Tubular Cell Line Research Articles

Deubiquitinase inhibitor bAP-15 suppresses renal epithelial to mesenchymal transition via inhibition of p300 stability

Renal fibrosis is an irreversible disease that is common in patients with chronic kidney disease. Elevated levels of the histone acetyltransferase p300 have been reported in various fibrotic diseases, including renal fibrosis, suggesting that p300 may be a promising therapeutic target. To investigate the specific deubiquitinase (DUB) involved in the regulation of p300 protein stability in renal epithelial cells, we tested 13 DUB inhibitors using a kidney tubular epithelial cell line. We found that the p300-specific DUB inhibitor, bAP-15 reduces p300 protein stability by targeting ubiquitin-specific protease 14 (USP14) and ubiquitin C-terminal hydrolase L5 (UCHL5). The mRNA levels of USP14 and UCHL5 were increased in patients with chronic kidney disease, and increased protein levels of USP14 and UCHL5 during fibrosis progression were validated using a mouse renal fibrosis model. Both USP14 and UCHL5 interacted with p300 in kidney tubular epithelial cells, with increased binding affinity in response to TGF-β. Moreover, bAP-15-induced p300 degradation inhibited epithelial-to-mesenchymal transition and reduced the expression of pro-fibrotic target genes. Our findings demonstrate an anti-fibrotic effect of bAP-15 through the regulation of p300 stability and suggest that bAP-15 may be a potential therapeutic agent for renal fibrosis.

Edaravone mitigates calcium oxalate-induced renal tubular epithelial cell injury by inhibiting autophagy-mediated ferroptosis.

Edaravone (EDA) has been found to exert protective effects on kidney injury. Nevertheless, the functions of EDA in kidney stones as well as the potential mechanism are vague. Calcium oxalate (CaOx) was used to induce kidney stones cell model with human renal tubular epithelial cell line HK-2. CCK-8 assay was employed to detect cell viability injury. Oxidative stress was measured by DCFH-DA staining and detection of MDA, SOD, and GSH. Staining of FerroOrange and western blot were applied for ferroptosis. In addition, autophagy was elucidated by western blot and immunofluorescence staining. The data showed that CaOx treatment aggravated HK-2 cell viability injury, increased the levels of ROS, MDA, and Fe2+ in HK-2 cells, and reduced the contents of SOD and GSH. Additionally, CaOx enhanced the expression of KIM1, TFR1, LC3II/LC31, and BECLIN1 in HK-2 cells, while resulting in a decrease in the expression of GPX4, SLC7A11, and P62. Pretreatment of EDA mitigated CaOx-induced oxidative stress and ferroptosis, as well as autophagy in renal tubular epithelial cells. However, autophagy inducer rapamycin (Rap) reversed the protective role of EDA on renal tubular epithelial cell injury, oxidative stress, and ferroptosis. In conclusion, EDA contributes to suppressing oxidative stress and ferroptosis in CaOx-induced HT22 cells by restraining autophagy, which may be a potential candidate for the treatment of kidney stones caused by renal tubular epithelial cell damage.

Synthesis, anticancer activity and mechanism of action of Fe(III) complexes.

To inhibit the growth and metastasis of triple-negative breast cancer (TNBC), two Fe(III) thiosemicarbazone complexes (Fe1 and Fe2) were designed and synthesized. The structures of the Fe(III) complexes were characterized by single crystal X-ray diffraction. The antiproliferative activity of Fe1 and Fe2 against four cancer lines (MDA-MB-231, T98G, HepG2, 143B) and human renal proximal tubular epithelial cell line (HK-2) was evaluated by MTT assay. Among all cells, Fe2 showed significant cytotoxicity to TNBC cells (MDA-MB-231), with an IC50 value of 12.38 μM. Furthermore, Fe2 showed less toxicity to HK-2 cells. The two Fe(III) complexes can produce excess of reactive oxygen species, decrease of mitochondrial membrane potential, and induce DNA damage, then lead to apoptosis of MDA-MB-231 cells. In addition, Fe1 and Fe2 can also inhibit migration and invasion of MDA-MB-231 cells. This study provides guidance for the development of metal complexes that inhibit the growth and metastasis of TNBC.

Dichloroacetate reduces cisplatin-induced apoptosis by inhibiting the JNK/14-3-3/Bax/caspase-9 pathway and suppressing caspase-8 activation via cFLIP in murine tubular cells

Cisplatin-induced injury to renal proximal tubular cells stems from mitochondrial damage-induced apoptosis and inflammation. Dichloroacetate (DCA), a pyruvate dehydrogenase kinase (PDK) inhibitor, a potential generator of ROS and ATP, protects against cisplatin-induced nephrotoxicity by promoting the TCA cycle. However, its effects on apoptotic pathways and ROS production in renal tubular cells remain unclear. Here, we investigated the detailed molecular mechanisms of the DCA’s effects by immunoblot, RT-PCR, RNA-sequencing, and RNA-silencing in a murine renal proximal tubular (mProx) cell line and mouse kidneys. In mProx cells, DCA suppressed cisplatin-induced apoptosis by attenuating the JNK/14-3-3/Bax/caspase-9 and death receptor/ligand/caspase-8 pathways without impeding inflammatory signaling. RNA-sequencing demonstrated that DCA increased the cisplatin-reduced expression of cFLIP, a caspase-8 inactivator, and decreased the expression of almost all oxidative phosphorylation (OXPHOS) genes. DCA also increased NF-kB activation and ROS production, probably enhancing the cFLIP induction and OXPHOS gene reduction, respectively. Furthermore, cFLIP silencing weakened the DCA’s anti-apoptotic effects. Finally, in mouse kidneys, DCA attenuated cisplatin-caused injuries such as functional and histological damages, caspase activation, JNK/14-3-3 activation, and cFLIP reduction. Conclusively, DCA mitigates cisplatin-induced nephrotoxicity by attenuating the JNK/14-3-3/Bax/caspase-9 pathway and inhibiting the caspase-8 pathways via cFLIP induction, probably outweighing the cisplatin plus DCA-derived cytotoxic effects including ROS.

Hypoxia Activates FGF-23-ERK/MAPK Signaling Pathway in Ischemia-Reperfusion-Induced Acute Kidney Injury

Introduction: Both hypoxia and fibroblast growth factor-23 (FGF-23) are key factors in ischemia-reperfusion (I/R)-induced acute kidney injury (AKI). This study aimed to explore the relationship between hypoxia and FGF-23 in AKI. Methods: An I/R-AKI animal model was established using male BALB/c mice. HK-2 cells, a part of the human proximal tubular epithelial cell line, were subjected to hypoxia/reoxygenation (H/R). qPCR was used to measure FGF-23 and HIF1α, and ELISA was used to measure inflammatory and oxidative stress cytokines. Western blotting was used to measure the phosphorylation of extracellular signal-regulated kinase (ERK) level. Results: In I/R mice, the levels of interleukin-6 (IL-6), tumor necrosis factor (TNF-α), malondialdehyde (MDA), and the phosphorylation of ERK (p-ERK) were increased, whereas the levels of interleukin-10 (IL-10), superoxide dismutase (SOD), glutathione peroxidase (GPx), and klotho were decreased, compared to the sham-operated mice. Silencing the FGF-23 expression in I/R mice normalized the levels of IL-6, IL-10, TNF-α, MDA, SOD, GPx, and p-ERK. In HK-2 cells, hypoxia-reperfusion (H/R) elevated the levels of IL-6, TNF-α, MDA, and p-ERK, but reduced IL-10, SOD, GPx, and klotho levels. Hypoxia induced apoptosis in HK-2 cells, but silencing of FGF-23 expression blocked the effects of hypoxia on cell apoptosis, pro-inflammatory factor levels, oxidative stress response, and p-ERK levels. Conclusion: FGF-23 is a key molecule in AKI, and hypoxia plays a crucial role in AKI by inducing cell apoptosis; however, its role is regulated by FGF-23. FGF-23 affects oxidative stress and the inflammatory response of kidney tissues by activating the ERK/mitogen-activated protein kinase (MAPK) signaling pathway.

Silencing LncRNA SNHG14 alleviates renal tubular injury via the miR-483-5p/HDAC4 axis in diabetic kidney disease.

This study explored the clinical value of long non-coding RNA small nucleolar RNA host gene 14 (SNHG14) in diabetic kidney disease (DKD) and the mechanism of renal tubular injury. Patients with DKD, type 2 diabetes mellitus (T2DM) and healthy individuals (HVs) were included, as well as the human proximal tubular epithelial cell line (HK-2) induced by high glucose was also included. The mRNA levels of SNHG14 in the serum and cells were detected using RT-qPCR. Diagnostic significance was examined using receiver operating characteristic (ROC) analysis. A commercial test kit, flow cytometry, and enzyme-linked immunosorbent assays were employed to assess reactive oxygen species (ROS) production, apoptosis, inflammatory factor secretion, and extracellular matrix protein levels in HK-2 cells. The dual-luciferase reporter assay and RNA immunoprecipitation were used to validate miR-483-5p concerning SNHG14 or histone deacetylase 4 (HDAC4). SNHG14 and HDAC4 levels were elevated in the serum of DKD patients and HG-induced HK-2 cells, while miR-483-5p levels were decreased (P < 0.001). SNHG14 increased HDAC4 levels by sponging miR-483-5p. Elevated SNHG14 levels significantly differentiated DKD patients from HVs (AUC = 0.944) and T2DM (AUC = 0.867). Silencing of SNHG14 alleviated HG-induced ROS production and apoptosis as well as the over-secretion of inflammatory factors and extracellular matrix proteins; however, this alleviation was typically suppressed by low expression of miR-483-5p (P < 0.001). Elevated miR-483-5p alleviates HG-induced renal tubular injury, but this alleviation is suppressed by HDAC4 overexpression. In summary, suppression of SNHG14 has been shown in our study to mitigate renal tubular injury in DKD by regulating apoptosis, oxidative stress, inflammation, and fibrosis through the miR-483-5p/HDAC4 axis.

Elevated serum GDF15 level as an early indicator of proximal tubular cell injury in acute kidney injury

Acute kidney injury (AKI) is a high-burden medical condition, and current diagnostic criteria can only assess AKI after full manifestation. Stress marker growth differentiation factor 15 (GDF15) was reported to have a role in kidney injury of critical patients. Herein, we evaluated dynamic changes in GDF15 across diverse AKI scenarios and explored the underlying mechanisms of its induction. Serum parameters and renal lesions were analyzed in mouse models of unilateral ischemia-reperfusion injury (uni-IRI) and unilateral ureteral obstruction (UUO). The human proximal tubular (HK−2) cell line was stimulated with various conditions, and induction of GDF15 expression was determined. Serum GDF15 levels were rapidly induced within hours after injury in both animal models and declined thereafter. Renal GDF15 expression exhibited a temporary and early increased induction and was mainly located in aquaporin 1-positive proximal tubules in both unilateral AKI model tissues. In cell experiments, rapid GDF15 production was highly induced by t-BHP and CoCl2. Treatment with either an antioxidant or mitogen-activated protein kinase inhibitors abolished t-BHP- and CoCl2-mediated GDF15 expression. In addition, silencing nuclear factor erythroid 2-related factor 2 expression also reduced the basal and t-BHP- or CoCl2-mediated GDF15 expression level in HK-2 cells. Our data showed that elevated serum GDF15 levels could be detected early in unilateral AKI models without notable alterations in kidney function parameters. GDF15 expression was associated with oxidative stress- and hypoxia-mediated proximal tubular cell injury. These data document that elevated serum GDF15 can possibly serve as an early biomarker for proximal tubular cell injury in AKI.

Coexposure to microplastic and Bisphenol A exhacerbates damage to human kidney proximal tubular cells

Microplastics (MPs) accumulate in tissues, including kidney tissue, while Bisphenol A (BPA) is a plasticizer of particular concern. At present, the combined effects of MPs and BPA are unexplored in human renal cells. Therefore, we exposed a proximal tubular cell line (PTECs) to polyethylene (PE)-MPs and BPA, both separately and in combination. When co-exposed, cells showed a significantly reduced cell viability (MTT test) and a pronounced pro-oxidant (MDA levels, NRF2 and NOX4 expression by Western blot) and pro-inflammatory response (IL1β, CCL/CCR2 and CCL/CCR5 mRNAs by RT-PCR), compared to those treated with a single compound. In addition, heat shock protein (HSP90), a chaperone involved in multiple cellular functions, was reduced (by Western Blot and immunocytochemistry), while aryl hydrocarbon receptor (AHR) expression, a transcription factor which binds environmental ligands, was increased (RT-PCR and immunofluorescence). Our research can contribute to the study of the nephrotoxic effects of pollutants and MPs and shed new light on the combined effects of BPA and PE-MPs.

Evaluation of the novel ALK5 inhibitor EW-7197 on therapeutic efficacy in renal fibrosis using a three-dimensional chip model.

EW-7197, a potent oral ALK5 inhibitor, was assessed for its impact on transforming growth factor beta 1 (TGF-β1)-induced fibrosis in a three-dimensional (3D) renal fibrosis-on-a-chip and a mouse model. The evaluation included tubular epithelial-mesenchymal transition, angiogenesis, and inflammatory cytokine expression. In a 3D renal fibrosis-on-a-chip model, three cell types (kidney fibroblasts, human proximal tubular cell line, and human umbilical vein endothelial cells) were cultured and treated with TGF-β1 and EW-7197. Expression of alpha smooth muscle actin (α-SMA) and keratin 8 (KRT-8) was assessed, angiogenesis was observed via confocal microscopy, and cytokine levels were measured using real-time polymerase chain reaction, immunoassay, and enzyme-linked immunosorbent assay. In a cisplatin-induced renal fibrosis mouse model, blood urea nitrogen levels, TGF-β, and Smad 2/3 were determined, and renal fibrosis was assessed with Masson's trichrome stain. The α-SMA expression was significantly lower in the EW-7197 group than in the TGF-β fibrosis group. TGF-β decreased the expression of the epithelial marker KRT-8, an effect that was reversed by EW-7197 and SB431542. In the TGF-β-induced fibrosis model, the length of the thick vessels was reduced, and the diameter of both thick and thin vessels was decreased, but EW-7197 reversed these effects. EW-7197 significantly reduced the messenger RNA expression of TGF-β and increased the levels of vascular endothelial growth factor receptor 2, interleukin (IL)-10, and IL-6. EW-7197 reduced the levels of secretory cytokines TGF-β1, TGF-β3, IL-1β. In the cisplatin-induced renal fibrosis mouse model, EW-7197 reduced renal fibrosis by down-regulating TGF-β signaling. EW-7197 attenuated the TGF-β1-induced fibrotic cellular response in the 3D chip model and animal model. These findings indicate the potential effect of EW-7197 in attenuating renal fibrosis.

Quercetin Protects Against Hypertensive Renal Injury by Attenuating Apoptosis: An Integrated Approach Using Network Pharmacology and RNA Sequencing.

Quercetin is known for its antihypertensive effects. However, its role on hypertensive renal injury has not been fully elucidated. In this study, hematoxylin and eosin staining, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining, and Annexin V staining were used to assess the pathological changes and cell apoptosis in the renal tissues of angiotensin II (Ang II)-infused mice and Ang II-stimulated renal tubular epithelial cell line (NRK-52E). A variety of technologies, including network pharmacology, RNA-sequencing, immunohistochemistry, and Western blotting, were performed to investigate its underlying mechanisms. Network pharmacology analysis identified multiple potential candidate targets (including TP53, Bcl-2, and Bax) and enriched signaling pathways (including apoptosis and p53 signaling pathway). Quercetin treatment significantly alleviated the pathological changes in renal tissues of Ang II-infused mice and reversed 464 differentially expressed transcripts, as well as enriched several signaling pathways, including those related apoptosis and p53 pathway. Furthermore, quercetin treatment significantly inhibited the cell apoptosis in renal tissues of Ang II-infused mice and Ang II-stimulated NRK-52E cells. In addition, quercetin treatment inhibited the upregulation of p53, Bax, cleaved-caspase-9, and cleaved-caspase-3 protein expression and the downregulation of Bcl-2 protein expression in both renal tissue of Ang II-infused mice and Ang II-stimulated NRK-52E cells. Moreover, the molecular docking results indicated a potential binding interaction between quercetin and TP53. Quercetin treatment significantly attenuated hypertensive renal injury and cell apoptosis in renal tissues of Ang II-infused mice and Ang II-stimulated NRK-52E cells and by targeting p53 may be one of the potential underlying mechanisms.

Wnt-5a–Receptor Tyrosine Kinase-Like Orphan Receptor 2 Signaling Provokes Metastatic Colonization and Angiogenesis in Renal Cell Carcinoma, and Prunetin Supresses the Axis Activation

Wnt-5a is a protein that is encoded by the WNT5A gene and is a ligand for the ROR2 receptor. However, its biological impact on clear cell renal cell carcinoma (ccRCC) remains unclear. In this study, the prognostic significance of concurrent WNT5A and ROR2 expression levels was observed to predict unfavorable overall survival and disease-specific survival. High Wnt-5a expression was detected in a ccRCC cell line panel but not in HK-2 cells, a normal proximal tubular cell line. Inhibition of DNA methyltransferase by 5-azaC in 786-O and Caki-2 cells resulted in Wnt-5a upregulation, indicating potential epigenetic modification. Furthermore, the results revealed the repression of cell movement in vitro and metastatic colonization in vivo upon WNT5A and ROR2 knockdown. The suppressions of angiogenesis in vivo and tubular-like structure formation in endothelial cells in vitro were also observed after silencing WNT5A and ROR2 expression. In addition, alteration in the downstream gene signature of the Wnt-5a-ROR2 signaling was discovered to be similar to that in MTA1-CTNNB1 axis. Moreover, prunetin treatment was found to reverse the gene signature derived from Wnt-5a-ROR2 signaling activation and to abolish ccRCC cell migration and proliferation. Overall, this study demonstrates the clinical and functional significance of the Wnt-5a-ROR2 axis and identify prunetin as a potential precision medicine for ccRCC patients harboring aberrant Wnt-5a-ROR2 signaling pathways.

Nrf2/FSP1/CoQ10 axis-mediated ferroptosis is involved in sodium aescinate-induced nephrotoxicity

Sodium aescinate (SA), an active compound found in horse chestnut seeds, is widely used in clinical practice. Recently, the incidence of SA-induced adverse events, particularly renal impairment, has increased. Our previous work demonstrated that SA causes severe nephrotoxicity via nephrocyte ferroptosis; however, the underlying mechanism remains to be fully elucidated. In the current study, we investigated additional molecular pathways involved in SA-induced nephrotoxicity. Our results showed that SA inhibited cell viability, disrupted cellular membrane integrity, and enhanced reactive oxygen species (ROS), ferrous iron (Fe2+), and malondialdehyde (MDA) levels, as well as lipid peroxidation in rat proximal renal tubular epithelial cell line (NRK-52E) cells. SA also depleted coenzyme Q10 (CoQ10, ubiquinone) and nicotinamide adenine dinucleotide (NADH) and reduced ferroptosis suppressor protein 1 (FSP1) and polyprenyltransferase (coenzyme Q2, COQ2) activity, triggering lipid peroxidation and ROS accumulation in mouse kidneys and NRK-52E cells. The overexpression of COQ2, FSP1, or CoQ10 (ubiquinone) supplementation effectively attenuated SA-induced ferroptosis, whereas iFSP1 or 4-formylbenzoic acid (4-CBA) pretreatment exacerbated SA-induced nephrotoxicity. Additionally, SA decreased nuclear factor-erythroid-2-related factor 2 (Nrf2) levels and inhibited Nrf2 binding to the −1170/-1180 bp ARE site in FSP1 promoter, resulting in FSP1 suppression. Overexpression of Nrf2 or its agonist dimethyl fumarate (DMF) promoted FSP1 expression, thereby improving cellular antioxidant capacity and alleviating SA-induced ferroptosis. These results suggest that SA-triggers renal injury through oxidative stress and ferroptosis, driven by the suppression of the Nrf2/FSP1/CoQ10 axis.

Gemigliptin mitigates TGF-β-induced renal fibrosis through FGF21-mediated inhibition of the TGF-β/Smad3 signaling pathway

Fibroblast growth factor 21 (FGF21), a well-known regulator of metabolic disorders, exhibits the potential to prevent renal fibrosis by negatively regulating the transforming growth factor β (TGF-β)/Smad3 signaling pathway. Gemigliptin and other dipeptidyl peptidase-4 inhibitors are frequently used for the management of patients with type 2 diabetes. However, the protective effect of gemigliptin against renal fibrosis, particularly its potential to upregulate the expression of FGF21, remains incompletely understood. This study assessed the renoprotective effects of gemigliptin against TGF-β-induced renal fibrosis by enhancing the expression of FGF21 in the cultured human proximal tubular epithelial cell line HK-2. Treatment with FGF21 effectively prevented TGF-β-induced renal fibrosis by attenuating the TGF-β/Smad3 signaling pathway. Similarly, gemigliptin exhibited protective effects against TGF-β-induced renal fibrosis by mitigating TGF-β/Smad3 signaling through the upregulation of FGF21 expression. However, the protective effects of gemigliptin were blocked when FGF21 expression was knocked down in TGF-β-treated HK-2 cells. These results indicate that gemegliptin has the potential to exhibit protective effects against TGF-β-induced renal fibrosis by elevating FGF21 expression levels in cultured human proximal tubular epithelial cells.

PDK4-mediated Nrf2 inactivation contributes to oxidative stress and diabetic kidney injury

Diabetic kidney disease (DKD) is often featured with redox dyshomeostatis. Pyruvate dehydrogenase kinase 4 (PDK4) is the hub for DKD development. However, the mechanism by which PDK4 mediates DKD is poorly understood. The current work aimed to elucidate the relationship between PDK4 and DKD from the perspective of redox manipulation. Oxidative stress was observed in the human proximal tubular cell line (HK-2 cells) treated with a high concentration of glucose and palmitic acid (HGL). The mechanistic study showed that PDK4 could upregulate Kelch-like ECH-associated protein 1 (Keap1) in HGL-treated HK-2 cells through the suppression of autophagy, resulting in the depletion of nuclear factor erythroid 2-related factor 2 (Nrf2), the master regulator of redox homeostasis. At the cellular level, pharmacological inhibition or genetic knockdown of PDK4 could boost Nrf2, followed by the increase of a plethora of antioxidant enzymes and ferroptosis-suppression enzymes. Meanwhile, the inhibition or knockdown of PDK4 remodeled iron metabolism, further mitigating oxidative stress and lipid peroxidation. The same trend was observed in the DKD mice model. The current work highlighted the role of PDK4 in the development of DKD and suggested that PDK4 might be a promising target for the management of DKD.

Chlorogenic Acid Alleviates High Glucose-induced HK-2 Cell Oxidative Damage through Activation of KEAP1/NRF2/ARE Signaling Pathway.

To investigate the alleviating effect of chlorogenic acid (CGA) on oxidative damage in high glucose (HG)-induced HK-2 cells and to explore its potential mechanisms. We cultured the human proximal tubular cell line HK-2 and divided them into the control group and different concentrations of CGA groups (0, 5, 10, 25, 50, 100, 200 μM). The trypan blue dye test was used to detect CGA's potential cytotoxicity on HK-2 cells. Then, we treated HK-2 with HG and CGA; the Cell Counting Kit-8 (CCK-8) method was used to detect the cell viability of HK-2 cells in each group. Flow cytometry was employed to measure the apoptosis rate of cells. Western blot was performed to detect the expression of apoptosis proteins B-cell lymphoma-2 (BCL-2), BCL-2-associated X protein (BAX), cysteinyl aspartate specific proteinase (CASPASE)-9, and CASPASE-3. In addition, enzymatic activities, including superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and lipid peroxide (LPO), were measured with the corresponding detection kits. 2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) assay and flow cytometry were performed to detect reactive oxygen species (ROS) production. Western blot analysis and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) were conducted to evaluate protein and mRNA expressions of the Kelch-like ECH-associated protein-1 (KEAP1)/Nuclear factor erythroid 2-related factor 2 (NRF2)/Antioxidant Response Elements (ARE) signaling pathway. The outcomes showed that, in a dose-dependent way, CGA dramatically increased the vitality of HK-2 induced by HG. Furthermore, CGA significantly reduced the HG-stimulated HK-2 cell apoptosis, which may be linked to the promotion of BCL-2 and the suppression of BAX, cleaved-CASPASE-3, and cleaved-CASPASE-9 expression. In HK-2 cells, CGA reduced the formation of ROS generated by HG levels and markedly boosted the activity of the antioxidant enzymes SOD, GSH-Px, and CAT. Furthermore, compared with the HG group, CGA significantly raised NRF2 nuclear expression and downregulated NRF2 cytosolic expression and increased the mRNA expression of NRF2 and its target genes, heme oxygenase-1 (HO-1), KEAP1, and NAD(P)H dehydrogenase quinone 1 (NQO1). These results show that CGA might be useful in managing oxidative damage in HG-induced HK-2 cells.

Mitochondrial calcium uniporter promotes kidney aging in mice through inducing mitochondrial calcium-mediated renal tubular cell senescence.

Renal tubular epithelial cell senescence plays a critical role in promoting and accelerating kidney aging and age-related renal fibrosis. Senescent cells not only lose their self-repair ability, but also can transform into senescence-associated secretory phenotype (SASP) to trigger inflammation and fibrogenesis. Recent studies show that mitochondrial dysfunction is critical for renal tubular cell senescence and kidney aging, and calcium overload and abnormal calcium-dependent kinase activities are involved in mitochondrial dysfunction-associated senescence. In this study we investigated the role of mitochondrial calcium overload and mitochondrial calcium uniporter (MCU) in kidney aging. By comparing the kidney of 2- and 24-month-old mice, we found calcium overload in renal tubular cells of aged kidney, accompanied by significantly elevated expression of MCU. In human proximal renal tubular cell line HK-2, pretreatment with MCU agonist spermine (10 μM) significantly increased mitochondrial calcium accumulation, and induced the production of reactive oxygen species (ROS), leading to renal tubular cell senescence and age-related kidney fibrosis. On the contrary, pretreatment with MCU antagonist RU360 (10 μM) or calcium chelator BAPTA-AM (10 μM) diminished D-gal-induced ROS generation, restored mitochondrial homeostasis, retarded cell senescence, and protected against kidney aging in HK-2 cells. In a D-gal-induced accelerated aging mice model, administration of BAPTA (100 μg/kg. i.p.) every other day for 8 weeks significantly alleviated renal tubuarl cell senescence and fibrosis. We conclude that MCU plays a key role in promoting renal tubular cell senescence and kidney aging. Targeting inhibition on MCU provides a new insight into the therapeutic strategy against kidney aging.

#1453 EZH2 is an epigenetic regulator of renal gluconeogenesis in chronic kidney disease through downregulation of PCK1

Abstract Background and Aims Impaired renal gluconeogenesis has been recently identified as a hallmark of chronic kidney diseases (CKD), and is tightly correlated with the progression of renal tubulointerstitial fibrosis. The methyltransferase enhancer of zeste homolog 2 (EZH2) is an epigenetic regulator playing an important role in renal tubulointerstitial fibrosis. Whether renal gluconeogenesis can be epigenetically regulated is currently unknown. In the current study, we studied EZH2-mediated epigenetic regulation of renal gluconeogenesis in three different mouse models of renal fibrosis. Method Mouse models of renal fibrosis were established by unilateral ureteral obstruction (UUO) operation, unilateral ischemia reperfusion injury or folic acid (FA) injection. Human proximal tubular cell line (HK2), primary tubular cells or primary tubules was used as an in vitro or ex vivo model. EZH2 was conditional knockout or inhibited by 3-DZNeP in mice. Inhibition of EZH2 by 3-DZNeP or overexpressed by adenovirus was performed in vitro. RNA sequencing and cleavage under targets and tagmentation (CUT&amp;Tag) sequencing analysis were performed and followed by quantitative PCR, CUT&amp;Tag and Western blotting analysis. Glucogenic metabolites were measured and systemic glucose metabolism was assessed by tolerance tests. Results By deletion of Ezh2 gene in mice through a ubiquitous expressed Cre/Esr1 system, we confirmed the pro-fibrotic effect of EZH2 in unilateral ureteral obstruction (UUO) kidneys. Through RNA sequencing and CUT&amp;Tag sequencing analysis, we found that the phosphoenolpyruvate carboxykinase 1 (PCK1), a critical enzyme in gluconeogenesis, is negatively regulated by the methyltransferase EZH2 in fibrotic kidneys, which was further confirmed by quantitative PCR, CUT&amp;Tag and Western blotting analysis in renal cells and UUO or FA kidneys. We further showed that pharmaceutical inhibition of EZH2 by 3-DZNeP enhances PCK1 expression in fibrotic kidneys of three mouse models. Moreover, the concentration of lactate, a glucogenic metabolic intermediate, was increased in mouse fibrotic kidneys, which was reduced by EZH2 inhibition. A further experiment showed that systemic glucose metabolism was impaired in UUO mice and restored by EZH2 inhibition as assessed by insulin or glucose tolerance test. Upregulated EZH2 in UUO kidneys is colocalized with the proximal tubular marker as shown by immunofluorescent staining. The direct anti-gluconeogenesis effect of EZH2 on renal proximal epithelial cells and primary renal tubules was shown by analysis of gluconeogenic gene expression and production of glucose or lactate in human HK2 cells treated with EZH2 inhibitor or exogeneous expression of EZH2 by adenovirus transfection. Importantly, we further confirmed the direct effect of tubular EZH2 on gluconeogenesis in tubular specific deleted EZH2 mice (Ezh2fl/fl;Cdh16-Cre). Moreover, inhibition of PCK1 by 3-mercaptopropionic acid abrogated the pro-gluconeogenic effect and anti-fibrotic effect of EZH2 inhibitor in renal cells and UUO kidneys. Finally, RNA sequencing data from kidney allograft biopsies showed that EZH2 is progressively increased in CKD patients. Conclusion We conclude that EZH2 promotes renal tubulointerstitial fibrosis through epigenetic inhibition of PCK1. Our study suggests that therapeutic restoration of the impaired renal gluconeogenesis is beneficial to CKD.

#1581 Anoctamin 3 inhibits gluconeogenesis and contributes to renal interstitial fibrosis through EZH2

Abstract Background and Aims Renal fibrosis is the common pathological pathway of various chronic kidney diseases progressing to the end stage of renal failure. Anoctamin 3 (ANO3) has been identified as a K-channel regulator and been extensively studied in the field of neurobiology. The role of ANO3 in kidney diseases is currently unknown. Method Mouse models of renal fibrosis were established by unilateral ureteral obstruction (UUO) operation or aristolochic acid I (AAI) injection. Human proximal tubular cell line (HK2) was used as an in vitro model. ANO3 was deleted in vitro by siRNA. Western blotting analysis was performed. Glucogenic metabolites were measured. Results We found that ANO3 is upregulated in mouse kidneys with unilateral ureteral obstruction (UUO) or aristolochic acid nephropathy (AAN), and tightly correlated with the progression of renal fibrosis and negatively correlated with expression of several rate-limiting enzymes of renal gluconeogenesis. Knockdown of ANO3 with siRNA increased the concentration of glucose and reduced the concentration of lactate in the supernatant of TGF-β stimulated renal proximal tubular (HK2) cells. In parallel, transfection of ANO3 siRNA significantly reduced the expression of epithelial-to-mesenchymal transition (EMT) and fibrotic markers in TGF-β stimulated HK2 cells. We further showed that ANO3 expression is positively correlated with the expression of a methyltransferase enhancer of zeste homologue 2 (EZH2) in UUO and AAN mouse kidneys by time. Transfection of ANO3 siRNA significantly reduced the expression of EZH2 in TGF-β stimulated HK2 cells. Overexpression of EZH2 by adenovirus reversed the pro-gluconeogenic and anti-fibrotic effect of ANO3 siRNA in TGF-β stimulated HK2 cells by measuring the concentration of glucose and lactate in supernatant and analyzing the expression of EMT or fibrotic markers. Conclusion In conclusion, ANO3 inhibits renal gluconeogenesis through EZH2 and contributes to renal fibrosis. ANO3 could be promising therapeutic target to inhibit renal fibrosis in chronic kidney disease patients.

HIF-1α participates in the regulation of S100A16-HRD1-GSK3β/CK1α pathway in renal hypoxia injury

S100 calcium-binding protein 16 (S100A16) is implicated in both chronic kidney disease (CKD) and acute kidney injury (AKI). Previous research has shown that S100A16 contributes to AKI by facilitating the ubiquitylation and degradation of glycogen synthase kinase 3β (GSK3β) and casein kinase 1α (CK1α) through the activation of HMG-CoA reductase degradation protein 1 (HRD1). However, the mechanisms governing S100A16-induced HRD1 activation and the upregulation of S100A16 expression in renal injury are not fully understood. In this study, we observed elevated expression of Hypoxia-inducible Factor 1-alpha (HIF-1α) in the kidneys of mice subjected to ischemia-reperfusion injury (IRI). S100A16 deletion attenuated the increased HIF-1α expression induced by IRI. Using a S100A16 knockout rat renal tubular epithelial cell line (NRK-52E cells), we found that S100A16 knockout effectively mitigated apoptosis during hypoxic reoxygenation (H/R) and cell injury induced by TGF-β1. Our results revealed that H/R injuries increased both protein and mRNA levels of HIF-1α and HRD1 in renal tubular cells. S100A16 knockout reversed the expressions of HIF-1α and HRD1 under H/R conditions. Conversely, S100A16 overexpression in NRK-52E cells elevated HIF-1α and HRD1 levels. HIF-1α overexpression increased HRD1 and β-catenin while decreasing GSK-3β. HIF-1α inhibition restored HRD1 and β-catenin upregulation and GSK-3β downregulation by cellular H/R injury. Notably, Chromatin immunoprecipitation (ChIP) and luciferase reporter assays demonstrated HIF-1α binding signals on the HRD1 promoter, and luciferase reporter gene assays confirmed HIF-1α‘s transcriptional regulation of HRD1. Additionally, we identified Transcription Factor AP-2 Beta (TFAP2B) as the upregulator of S100A16. ChIP and luciferase reporter assays confirmed TFAP2B as a transcription factor for S100A16. In summary, this study identifies TFAP2B as the transcription factor for S100A16 and demonstrates HIF-1α regulation of HRD1 transcription within the S100A16-HRD1-GSK3β/CK1α pathway during renal hypoxia injury. These findings provide crucial insights into the molecular mechanisms of kidney injury, offering potential avenues for therapeutic intervention.

Mitophagy mediated by HIF-1α/FUNDC1 signaling in tubular cells protects against renal ischemia/reperfusion injury

Acute kidney injury (AKI) is associated with a high mortality rate. Pathologically, renal ischemia/reperfusion injury (RIRI) is one of the primary causes of AKI, and hypoxia-inducible factor (HIF)-1α may play a defensive role in RIRI. This study assessed the role of hypoxia-inducible factor 1α (HIF-1α)-mediated mitophagy in protection against RIRI in vitro and in vivo. The human tubular cell line HK-2 was used to assess hypoxia/reoxygenation (H/R)-induced mitophagy through different in vitro assays, including western blotting, immunofluorescence staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and reactive oxygen species (ROS) measurement. Additionally, a rat RIRI model was established for evaluation by renal histopathology, renal Doppler ultrasound, and transmission electron microscopy to confirm the in vitro data. The selective HIF-1α inhibitor LW6 reduced H/R-induced mitophagy but increased H/R-induced apoptosis and ROS production. Moreover, H/R treatment enhanced expression of the FUN14 domain-containing 1 (FUNDC1) protein. Additionally, FUNDC1 overexpression reversed the effects of LW6 on the altered expression of light chain 3 (LC3) BII and voltage-dependent anion channels as well as blocked the effects of HIF-1α inhibition in cells. Pretreatment of the rat RIRI model with roxadustat, a novel oral HIF-1α inhibitor, led to decreased renal injury and apoptosis in vivo. In conclusion, the HIF-1α/FUNDC1 signaling pathway mediates H/R-promoted renal tubular cell mitophagy, whereas inhibition of this signaling pathway protects cells from mitophagy, thus aggravating apoptosis, and ROS production. Accordingly, roxadustat may protect against RIRI-related AKI.