Teatime: epigallocatechin gallate targets fibroblast-epithelial cell crosstalk to combat lung fibrosis.

Epigallocatechin gallate (EGCG) is a polyphenol plant metabolite abundant in tea that has demonstrated antifibrotic properties in the lung. In this issue of the JCI, Cohen, Brumwell, and colleagues interrogated the mechanistic action of EGCG by investigating lung biopsies of patients with mild interstitial lung disease (ILD) who had undergone EGCG treatment. EGCG targeted the WNT inhibitor SFRP2, which was enriched in fibrotic fibroblasts and acted as a TGF-ß target, with paracrine effects leading to pathologic basal metaplasia of alveolar epithelial type 2 cells. This study emphasizes the epithelial-mesenchymal trophic unit as a central signaling hub in lung fibrosis. Understanding and simultaneous targeting of interlinked signaling pathways, such as TGF-ß and WNT, paves the road for future treatment options for pulmonary fibrosis.

HSPA12A promotes c-Myc lactylation-mediated proliferation of tubular epithelial cells to facilitate renal functional recovery from kidney ischemia/reperfusion injury.

Proliferation of renal tubular epithelial cells (TEC) is essential for restoring tubular integrity and thereby to support renal functional recovery from kidney ischemia/reperfusion (KI/R) injury. Activation of transcriptional factor c-Myc promotes TEC proliferation following KI/R; however, the mechanism regarding c-Myc activation in TEC is incompletely known. Heat shock protein A12A (HSPA12A) is an atypic member of HSP70 family. In this study, we found that KI/R decreased HSPA12A expression in mouse kidneys and TEC, while ablation of HSPA12A in mice impaired TEC proliferation and renal functional recovery following KI/R. Gain-of-functional studies demonstrated that HSPA12A promoted TEC proliferation upon hypoxia/reoxygenation (H/R) through directly interacting with c-Myc and enhancing its nuclear localization to upregulate expression of its target genes related to TEC proliferation. Notably, c-Myc was lactylated in TEC after H/R, and this lactylation was enhanced by HSPA12A overexpression. Importantly, inhibition of c-Myc lactylation attenuated the HSPA12A-induced increases of c-Myc nuclear localization, proliferation-related gene expression, and TEC proliferation. Further experiments revealed that HSPA12A promoted c-Myc lactylation via increasing the glycolysis-derived lactate generation in a Hif1α-dependent manner. The results unraveled a role of HSPA12A in promoting TEC proliferation and facilitating renal recovery following KI/R, and this role of HSPA12A was achieved through increasing lactylation-mediated c-Myc activation. Therefore, targeting HSPA12A in TEC might be a viable strategy to promote renal functional recovery from KI/R injury in patients.

Malate dehydrogenase-2 inhibition shields renal tubular epithelial cells from anoxia-reoxygenation injury by reducing reactive oxygen species.

Ischemia-reperfusion (I-R) injury is the most common cause of acute kidney injury. In experiments involving primary human renal proximal tubular epithelial cells (RPTECs) exposed to anoxia-reoxygenation, we explored the hypothesis that mitochondrial malate dehydrogenase-2 (MDH-2) inhibition redirects malate metabolism from the mitochondria to the cytoplasm, towards the malate-pyruvate cycle and reversed malate-aspartate shuttle. Colorimetry, fluorometry, and western blotting showed that MDH2 inhibition accelerates the malate-pyruvate cycle enhancing cytoplasmic NADPH, thereby regenerating the potent antioxidant reduced glutathione. It also reversed the malate-aspartate shuttle and potentially diminished mitochondrial reactive oxygen species (ROS) production by transferring electrons, in the form of NADH, from the mitochondria to the cytoplasm. The excessive ROS production induced by anoxia-reoxygenation led to DNA damage and protein modification, triggering DNA damage and unfolded protein response, ultimately resulting in apoptosis and senescence. Additionally, ROS induced lipid peroxidation, which may contribute to the process of ferroptosis. Inhibiting MDH-2 proved effective in mitigating ROS overproduction during anoxia-reoxygenation, thereby rescuing RPTECs from death or senescence. Thus, targeting MDH-2 holds promise as a pharmaceutical strategy against I-R injury.

The presence of cancer-associated fibroblast in breast cavity side margins is in correlation with the expression of oncoproteins by adjacent epithelial cells: a new era in cancerous potential.

PURPOSE: Cancer-associated fibroblasts (CAFs) are one of the most critical cells in the tumor environment, with crucial roles in cancer progression and metastasis. Due to Field-Effect phenomena (also called field cancerization), the adjacent cavity side area of the margin is histologically normal, but it has been entered into neoplastic transformation due to MCT4 and MCT1 pathways activated by H2O2/ROS oxidative stress agents secreted by CAF in adjacent tumor bed microenvironment. This paper specifically focused on the role of cancer-associated fibroblast in breast tumor beds and its correlation with the presence of scattered cancer cells or onco-protein-activated cells (may be high risk but not completely transformed cancer cells) in the cavity side margins. METHODS: In this study, the glycolytic behavior of non-tumoral cavity side margins was examined using carbon nanotube-based electrochemical biosensors integrated into a cancer diagnostic probe. This method enabled the detection of CAF accumulation sites in non-cancerous neighboring tissues of tumors, with a correlation to CAF concentration. Subsequently, RT-PCR, fluorescent, histopathological, and invasion assays were conducted on hyperglycolytic lesions to explore any correlation between the abundance of CAFs and the electrochemical responses of the non-cancerous tissues surrounding the tumor, as well as their neoplastic potential. RESULTS: We observed overexpression of cancer-associated transcriptomes as well as the presence and hyperactivation of CAFs in cavity-side regions in which glycolytic metabolism was recorded, independent of the histopathological state of the lesion. At mean 70.4%, 66.7%, 70.4%, and 44.5% increments were observed in GLUT-1, MMP-2, N-cadherin, and MMP-9 transcriptomes by highly glycolytic but histologically cancer-free expression samples in comparison with negative controls (histologically non-cancer lesions with low glycolytic behavior). CONCLUSION: The presence of CAFs is correlated with the presence of high glycolytic metabolism in the cavity margin lesion, high ROS level in the lesion, and finally aggressive cancer-associated proteins (such as MMP2, …) in the margin while these metabolomes, molecules, and proteins are absent in the margins with negatively scored CDP response and low ROS level. So, it seems that when we observe CAFs in glycolytic lesions with high ROS levels, some high-risk epithelial breast cells may exist while no histological trace of cancer cells was observed. Further research on CAFs could provide valuable insights into the local recurrence of malignant breast diseases. Hence, real-time sensors can be used to detect and investigate CAFs in the non-tumoral regions surrounding tumors in cancer patients, potentially aiding in the prevention of cancer recurrence.

Artemisinin conferred cytoprotection to human retinal pigment epithelial cells exposed to amiodarone-induced oxidative insult by activating the CaMKK2/AMPK/Nrf2 pathway.

BACKGROUND: Ocular toxicity is a severe adverse effect that limits the chronic clinical use of the antiarrhythmic drug amiodarone. Here, we aimed to evaluate the cytoprotective effect of artemisinin and explore the potential signalling pathways in human retinal pigment epithelial (RPE) cell cultures. METHODS: D407 cell cultures were exposed to amiodarone and the impact of artemisinin was evaluated. The key parameters included lactate dehydrogenase (LDH) release, intracellular reactive oxygen species (ROS) generation, and the mitochondrial membrane potential (MMP). We also assessed the protein levels of cleaved caspase-3, cleaved poly (ADP-ribose) polymerase (PARP), phosphorylated adenosine monophosphate-activated protein kinase (AMPK)ɑ (p-AMPK), calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2), and nuclear factor erythroid 2-related factor 2 (Nrf2). RESULTS: Artemisinin reduced the cytotoxicity induced by amiodarone, as reflected by decreased LDH release, ROS generation, and MMP disruption. Additionally, artemisinin increased p-AMPK, CaMKK2, and Nrf2 protein levels. Inhibition of AMPK, CaMKK2, or Nrf2 abolished the cytoprotective effect of artemisinin. AMPK activation and Nrf2 knockdown further supported its protective role. CONCLUSIONS: Artemisinin protected RPE cells from amiodarone-induced damage via the CaMKK2/AMPK/Nrf2 pathway. The in vivo experiments in mice confirmed its efficacy in preventing retinal injury caused by amiodarone. These results suggest that an artemisinin-based eye formulation could be repurposed for treating amiodarone-induced ocular toxicity.

P4HB regulates the TGFß/SMAD3 signaling pathway through PRMT1 to participate in high glucose-induced epithelial-mesenchymal transition and fibrosis of renal tubular epithelial cells.

BACKGROUND: Diabetic nephropathy (DN) is a common complication of diabetes mellitus, and Prolyl 4-Hydroxylase Subunit Beta (P4HB) expression is increased in high glucose (HG)-induced renal tubular epithelial cells (TECs). But it's role in HG-induced TECs remains to be elucidated. METHODS: The HK-2 cells were induced using HG and transfected with SiRNA-P4HB. DCFH-DA staining was utilized for the detection of cellular levels of ROS. WB and immunofluorescence were utilized to detect the expression of P4HB, epithelial-mesenchymal transition (EMT), fibrosis, and TGFß/SMAD3-related proteins in HK-2 cells. Online databases were utilized for predicting the interaction target of P4HB, and immunoprecipitation (IP) experiments were employed to validate the binding of P4HB with the target. SiRNA and overexpression vectors of target gene were used to verify the mechanism of action of P4HB. RESULTS: HG induced an increase in the expression of P4HB and TGFß, p-SMAD3, and ROS in HK-2 cells. Furthermore, HG downregulated the expression of E-cadherin and upregulated the expression of N-cadherin, Vimentin, α-SMA, Fibronectin, Collagen IV, SNAIL, and SLUG in HK-2 cells. Interfering with P4HB significantly reversed the expression of these proteins. Database predictions and IP experiments showed that P4HB interacts with PRMT1, and the expression of PRMT1 was increased in HG-induced HK-2 cells. Interfering with PRMT1 inhibited the changes in expression of EMT and fibrosis related proteins induced by HG. However, overexpression of PRMT1 weakened the regulatory effect of P4HB interference on the EMT, fibrosis, and TGFß/SMAD3-related proteins in HK-2 cells. CONCLUSION: P4HB regulated the TGFß/SMAD3 signaling pathway through PRMT1 and thus participates in HG-induced EMT and fibrosis in HK-2 cells.

A lipidomic study on the lens epithelial cells of patients with age related cataracts.

Age related cataracts (ARC) represent the main reason for blindness globally. The lens epithelial cells (LECs) participate not only in the metabolism of many substances in the lens but also in maintaining lens transparency. This study used lipidomics to investigate the metabolic differences in LECs of ARC patients with different severity, aiming at identifying potential metabolic biomarkers of ARC. Patients diagnosed with ARC and underwent cataract surgery at Shanghai Tongren Hospital were selected to participate in this study, which were classified as mild ARC group and severe ARC group. During their cataract surgery, anterior lens capsules(LCs) containing LECs were obtained. The lipidomics of LECs were analyzed using the liquid chromatography­mass spectrometry (LC-MS). Potential pathways of lipids were searched for using databases such as the Kyoto Encyclopedia of Genes and Genomes (KEGG) and MetaboAnalyst platform. In LEC lipids, 26 lipids have been identified as potential biomarkers between mild ARC and severe ARC, with AUC values of 0.67-0.94. The pathway analysis results revealed that the Glycerophospholipid (GPL) metabolism was significantly influenced, indicating that these metabolic markers contribute significantly to regulating this pathway. The LEC metabolic spectrum demonstrates a proficient ability to differentiate between patients with varying levels of cataracts. Herein, we have successfully identified potential metabolic biomarkers and pathways that have proven to be valuable in enhancing our understanding of ARC pathogenesis. The finding has translational value for developing new cataract treatment methods in the future.

Osteopontin deletion attenuates cyst growth but exacerbates fibrosis in mice with cystic kidney disease.

Osteopontin (OPN) is a multi-functional glycoprotein that coordinates the innate immune response, prevents nanocrystal formation in renal tubule fluid, and is a biomarker for kidney injury. OPN expression is markedly increased in cystic epithelial cells of polycystic kidney disease (PKD) kidneys; however, its role in PKD progression remains unclear. We investigated the in vitro effects of recombinant OPN on the proliferation of tubular epithelial cells from PKD and normal human kidneys and in vivo effects of OPN deletion on kidney cyst formation, fibrosis, and mineral metabolism in pcy/pcy mice, a non-orthologous model of autosomal-dominant PKD. In vitro studies revealed that OPN enhanced the proliferation of PKD cells but had no effect on normal kidney cells. Deletion of OPN in pcy/pcy mice significantly reduced kidney cyst burden; however, this was accompanied by increased fibrosis and no change in kidney function. The loss of OPN had no effect on kidney macrophage numbers, cyst epithelial cell proliferation, or apoptosis. Furthermore, there was no difference in kidney mineral deposition or mineral metabolism parameters between pcy/pcy mice with and without OPN expression. Global deletion of OPN reduced kidney cyst burden, while paradoxically exacerbating kidney fibrosis in mice with cystic kidney disease.

PARVB deficiency alleviates cisplatin-induced tubular injury by inhibiting TAK1 signaling.

Cisplatin-induced renal tubular injury largely restricts the wide-spread usage of cisplatin in the treatment of malignancies. Identifying the key signaling pathways that regulate cisplatin-induced renal tubular injury is thus clinically important. PARVB, a focal adhesion protein, plays a crucial role in tumorigenesis. However, the function of PARVB in kidney disease is largely unknown. To investigate whether and how PARVB contributes to cisplatin-induced renal tubular injury, a mouse model (PARVB cKO) was generated in which PARVB gene was specifically deleted from proximal tubular epithelial cells using the Cre-LoxP system. In this study, we found depletion of PARVB in proximal tubular epithelial cells significantly attenuates cisplatin-induced renal tubular injury, including tubular cell death and inflammation. Mechanistically, PARVB associates with transforming growth factor-ß-activated kinase 1 (TAK1), a central regulator of cell survival and inflammation that is critically involved in mediating cisplatin-induced renal tubular injury. Depletion of PARVB promotes cisplatin-induced TAK1 degradation, inhibits TAK1 downstream signaling, and ultimately alleviates cisplatin-induced tubular cell damage. Restoration of PARVB or TAK1 in PARVB-deficient cells aggravates cisplatin-induced tubular cell injury. Finally, we demonstrated that PARVB regulates TAK1 protein expression through an E3 ligase ITCH-dependent pathway. PARVB prevents ITCH association with TAK1 to block its ubiquitination. Our study reveals that PARVB deficiency protects against cisplatin-induced tubular injury through regulation of TAK1 signaling and indicates targeting this pathway may provide a novel therapeutic strategy to alleviate cisplatin-induced kidney damage.

Deficiency of geranylgeranyl biphosphate synthase in kidney tubules causes cystic kidney disease.

Polycystic kidney disease (PKD) is a common hereditary kidney disease. Although PKD occurrence is associated with certain gene mutations, its onset regulatory mechanisms are still not well understood. Here, we first report that the key enzyme geranylgeranyl diphosphate synthase (GGPPS) is specifically expressed in renal tubular epithelial cells of mouse kidneys. We aimed to explore the role of GGPPS in PKD. In this study, we established a Ggppsfl/fl:Cdh16cre mouse model and compared its phenotype with that of wild-type mice. A Ggpps-downregulation HK2 cell model was also used to further determine the role of GGPPS. We found that GGPPS was specifically expressed in renal tubular epithelial cells of mouse kidneys. Its expression also increased with age. Low GGPPS expression was observed in human ADPKD tissues. In the Ggppsfl/fl:Cdh16cre mouse model, Ggpps deletion in renal tubular epithelial cells induced the occurrence and development of renal tubule cystic dilation and caused the death of mice after birth due to abnormal renal function. Enhanced proliferation of cyst-lining epithelial cells was also observed after the knockout of Ggpps. These processes were related to the increased rate of Rheb on membrane/cytoplasm and hyperactivation of mTORC1 signaling. In conclusion, the deficiency of GGPPS in kidney tubules induced the formation of renal cysts. It may play a critical role in PKD pathophysiology. A novel therapeutic strategy could be designed according to this work.

Single-cell RNA sequencing in endometrial cancer: exploring the epithelial cells and the microenvironment landscape.

Single-cell RNA sequencing (scRNA-seq) technology has emerged as a powerful tool for dissecting cellular heterogeneity and understanding the intricate biology of diseases, including cancer. Endometrial cancer (EC) stands out as the most prevalent gynecological malignancy in Europe and the second most diagnosed worldwide, yet its cellular complexity remains poorly understood. In this review, we explore the contributions of scRNA-seq studies to shed light on the tumor cells and cellular landscape of EC. We discuss the diverse tumoral and microenvironmental populations identified through scRNA-seq, highlighting the implications for understanding disease progression. Furthermore, we address potential limitations inherent in scRNA-seq studies, such as technical biases and sample size constraints, emphasizing the need for larger-scale research encompassing a broader spectrum of EC histological subtypes. Notably, a significant proportion of scRNA-seq analyses have focused on primary endometrioid carcinoma tumors, underscoring the need to incorporate additional histological and aggressive types to comprehensively capture the heterogeneity of EC. By critically evaluating the current state of scRNA-seq research in EC, this review underscores the importance of advancing towards more comprehensive studies to accelerate our understanding of this complex disease.

Missing regulatory effects and viral triggers explored for childhood-onset asthma.

Missing regulatory effects of asthma genetic risks might be hidden within specific cell states. In this issue of Cell Genomics, Djeddi et al.1 uncover how airway epithelial cells, when activated by rhinovirus, influence genetic susceptibility to childhood-onset asthma, and this preview emphasizes the need to address these missing regulatory effects across diverse cell states.

Identification and multimodal characterization of a specialized epithelial cell type associated with Crohn's disease.

Crohn's disease (CD) is a complex chronic inflammatory disorder with both gastrointestinal and extra-intestinal manifestations associated immune dysregulation. Analyzing 202,359 cells from 170 specimens across 83 patients, we identify a distinct epithelial cell type in both terminal ileum and ascending colon (hereon as 'LND') with high expression of LCN2, NOS2, and DUOX2 and genes related to antimicrobial response and immunoregulation. LND cells, confirmed by in-situ RNA and protein imaging, are rare in non-IBD controls but expand in active CD, and actively interact with immune cells and specifically express IBD/CD susceptibility genes, suggesting a possible function in CD immunopathogenesis. Furthermore, we discover early and late LND subpopulations with different origins and developmental potential. A higher ratio of late-to-early LND cells correlates with better response to anti-TNF treatment. Our findings thus suggest a potential pathogenic role for LND cells in both Crohn's ileitis and colitis.

Vitamin D ameliorates particulate matter induced mitochondrial damages and calcium dyshomeostasis in BEAS-2B human bronchial epithelial cells.

BACKGROUND: Mitochondria is prone to oxidative damage by endogenous and exogenous sources of free radicals, including particulate matter (PM). Given the role of mitochondria in inflammatory disorders, such as asthma and chronic obstructive pulmonary disease, we hypothesized that supplementation of vitamin D may play a protective role in PM-induced mitochondrial oxidative damages of human bronchial epithelial BEAS-2B cells. METHODS: BEAS-2B cells were pretreated with 1,25(OH)2D3, an active form of vitamin D, for 1 h prior to 24-hour exposure to PM (SRM-1648a). Oxidative stress was measured by flow cytometry. Mitochondrial functions including mitochondrial membrane potential, ATP levels, and mitochondrial DNA copy number were analyzed. Additionally, mitochondrial ultrastructure was examined using transmission electron microscopy. Intracellular and mitochondrial calcium concentration changes were assessed using flow cytometry based on the expression of Fluo-4 AM and Rhod-2 AM, respectively. Pro-inflammatory cytokines, including IL-6 and MCP-1, were quantified using ELISA. The expression levels of antioxidants, including SOD1, SOD2, CAT, GSH, and NADPH, were determined. RESULTS: Our findings first showed that 24-hour exposure to PM led to the overproduction of reactive oxygen species (ROS) derived from mitochondria. PM-induced mitochondrial oxidation resulted in intracellular calcium accumulation, particularly within mitochondria, and alterations in mitochondrial morphology and functions. These changes included loss of mitochondrial membrane integrity, disarrayed cristae, mitochondrial membrane depolarization, reduced ATP production, and increased mitochondrial DNA copy number. Consequently, PM-induced mitochondrial damage triggered the release of certain inflammatory cytokines, such as IL-6 and MCP-1. Similar to the actions of mitochondrial ROS inhibitor MitoTEMPO, 1,25(OH)2D3 conferred protective effects on mtDNA alterations, mitochondrial damages, calcium dyshomeostasis, thereby decreasing the release of certain inflammatory cytokines. We found that greater cellular level of 1,25(OH)2D3 upregulated the expression of enzymatic (SOD1, SOD2, and CAT) and non-enzymatic (GSH and NADPH) antioxidants to modulate cellular redox homeostasis. CONCLUSION: Our study provides new evidence that 1,25(OH)2D3 acts as an antioxidant, enhancing BEAS-2B antioxidant responses to regulate mitochondrial ROS homeostasis and mitochondrial function, thereby enhancing epithelial defense against air pollution exposure.

Genotoxicity and Micronucleus Formation as a Result of Panoramic Radiography in Epithelial Cells of the Buccal Mucosa: A Cross-sectional Study in Adults.

OBJECTIVES: To determine the genetic effects of panoramic radiography on the epithelial cells of the buccal mucosa by examining the micronucleus formation in these cells. MATERIALS AND METHODS: In this cross-sectional study, exfoliative cytology samples were prepared from the buccal mucosa of 36 patients immediately before and 10 days after panoramic radiography. The samples were prepared using liquid-based cytology with Papanicolaou staining. The slides were simultaneously evaluated by two expert pathologists and the ratio of the number of cells with micronuclei to the total number of cells on the slide was reported as a percentage. Data analysis was done using paired-samples T test, Pearson's correlation coefficient, and covariance analysis (α = 0.05). RESULTS: The study sample consisted of 24 (66.67%) males and 12 females (33.33%) with a mean (SD) age of 27.36 (8.19) years. The frequency of cells with micronucleus before and after panoramic radiography was not statistically different (p = 0.468). Additionally, the frequency of micronucleated cells was not correlated with age (p = 0.737) and sex (p = 0.211). CONCLUSION: Panoramic exposure slightly increased the frequency of cells with micronucleus in epithelial cells of the buccal mucosa. However, this increase was not statistically significant.

Gamma-glutamyl transferase secreted by Helicobacter pylori promotes the development of gastric cancer by affecting the energy metabolism and histone methylation status of gastric epithelial cells.

BACKGROUND: Helicobacter pylori (H. pylori) infection is critical in the development and occurrence of gastric cancer. H. pylori secretes gamma-glutamyl transferase (GGT), which affects energy metabolism and histone methylation in mesenchymal stem cells. However, its effect on human gastric epithelial cells remains unclear. This study aimed to investigate the effects of GGT on energy metabolism and histone methylation in gastric epithelial cells and determine its role in the development and progression of H. pylori-induced gastric cancer. METHODS: A GGT knockout H. pylori strain and mouse gastric cancer model were constructed, and alpha-ketoglutarate (α-KG) was added. The underlying mechanism was investigated using proteomics, immunohistochemistry, Western blotting, and other experimental assays. RESULTS: H. pylori can colonize the host's stomach and destroy the gastric epithelium. GGT secreted by H. pylori decreased the concentration of glutamine in the stomach and increased H3K9me3 and H3K27me3 expression, which promoted the proliferation and migration of gastric epithelial cells. Additionally, α-KG reversed this effect. GGT increased the tumorigenic ability of nude mice. GGT, secreted by H. pylori, promoted the expression of ribosomal protein L15 (RPL15), while GGT knockout and supplementation with α-KG and trimethylation inhibitors reduced RPL15 expression and Wnt signaling pathway expression. CONCLUSIONS: H. pylori secreted GGT decreased the expression of glutamine and α-KG in gastric epithelial cells, increased the expression of histones H3K9me3 and H3K27me3, and activated the Wnt signaling pathway through RPL15 expression, ultimately changing the biological characteristics of the gastric epithelium and promoting the occurrence of gastric cancer. Altered energy metabolism and histone hypermethylation are important factors involved in this process.

Targeting alternative splicing of fibronectin in human renal proximal tubule epithelial cells with antisense oligonucleotides to reduce EDA+ fibronectin production and block an autocrine loop that drives renal fibrosis.

TGFß1 is a powerful regulator of fibrosis; secreted in a latent form, it becomes active after release from the latent complex. During tissue fibrosis, the EDA + isoform of cellular fibronectin is overexpressed. In pulmonary fibrosis it has been proposed that the fibronectin splice variant including an EDA domain (FN EDA+) activates latent TGFß. Our work investigates the potential of blocking the 'splicing in' of EDA with antisense oligonucleotides to inhibit TGFß1-induced EDA + fibronectin and to prevent the cascade of events initiated by TGFß1 in human renal proximal tubule cells (PTEC). Human primary PTEC were treated with TGFß1 for 48 h, medium removed and the cells transfected with RNase H-independent antisense oligonucleotides (ASO) designed to block EDA exon inclusion (ASO5). The efficacy of ASO to block EDA exon inclusion was assessed by EDA + fibronectin RNA and protein expression; the expression of TGFß, αSMA (α smooth muscle actin), MMP2 (matrix metalloproteinse-2), MMP9 (matrix metalloproteinse-9), Collagen I, K Cadherin and connexin 43 was analysed. Targeting antisense oligonucleotides designed to block EDA exon inclusion in fibronectin pre mRNA were effective in reducing the amount of TGFß1 -induced cellular EDA + fibronectin RNA and secreted EDA + fibronectin protein (assessed by western immunoblotting and immunocytochemistry) in human proximal tubule cells in an in vitro cell culture model. The effect was selective for EDA + exon with no effect on EDB + fibronectin RNA and total fibronectin mRNA. Exogenous TGFß1 induced endogenous TGFß, αSMA, MMP2, MMP9 and Col I mRNA. TGFß1 treatment for 48h reduced the expression of K-Cadherin and increased the expression of connexin-43. These TGFß1-induced pro-fibrotic changes were attenuated by ASO5 treatment. 48 h after the removal of exogenous TGFß, further increases in αSMA, MMP2, MMP9 was observed; ASO5 significantly inhibited this subsequent increase. ASO5 treatment also significantly inhibited ability of the cell culture medium harvested at the end of the experiment (96h) to stimulate SMAD3 reporter cells. The role of endogenous TGFß1 was confirmed by the use of a TGFß receptor inhibitor. Our results demonstrate a critical role of FN EDA+ in a cycle of TGFß driven pro-fibrotic responses in human PTEC and blocking its production with ASO technology offers a potential therapy to interrupt this vicious circle and hence limit the progression of renal fibrosis.

LincRNA-p21/AIF-1/CMPK2/NLRP3 pathway promoted inflammation, autophagy and apoptosis of human tubular epithelial cell induced by urate via exosomes.

Urate nephropathy, a common complication of hyperuricemia, has garnered increasing attention worldwide. However, the exact pathogenesis of this condition remains unclear. Currently, inflammation is widely accepted as the key factor in urate nephropathy. Therefore, the aim of this study was to elucidate the interaction of lincRNA-p21/AIF-1/CMPK2/NLRP3 via exosomes in urate nephropathy. This study evaluated the effect of lincRNA-p21/AIF-1/CMPK2/NLRP3 using clinical data collected from patients with urate nephropathy and human renal tubular epithelial cells (HK2) cultured with different concentrations of urate. In clinical research section, the level of lincRNA-p21/AIF-1 in exosomes of urine in patients with hyperuricemia or urate nephropathy was found to be increased, particularly in patients with urate nephropathy. In vitro study section, the level of exosomes, inflammation, autophagy, and apoptosis was increased in HK2 cells induced by urate. Additionally, the expression of lincRNA-p21, AIF-1, CMPK2, and NLRP3 was upregulated in exosomes and HK2 cells. Furthermore, manipulating the activity of lincRNA-p21, AIF-1, CMPK2, and NLRP3 through overexpression or interference vectors regulated the level of inflammation, autophagy, and apoptosis in HK2 cells. In conclusion, the pathway of lincRNA-p21/AIF-1/CMPK2/NLRP3 contributed to inflammation, autophagy, and apoptosis of human renal tubular epithelial cell induced by urate via exosomes. Additionally, the specific exosomes in urine might serve as novel biomarkers for urate nephropathy.

ALTERATIONS IN HISTIDINE METABOLISM IS A FEATURE OF EARLY AUTOSOMAL DOMINANT POLYCYSTIC KIDNEY DISEASE (ADPKD).

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by epithelial proliferation and progressive cyst enlargement. Using a non-targeted high-resolution metabolomics approach, we analyzed biofluids from 36 ADPKD and 18 healthy controls with estimated glomerular filtration rate (eGFR) > 60 ml/min to identify features specific to ADPKD or that associate with disease severity [eGFR or height-corrected total kidney volume (htTKV)]. Multiple pathways differed between ADPKD subjects and controls, with the histidine pathway being the most highly represented. Plasma histidine, urinary N-methylhistamine, methylimidazole-acetaldehyde, and imidazole-acetaldehyde, as well as 3-methylhistidine and anserine were increased, while plasma N-acetylhistamine and urinary imidazole-acetic acid were decreased in ADPKD compared to controls. In ADPKD, urinary histidine and a histidine derivative, urocanate (a precursor of glutamate), were significantly associated. HtTKV and eGFR were inversely associated with urinary glutamine and plasma 4-imidazolone-5-propionic acid, respectively. Supernatant from cultured human ADPKD renal cystic epithelia demonstrated increased aspartate and glutamate levels at 8 and 24 hours compared to primary tubular epithelia (p < 0.001). Following exposure over 48 hours to α-fluromethylhistidine, an inhibitor of histamine production, primary human PKD1 cyst epithelia proliferation increased significantly from baseline (p < 0.01) and greater than non-cystic epithelia (p < 0.05). The histidine ammonia lyase inhibitor nitromethane reversed α-fluromethylhistidine-induced cyst epithelia proliferation indicating a role for glutamate in cyst growth. In conclusion, histidine metabolism is altered preferentially leading to glutamate production and epithelial proliferation in ADPKD and associates with disease severity.

p27 specifically decreases in squamous carcinoma, and mediates NNK-induced transformation of human bronchial epithelial cells.

Lung cancer remains the leading cause of cancer-related deaths, with cigarette smoking being the most critical factor, linked to nearly 90% of lung cancer cases. NNK, a highly carcinogenic nitrosamine found in tobacco, is implicated in the lung cancer-causing effects of cigarette smoke. Although NNK is known to mutate or activate certain oncogenes, its potential interaction with p27 in modulating these carcinogenic effects is currently unexplored. Recent studies have identified specific downregulation of p27 in human squamous cell carcinoma, in contrast to adenocarcinoma. Additionally, exposure to NNK significantly suppresses p27 expression in human bronchial epithelial cells. Subsequent studies indicates that the downregulation of p27 is pivotal in NNK-induced cell transformation. Mechanistic investigations have shown that reduced p27 expression leads to increased level of ITCH, which facilitates the degradation of Jun B protein. This degradation in turn, augments miR-494 expression and its direct regulation of JAK1 mRNA stability and protein expression, ultimately activating STAT3 and driving cell transformation. In summary, our findings reveal that: (1) the downregulation of p27 increases Jun B expression by upregulating Jun B E3 ligase ITCH, which then boosts miR-494 transcription; (2) Elevated miR-494 directly binds to 3'-UTR of JAK1 mRNA, enhancing its stability and protein expression; and (3) The JAK1/STAT3 pathway is a downstream effector of p27, mediating the oncogenic effect of NNK in lung cancer. These findings provide significant insight into understanding the participation of mechanisms underlying p27 inhibition of NNK induced lung squamous cell carcinogenic effect.