Renal Tubular Epithelial Cell Line Research Articles

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.

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.

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.

MiR-92a-3p Knockdown Attenuates Transforming Growth Factor-β1-induced Tubulointerstitial Fibrosis by Targeting LIN28A-mediated EMT Pathway.

The role of microRNAs in regulating tubulointerstitial fibrosis, a key feature of progressive chronic kidney disease, is of significant importance. LIN28A has been reported to attenuate renal fibrosis in obstructive nephropathy. Here, our objective was to investigate the precise biological function of the miR-92a-3p/LIN28A axis in tubulointerstitial fibrosis. The human renal proximal tubular epithelial (HK-2) cell line was exposed to transforming growth factor (TGF)-β1, establishing an in vitro model mimicking tubulointerstitial fibrosis. Luciferase reporter assay was utilized to investigate the relationship between miR-92a-3p and LIN28A. Cell transfection techniques were employed to modify the expression of miR-92a-3p and LIN28A. An in vivo model of tubulointerstitial fibrosis was created by inducing unilateral ureteral obstruction (UUO) in C57BL/6N mice. Our initial observations showed that TGF-β1 treatment of HK-2 cells and the UUO mice model led to an increase in miR-92a-3p expression and a decrease in LIN28A expression. We confirmed that miR-92a-3p directly targeted LIN28A in HK-2 cells. In TGF-β1-stimulated HK-2 cells, knocking down miR-92a-3p notably reduced the levels of alpha smooth muscle actin and vimentin and concurrently enhanced the expression of E-cadherin. These changes were counteracted upon transfection with si-LIN28A. Thus, directing interventions toward miR-92a-3p holds the potential to emerge as a viable therapeutic approach for addressing tubulointerstitial fibrosis.

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.

PDCD4-induced oxidative stress through FGR/NF-κB axis in rectal cancer radiotherapy-induced AKI

This study aimed to investigate the molecular mechanism of the effect of PDCD4 on radiotherapy-induced acute kidney injury (AKI) in rectal cancer through the regulation of FGR/NF-κB signaling. Differentially expressed genes were identified using Gene Expression Omnibus (GEO) datasets (GSE90627 for rectal cancer and GSE145085 for AKI) and R software. The human renal tubular epithelial cell line, HK-2, was used to establish an in vitro model of radiotherapy-induced AKI. RT-qPCR and western blotting were used to detect gene and protein expression levels, respectively. Cell proliferation and apoptosis were assessed using the CCK-8 assay and flow cytometry, respectively. The malondialdehyde and superoxide dismutase levels in the cell culture supernatants were determined. Additionally, an in vivo AKI model was established using BALB/c mice, and kidney tissue morphology, expression of the renal injury molecule KIM-1, apoptosis of renal tubular cells, and TAS and TOS in serum were evaluated. Bioinformatics analysis revealed the upregulated expression of PDCD4 in AKI. In vitro experiments demonstrated that PDCD4 induced apoptosis in renal tubular cells by promoting FGR expression, which activated the NF-κB signaling pathway and triggered an oxidative stress response. In vivo animal experiments confirmed that PDCD4 promoted oxidative stress response and radiotherapy-induced AKI through the activation of the FGR/NF-κB signaling pathway. Silencing PDCD4 attenuated radiotherapy-induced AKI. Our findings suggest that PDCD4 may induce radiotherapy-induced AKI in rectal cancer by promoting FGR expression, activating the NF-κB signaling pathway, and triggering an oxidative stress response.

Baicalin inhibits monosodium urate crystal-induced pyroptosis in renal tubular epithelial cell line through Panx-1/P2X7 pathways: Molecular docking, molecular dynamics, and invitro experiments.

Pyroptosis is a programmed cell death process that frequently occurs in many diseases, including hyperuricemic nephropathy (HN). In HN, a range of stimuli mediates inflammation, leading to the activation of inflammasomes and the production of gasdermin D (GSDMD). Baicalin (BA), a natural flavonoid renowned for its antioxidant and anti-inflammatory properties, was investigated for its role in HN in this study. Initially, HN-like inflammation and pyroptosis were induced in HK-2 cells with treatment of monosodium urate (MSU), followed by the BA treatment. The expression of pyroptosis-associated genes, Panx-1 and P2X7, at both mRNA and protein levels was assessed through real-time polymerase chain reaction (RT-qPCR) and Western blotting (WB) without or with BA treatment. The results showed that expression of Panx-1 and P2X7 at mRNA and protein levels was increased in MSU-treated HK-2 cells, which subsequently decreased upon the BA treatment. Further experiments showed that BA could combine NLRP3 inflammasome and GSDMD, destabilizing GSDMD protein. Moreover, BA protected the cell membrane from MSU-induced damage, as evidenced by Hoechst 33342 and PI double staining, lactate dehydrogenase (LDH) assays, and electron microscopy observations. These results suggest that BA is involved in the regulating Panx-1/P2X7 pathways and thus inhibits pyroptosis, highlighting its potential therapeutic effect for HN.

Sleeve gastrectomy links the attenuation of diabetic kidney disease to the inhibition of renal tubular ferroptosis through down-regulating TGF-β1/Smad3 signaling pathway

PurposeTo investigate how sleeve gastrectomy (SG), a typical operation of bariatric surgery, attenuated symptom, and progression of diabetic kidney disease (DKD).MethodsDKD model was induced by high-fat diet (HFD) combined with streptozocin in Wistar rats. SG was performed, and the group subjected to sham surgery served as control. The animals were euthanized 12 weeks after surgery, followed by sample collection for the subsequent experiment. The HK-2, a renal proximal tubular epithelial cell line derived from human, was utilized to investigate the potential mechanisms.ResultsSG improved metabolic parameters and glucose homeostasis, and could alleviate DKD in terms of renal function indices as well as histological and morphological structures in DM rats, accompanied with a significant reduction in renal tubular injury. Compared with sham group, SG reduced the renal tubular ferroptosis. To further clarify the mechanism involved, in vitro experiments were performed. In the presence of high glucose, renal tubular TGF-β1 secretion was significantly increased in HK-2 cell line, which led to activation of ferroptosis through TGF-β1/Smad3 signaling pathway. Inhibition of TGF-β1 receptor and phosphorylation of Smad3 significantly ameliorated TGF-β1-mediated ferroptosis. In vivo experiments also found that SG improved the hyperglycemic environment, reduced renal TGF-β1 concentrations, and down-regulated the TGF-β1/Smad3 signaling pathway.ConclusionsWith the capacity to lower the glucose, SG could attenuate the ferroptosis by inhibiting TGF-β1/Smad3 signaling pathway in DKD rats, and eventually attenuated DKD.

Proteomic analysis of mitochondria associated membranes in renal ischemic reperfusion injury

BackgroundThe mitochondria and endoplasmic reticulum (ER) communicate via contact sites known as mitochondria associated membranes (MAMs). Many important cellular functions such as bioenergetics, mitophagy, apoptosis, and calcium signaling are regulated by MAMs, which are thought to be closely related to ischemic reperfusion injury (IRI). However, there exists a gap in systematic proteomic research addressing the relationship between these cellular processes.MethodsA 4D label free mass spectrometry-based proteomic analysis of mitochondria associated membranes (MAMs) from the human renal proximal tubular epithelial cell line (HK-2 cells) was conducted under both normal (N) and hypoxia/reperfusion (HR) conditions. Subsequent differential proteins analysis aimed to characterize disease-relevant signaling molecules. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was applied to total proteins and differentially expressed proteins, encompassing Biological Process (BP), Cell Component (CC), Molecular Function (MF), and KEGG pathways. Further, Protein–Protein Interaction Network (PPI) exploration was carried out, leading to the identification of hub genes from differentially expressed proteins. Notably, Mitofusion 2 (MFN2) and BCL2/Adenovirus E1B 19-kDa interacting protein 3(BNIP3) were identified and subsequently validated both in vitro and in vivo. Finally, the impact of MFN2 on MAMs during hypoxia/reoxygenation was explored through regulation of gene expression. Subsequently, a comparative proteomics analysis was conducted between OE-MFN2 and normal HK-2 cells, providing further insights into the underlying mechanisms.ResultsA total of 4489 proteins were identified, with 3531 successfully quantified. GO/KEGG analysis revealed that MAM proteins were primarily associated with mitochondrial function and energy metabolism. Differential analysis between the two groups showed that 688 proteins in HR HK-2 cells exhibited significant changes in expression level with P-value < 0.05 and HR/N > 1.5 or HR/N < 0.66 set as the threshold criteria. Enrichment analysis of differentially expressed proteins unveiled biological processes such as mRNA splicing, apoptosis regulation, and cell division, while molecular functions were predominantly associated with energy metabolic activity. These proteins play key roles in the cellular responses during HR, offering insights into the IRI mechanisms and potential therapeutic targets. The validation of hub genes MFN2 and BNIP3 both in vitro and vivo was consistent with the proteomic findings. MFN2 demonstrated a protective role in maintaining the integrity of mitochondria associated membranes (MAMs) and mitigating mitochondrial damage following hypoxia/reoxygenation injury, this protective effect may be associated with the activation of the PI3K/AKT pathway.ConclusionsThe proteins located in mitochondria associated membranes (MAMs) are implicated in crucial roles during renal ischemic reperfusion injury (IRI), with MFN2 playing a pivotal regulatory role in this context.

MicroRNA-5010-5p ameliorates high-glucose induced inflammation in renal tubular epithelial cells by modulating the expression of PPP2R2D

IntroductionWe previously reported the significant upregulation of eight circulating exosomal microRNAs (miRNAs) in patients with diabetic kidney disease (DKD). However, their specific roles and molecular mechanisms in the kidney remain...

IFN-β Pretreatment Alleviates Allogeneic Renal Tubular Epithelial Cell-Induced NK Cell Responses via the IRF7/HLA-E/NKG2A Axis.

Immune checkpoint molecules are promising targets for suppressing the immune response but have received little attention in immune tolerance induction in organ transplantation. In this study, we found that IFN-β could induce the expression of HLA-E as well as PD-L1 on human renal tubular epithelial cell line HK-2 and renal tissue of the C57BL/6 mouse. The JAK/STAT2 pathway was necessary for this process. Upregulation of both HLA-E and PD-L1 was fully abrogated by the JAK1/2 inhibitor ruxolitinib. Signaling pathway molecules, including STAT1, STAT2, mTOR, Tyk2, and p38 MAPK, were involved in HLA-E and PD-L1 upregulation. IRF7 is the key transcription factor responsible for the activation of HLA-E and PD-L1 promoters. Through screening an epigenetic regulation library, we found a natural compound, bisdemethoxycurcumin, enhanced IFN-β-induced HLA-E and PD-L1 expression invitro and invivo. In PBMC-derived CD56+ NK cells, we found that NKG2A but not PD1 was constitutively expressed, indicating HLA-E/NKG2A as a more potent target to induce tolerance to innate immune cells. Pretreating HK-2 cells by IFN-β significantly attenuated the degranulation of their coincubated NK cells and protected cells from NK-mediated lysis. In conclusion, IFN-β pretreatment could activate HLA-E and PD-L1 transcription through the JAK/STAT/IRF7 pathway and then could protect renal tubular epithelial cells from allogeneic immune attack mediated by NK cells.

DACH1 attenuated PA-induced renal tubular injury through TLR4/MyD88/NF-κB and TGF-β/Smad signalling pathway.

Palmitic acid (PA), the major saturated fatty acid in the blood, often induces the initiation and progression of diabetic kidney disease (DKD). However, the underlying mechanism remains unclear. DACH1 is an important regulator of kidney functions. Herein, we investigated the roles of DACH1 in PA-induced kidney injury. Clinical data from the NHANES database were subjected to analyse the association between serum PA (sPA), blood glucose and kidney function. Molecular docking of PA was performed with DACH1. Immunohistochemistry, cell viability, annexin V/7-AAD double staining, TUNEL assay, immunofluorescent staining, autophagic flux analysis, qRT-PCR and western blot were performed. Clinical data confirmed that sPA was increased significantly in the pathoglycemia individuals compared with controls and correlated negatively with renal function. Our findings suggested that PA could dock with DACH1. DACH1 enhances cell viability by inhibiting apoptosis and attenuating autophagy blockage induced by PA. Furthermore, the results demonstrated that DACH1 ameliorated inflammation and fibrosis through TLR4/MyD88/NF-κB and TGF-β/Smad signalling pathway in PA-treated renal tubular epithelial cell line (HK-2). This study proved that sPA presents a risk factor for kidney injuries and DACH1 might serve as a protective target against renal function deterioration in diabetic patients.

Klotho activation of Nrf2 inhibits the ferroptosis signaling pathway to ameliorate sepsis-associated acute kidney injury.

Sepsis-associated acute kidney injury (SA-AKI) is a common complication of sepsis and greatly increases patient mortality. Recombinant human Klotho protein (Klotho) is a protective protein that can be secreted by the kidney. The aim of this study was to explore the protective effect of Klotho on SA-AKI and its molecular mechanism. In vivo, a mouse SA-AKI model was constructed by cecum ligation perforation (CLP). In vitro, a human renal tubular cell epithelial cell line (HK2) was induced with lipopolysaccharide (LPS) in the SA-AKI model. Determine renal injury markers, inflammatory factors, oxidative stress and molecular proteins related to the ferroptosis signaling pathway. Klotho reduced the release of renal injury markers and inflammatory cytokines, decreased oxidative stress, improved renal histopathological changes, ameliorated mitochondrial damage in mouse renal tubular epithelial cells, increased HK2 cell viability and reduced reactive oxygen species (ROS) accumulation. Exogenous supplementation with Klotho increased the Klotho content in circulating blood, renal tissue and HK2 cells. In the SA-AKI model, Klotho attenuated renal tissue injury, increased HK2 cell viability, decreased inflammatory factor expression and oxidative stress, restored tubular epithelial mitochondrial function, and increased its level in circulating blood, renal tissue and HK2 cells. Klotho probably exerts its protective effects by activating Nrf2 to inhibit the ferroptosis signaling pathway.

Cadmium exposure exacerbates kidney damage by inhibiting autophagy in diabetic rats

The incidence of diabetes mellitus (DM) is gradually increasing, making it a widespread global health concern. Cadmium (Cd) is a common toxic heavy metal in the environment, and cadmium exposure may be associated with diabetic nephropathy (DN). However, the mechanism of Cd-induced DN remains unclear. In this study, we aimed to determine the effect of cadmium on diabetic kidney injury and the underlying mechanism in diabetic rats and a renal tubular epithelial cell line (NRK-52E cells). Our results could provide novel insights on the nephrotoxic mechanism of cadmium. HE, PAS, and Masson staining were used to observe pathological renal injury. COL-I, COL-IV, CTSB, and CTSD protein levels were detected by immunohistochemistry and western blotting. Immunofluorescence was used to detect the fluorescence intensity of p62 and LC3 proteins in kidney tissue. TEM was used to observe the ultrastructure of mitochondria and number of autophagosomes. After cadmium exposure, DM rats showed a dramatic decrease in body weight compared to the unexposed DM group. Relative kidney weight showed a contrasting trend after cadmium exposure. Urinary microalbumin/creatinine significantly increased in normal and DM rats after cadmium exposure. However, the trend was clearer in the DM groups than in the control groups. Endogenous creatinine clearance exhibited a contrasting trend. After cadmium exposure in DM rats, MDA content significantly increased and GSH, CAT, SOD, and GSH-PX activation reduced compared to normal controls. Pathological damage was more pronounced, and the expression of autophagy related proteins and apoptosis and fibrosis proteins was significantly higher in vivo and vitro in the cadmium-exposed groups than in unexposed controls. Further, lysosomal protein levels were lower, and ROS content and autophagosome count significantly higher in the cadmium exposed groups compared to the unexposed controls. Therefore, Cadmium exposure aggravates diabetic kidney injury via autophagy inhibition.

Role of myo-inositol in acute kidney injury induced by cisplatin

There is an increasing evidence suggesting that myo-inositol (MI) may be a renoprotective factor. Our previous study revealed that decreased MI concentrations and increased excretion are often observed in animal models of renal injury and in patients with nephropathy. However, the role of MI supplementation in renal injury remains unclear. In this study, we aimed to explore the role of MI in cisplatin-induced acute kidney injury (AKI). We established a model of acute kidney injury caused by cisplatin (CDDP). Male Kunming mice were randomly divided into six groups: Sham (normal saline), CDDP (15 mg/kg), + MI (150 mg/kg), + MI (300 mg/kg), + MI (600 mg/kg) and MI (600 mg/kg). Human renal tubular epithelial cell line HK-2 cells were likewise separated into six groups at random: Control (normal saline), CDDP (20 µM), + MI (200 µM), + MI (400 µM), + MI (800 µM) and MI (800 µM). After the model was established, renal function indexes were subsequently detected, and experiments such as pathological staining analysis and protein expression analysis were performed. Our results showed that cisplatin administration led to AKI and apoptosis in mice and HK-2 cells, accompanied by markedly increased levels of MIOX, kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL), whereas exogenous MI significantly attenuated kidney injury and HK-2 cell damage induced by cisplatin both in vivo and in vitro by inhibiting excessive apoptosis. Overall, our findings demonstrate that exogenous MI can reduce excessive apoptosis, thus playing a protective role in cisplatin-induced AKI, indicating that exogenous MI may be used as an adjunctive treatment modality in cisplatin-induced AKI.

Gastrodin alleviates cisplatin nephrotoxicity by inhibiting ferroptosis via the SIRT1/FOXO3A/GPX4 signaling pathway

Ethnopharmacological relevanceCisplatin (CP) results in acute kidney injury (AKI) and negatively affects patients’ therapy and survival. The dried rhizome of Gastrodia elata Blume has been used to treat clinical kidney diseases. Gastrodin (GAS) is an active ingredient of the G. elata tuber. It is unknown whether GAS can alleviate CP-induced AKI. Aim of the studyThis study aimed to investigate whether GAS, an active ingredient of G. elata Blume, can alleviate CP-induced AKI and to explore its underlying mechanisms. Materials and methodsExperiments were conducted with a CP-induced AKI mouse model and an immortalized human renal tubular epithelial cell line (HK-2). Serum creatinine, Periodic acid–Schiff staining, tissue iron, glutathione, malondialdehyde, and 4-Hydroxynonenal were detected in serum and kidney samples to observe whether GAS inhibits CP-induced tubule ferroptosis. The drug target was verified by detecting the effects of GAS on sirtuin-1 (SIRT1) activity in vitro. Transcriptional regulation of glutathione peroxidase 4 (GPX4) by forkhead box O3A (FOXO3A) was verified by siRNA knockdown, overexpression, and chromatin immunoprecipitation. The effects of FOXO3A, SIRT1, and GAS on CP-induced ferroptosis were measured with propidium iodide, dihydroethidium, monobromobimane, and dipyrromethene boron difluoride staining in HK-2 cells. The relationship between GAS and the SIRT1/FOXO3A/GPX4 pathway was studied using Western blotting. ResultsGAS treatment inhibited CP-induced reactive oxygen species, lipid peroxidation, and tubule death in the cell and animal models. GAS activated SIRT1 in vitro. The SIRT1 inhibitor blocked the protective role of GAS in reducing lipid peroxidation in HK-2 cells. FOXO3A transcriptionally regulated GPX4 expression and inhibited CP-induced cell ferroptosis. Compared to CP-damaged mouse kidneys, GAS-treated mice demonstrated significantly increased SIRT1 and GPX4 expression levels, decreased CP-induced acetylation of FOXO3A, and inhibited lipid peroxidation and cell death. ConclusionsGAS alleviated CP-induced AKI by inhibiting ferroptosis via the SIRT1/FOXO3A/GPX4 signaling pathway. The results offer new insights into the development of new anti-AKI drugs from traditional Chinese medicine.

YAP/ACSL4 Pathway-Mediated Ferroptosis Promotes Renal Fibrosis in the Presence of Kidney Stones.

The potential association between calcium oxalate stones and renal fibrosis has been extensively investigated; however, the underlying mechanisms remain unclear. Ferroptosis is a novel form of cell death characterized by iron-dependent lipid peroxidation and regulated by acyl coenzyme A synthase long-chain family member 4 (ACSL4). Yes-associated protein (YAP), a transcriptional co-activator in the Hippo pathway, promotes ferroptosis by modulating ACSL4 expression. Nevertheless, the involvement of YAP-ACSL4 axis-mediated ferroptosis in calcium oxalate crystal deposition-induced renal fibrosis and its molecular mechanisms have not been elucidated. In this study, we investigated ACSL4 expression and ferroptosis activation in the kidney tissues of patients with calcium oxalate stones and in mice using single-cell sequencing, transcriptome RNA sequencing, immunohistochemical analysis, and Western blot analysis. In vivo and in vitro experiments demonstrated that inhibiting ferroptosis or ACSL4 mitigated calcium oxalate crystal-induced renal fibrosis. Furthermore, YAP expression was elevated in the kidney tissues of patients with calcium oxalate stones and in calcium oxalate crystal-stimulated human renal tubular epithelial cell lines. Mechanistically, in calcium oxalate crystal-stimulated human renal tubular epithelial cell lines, activated YAP translocated to the nucleus and enhanced ACSL4 expression, consequently inducing cellular ferroptosis. Moreover, YAP silencing suppressed ferroptosis by downregulating ACSL4 expression, thereby attenuating calcium oxalate crystal-induced renal fibrosis. Conclusively, our findings suggest that YAP-ACSL4-mediated ferroptosis represents an important mechanism underlying the induction of renal fibrosis by calcium oxalate crystal deposition. Targeting the YAP-ACSL4 axis and ferroptosis may therefore hold promise as a potential therapeutic approach for preventing renal fibrosis in patients with kidney stones.

HK-2 cell response to TGF-β highly depends on cell culture medium formulations

The immortalized human renal proximal tubular epithelial cell line HK-2 is most commonly used to study renal cell physiology and human kidney diseases with tubulointerstitial fibrosis such as diabetic nephropathy, obstructive uropathy or allograft fibrosis. Epithelial-to-mesenchymal transition (EMT) is the main pathological process of tubulointerstitial fibrosis in vitro. Transforming growth factor-beta (TGF-β) is a key inducer of EMT. Several pro-fibrotic gene expression differences have been observed in a TGF-β-induced EMT model of HK-2 cells. However, growth conditions and medium formulations might greatly impact these differences. We investigated gene and protein expression of HK-2 cells cultured in six medium formulations. TGF-β1 increased the expression of ACTA2, TGFB1, COL4A1, EGR2, VIM and CTGF genes while reducing PPARG in all medium formulations. Interestingly, TGF-β1 treatment either increased or decreased EGR1, FN, IL6 and C3 gene expression, depending on medium formulations. The cell morphology was slightly affected, but immunoblots revealed TGFB1 and vimentin protein overexpression in all media. However, fibronectin expression as well as the nuclear translocation of EGR1 was medium dependent. In conclusion, our study demonstrates that, using the HK-2 in vitro model of EMT, the meticulous selection of appropriate cell culture medium formulation is essential to achieve reliable scientific results.

Proteasomes Are Critical for Maintenance of CD133+CD24+ Kidney Progenitor Cells.

Kidney progenitor cells, although rare and dispersed, play a key role in the repair of renal tubules after acute kidney damage. However, understanding these cells has been challenging due to the limited access to primary renal tissues and the absence of immortalized cells to model kidney progenitors. Previously, our laboratory utilized the renal proximal tubular epithelial cell line, RPTEC/TERT1, and the flow cytometry technique to sort and establish a kidney progenitor cell model called Human Renal Tubular Precursor TERT (HRTPT) which expresses CD133 and CD24 and exhibits the characteristics of kidney progenitors, such as self-renewal capacity and multi-potential differentiation. In addition, a separate cell line was established, named Human Renal Epithelial Cell 24 TERT (HREC24T), which lacks CD133 expression and shows no progenitor features. To further characterize HRTPT CD133+CD24+ progenitor cells, we performed proteomic profiling which showed high proteasomal expression in HRTPT kidney progenitor cells. RT-qPCR, Western blot, and flow cytometry analysis showed that HRTPT cells possess higher proteasomal expression and activity compared to HREC24T non-progenitor cells. Importantly, inhibition of the proteasomes with bortezomib reduced the expression of progenitor markers and obliterated the potential for self-renewal and differentiation of HRTPT progenitor cells. In conclusion, proteasomes are critical in preserving progenitor markers expression and self-renewal capacity in HRTPT kidney progenitors.

Dietary docosahexaenoic acid plays an opposed role in ferroptotic and non-ferroptotic acute kidney injury

Ferroptosis due to polyunsaturated fatty acid (PUFA) peroxidation has been implicated in the pathogenesis of acute kidney injury (AKI), suggesting the risk of dietary intake of PUFA for people susceptible to AKI. Clinically, however, in addition to ferroptosis, other mechanisms also contribute to different types of AKI such as inflammation associated necroptosis and pyroptosis. Therefore, the role of PUFA, especially ω3 PUFA which is a common food supplement, in various AKIs deserves further evaluation. In this study, rhabdomyolysis- and folic acid-induced AKI (Rha-AKI and FA-AKI) were established in mice fed with different fatty acids Histology of kidney, blood urea nitrogen and creatinine, lipid peroxidation, and inflammatory factors were examined. Results showed that these two types of AKIs had diametrically different pathogenesis indicated by that ferrostatin-1 (Fer-1), a lipid antioxidant, can attenuate FA-AKI rather than Rha-AKI. Further, dietary DHA (provided by fish oil) reduced tubular injury and renal lesion by inhibiting peroxidation and inflammation in mice with Rha-AKI while increasing cell death, tissue damage, peroxidation and inflammation in mice with FA-AKI. In human renal tubular epithelial cell line HK-2, MTT assay and DHE staining showed that both myoglobin and ferroptosis inducers can cause cell death and oxidative stress. Ferroptosis inducer-induced cell death was promoted by DHA, while such result was not observed in myoglobin-induced cell death when adding DHA. This study illustrates that the mechanisms of AKI might be either ferroptosis dependent or -independent and the deterioration effect of dietary DHA depends on whether ferroptosis is involved.