Seurat. Cell cluster annotation was performed to further analyze cell‒cell communication. The differentially expressed genes (DEGs) in RPEs were explored through pathway enrichment analysis and the protein‒ protein interaction (PPI) network. Core genes in the PPI were verified by quantitative PCR in ARPE-19 cells. RESULTS: We observed an increased proportion of RPEs in STZ mice. Although some overall intercellular communication pathways did not differ significantly in the STZ and control groups, RPEs relayed significantly more signals in the STZ group. In addition, THBS1, ITGB1, COL9A3, ITGB8, VTN, TIMP2, and FBN1 were found to be the core DEGs of the PPI network in RPEs. qPCR results showed that the expression of ITGB1, COL9A3, ITGB8, VTN, TIMP2, and FBN1 was higher and consistent with scRNA-seq results in ARPE-19 cells under hyperglycemic conditions. CONCLUSION: Our study, for the first time, investigated how signals that RPEs relay to and from other cells underly the progression of DR based on scRNA-seq. These signaling pathways and hub genes may provide new insights into DR mechanisms and therapeutic targets.

Nintedanib Mitigates Radiation-Induced Pulmonary Fibrosis by Suppressing Epithelial Cell Inflammatory Response and Inhibiting Fibroblast-to-Myofibroblast Transition.

Radiation-induced pulmonary fibrosis (RIPF) represents a serious complication observed in individuals undergoing thoracic radiation therapy. Currently, effective interventions for RIPF are unavailable. Prior research has demonstrated that nintedanib, a Food and Drug Administration (FDA)-approved anti-fibrotic agent for idiopathic pulmonary fibrosis, exerts therapeutic effects on chronic fibrosing interstitial lung disease. This research aimed to investigate the anti-fibrotic influences of nintedanib on RIPF and reveal the fundamental mechanisms. To assess its therapeutic impact, a mouse model of RIPF was established. The process involved nintedanib administration at various time points, both prior to and following thoracic radiation. In the RIPF mouse model, an assessment was conducted on survival rates, body weight, computed tomography features, histological parameters, and changes in gene expression. In vitro experiments were performed to discover the mechanism underlying the therapeutic impact of nintedanib on RIPF. Treatment with nintedanib, administered either two days prior or four weeks after thoracic radiation, significantly alleviated lung pathological changes, suppressed collagen deposition, and improved the overall health status of the mice. Additionally, nintedanib demonstrated significant mitigation of radiation-induced inflammatory responses in epithelial cells by inhibiting the PI3K/AKT and MAPK signaling pathways. Furthermore, nintedanib substantially inhibited fibroblast-to-myofibroblast transition by suppressing the TGF-ß/Smad and PI3K/AKT/mTOR signaling pathways. These findings suggest that nintedanib exerts preventive and therapeutic effects on RIPF by modulating multiple targets instead of a single anti-fibrotic pathway and encourage the further clinical trials to determine the efficacy of nintedanib in patients with RIPF.

Mechanisms underlying the direct programming of mouse embryonic fibroblasts to thymic epithelial cells by FOXN1.

Thymic epithelial cells (TECs) are a critical functional component of the thymus's ability to generate T cells for the adaptive immune system in vertebrates. However, no in vitro system for studying TEC function exists. Overexpressing the transcription factor FOXN1 initiates transdifferentiation of fibroblasts into TEC-like cells (iTECs) that support T cell differentiation in culture or after transplant. In this study, we characterized iTEC programming at the cellular and molecular level to determine how it proceeds and identified mechanisms that can be targeted for improving this process. These data showed that iTEC programming consisted of discrete gene expression changes that differed early and late in the process, and that iTECs upregulated markers of both cortical and medullary TEC (cTEC and mTEC) lineages. We demonstrated that promoting proliferation enhanced iTEC generation, and that Notch inhibition allowed induction of mTEC differentiation. Finally, we showed that MHCII expression was the major difference between iTECs and fetal TECs. MHCII expression was improved by co-culturing iTECs with fetal double-positive T-cells. This study supports future efforts to improve iTEC generation for both research and translational uses.

The role and mechanism of action of miR-483-3p in mediating the effects of IGF-1 on human renal tubular epithelial cells induced by high glucose.

This study aimed to elucidate the influence of miR-483-3p on human renal tubular epithelial cells (HK-2) under high glucose conditions and to understand its mechanism. Human proximal tubular epithelial cells (HK-2) were exposed to 50 mmol/L glucose for 48 h to establish a renal tubular epithelial cell injury model, denoted as the high glucose group (HG group). Cells were also cultured for 48 h in a medium containing 5.5 mmol/L glucose, serving as the low glucose group. Transfection was performed in various groups: HK-2 + low glucose (control group), high glucose (50 mM) (HG group), high glucose + miR-483-3p mimics (HG + mimics group), high glucose +miR-483-3p inhibitor (HG + inhibitor group), and corresponding negative controls. Real-time quantitative polymerase chain reaction (qPCR) assessed the mRNA expression of miR-483-3p, bax, bcl-2, and caspase-3. Western blot determined the corresponding protein levels. Proliferation was assessed using the CCK-8 assay, and cell apoptosis was analyzed using the fluorescence TUNEL method. Western blot and Masson's staining were conducted to observe alterations in cell fibrosis post miR-483-3p transfection. Furthermore, a dual-luciferase assay investigated the targeting relationship between miR-483-3p and IGF-1. The CCK8 assay demonstrated that the HG + mimics group inhibited HK-2 cell proliferation, while the fluorescent TUNEL method revealed induced cell apoptosis in this group. Conversely, the HG + inhibitor group promoted cell proliferation and suppressed cell apoptosis. The HG + mimics group upregulated mRNA and protein expression of pro-apoptotic markers (bax and caspase-3), while downregulating anti-apoptotic marker (bcl-2) expression. In contrast, the HG + inhibitor group showed opposite effects. Collagen I and FN protein levels were significantly elevated in the HG + mimics group compared to controls (P < 0.05). Conversely, in the HG + inhibitor group, the protein expression of Collagen I and FN was notably reduced compared to the HG group (P < 0.05). The dual luciferase reporter assay confirmed that miR-483-3p could inhibit the luciferase activity of IGF-1's 3'-UTR region (P < 0.05). miR-483-3p exerts targeted regulation on IGF-1, promoting apoptosis and fibrosis in renal tubular epithelial cells induced by high glucose conditions.

Membranous translocation of murine double minute 2 promotes the increased renal tubular immunogenicity in ischemia-reperfusion-induced acute kidney injury.

Kidneys from donors with prolonged warm and cold ischemia are prone to post-transplant T cell-mediated rejection (TCMR) due to ischemia-reperfusion injury (IRI). However, the precise mechanisms still remain obscure. Renal tubular epithelial cells (TECs) are the main target during IRI. Meanwhile, we reported previously that murine double minute 2 (MDM2) actively participates in TEC homeostasis during IRI. In this study, we established a murine model of renal IRI and a cell model of hypoxia/reoxygenation by culturing immortalized rat renal proximal tubule cells (NRK-52E) in a hypoxic environment for different time points followed by 24 hours of reoxygenation or incubating NRK-52E cells in a chemical anoxia/recovery environment. We found that during renal IRI, MDM2 expression increased on the membrane of TECs and aggregated mainly on the basolateral side. This process was accompanied by a reduction of a transmembrane protein programmed death-ligand 1 (PD-L1), a co-inhibitory second signal for T cells in TECs. By using mutant plasmids of MDM2 to anchor MDM2 on the cell membrane or nuclei, we found that the upregulation of membrane MDM2 could promote the ubiquitination of PD-L1 and lead to its ubiquitination-proteasome degradation. Lastly, we set up a co-culture system of TECs and CD4+ T cells in vitro; our results revealed that the immunogenicity of TECs was enhanced during IRI. In conclusion, our findings suggest that the increased immunogenicity of TECs during IRI may be related to ubiquitinated degradation of PD-L1 by increased MDM2 on the cell membrane, which consequently results in T cell activation and TCMR.

Nemo mRNA vaccination improves airway barrier function in mice with airway allergy.

Epithelial barrier dysfunction plays an important role in the pathogenesis of Th2 bias. The mechanism requires further clarification. NEMO is associated with regulating apoptotic activities in the cell. The purpose of this study is to investigate the role of insufficient Nemo signals in developing Th2 bias in the respiratory tract. Nemof/fEpcam-Cre mice (A mouse strain carrying NEMO-deficient epithelial cells. NemoKO mice, in short) was generated. An airway Th2 bias mouse model was established with the ovalbumin/alum protocol. The NemoKO mice exhibited spontaneous airway Th2 bias. Respiratory tract epithelial barrier integrity was compromised in NemoKO mice. Apoptosis was found in approximately 10% of the epithelial cells of the respiratory tract in NemoKO mice. The reconstruction of the Nemo expression restored homeostasis within the epithelial barrier of the airways. Restoration of Nemo gene expression in epithelial cells by Nemo mRNA vaccination alleviated Th2 bias in mice with airway allergy. To sum up, NEMO plays an important role in maintaining the integrity of the epithelial barrier in the respiratory tract. Administration of NEMO mRNA vaccines can restore epithelial barrier functions and alleviate Th2 bias in the airways.