[Dexmedetomidine inhibits ferroptosis of human renal tubular epithelial cells by activating the Nrf2/HO-1/GPX4 pathway].

OBJECTIVE: To investigate the protective effect of dexmedetomidine (DEX) against erastin-induced ferroptosis in human renal tubular epithelial cells (HK-2 cells) and explore the underlying mechanism. METHODS: HK-2 cells were treated with erastin alone or in combination with different concentrations (2.5, 5.0 and 10 µmol/L) of DEX, and the changes in cell viability were observed using CCK-8 assay. To explore the mechanism by which DEX inhibits erastin-induced ferroptosis, HK-2 cells were treated with erastin, erastin+10 µmol/L DEX, or erastin+10 µmol/L DEX+ML385 (a Nrf2 inhibitor), after which the cell viability was assessed. The level of intracellular Fe2+ was detected by cell ferrous iron colorimetric assay kit, and flow cytometry was performed to detect reactive oxygen species (ROS); MDA and reduced glutathione assay kits were used to detect the contents of MDA and GSH in the cells; The expressions of Nrf2, HO-1 and GPX4 proteins were detected by Western blotting. RESULTS: Erastin treatment significantly inhibited the viability of the cells, decreased GSH content, and increased intracellular levels of Fe2+, ROS and MDA. The combined treatment with 10 µmol/L DEX markedly increased the viability of the cells, increased GSH content, reduced the levels of Fe2+, ROS and MDA, and upregulated the protein expressions of Nrf2, HO-1 and GPX4 in the cells. The application of ML385 obviously blocked the protective effect of DEX and caused significant inhibition of the Nrf2/HO-1/GPX4 pathway, decreased the cell viability and GSH content, and increased the levels of Fe2+, ROS and MDA in HK-2 cells. CONCLUSION: The protective effect of DEX against erastin-induced ferroptosis of HK-2 cells is probably mediated by activation of the Nrf2/HO-1/GPX4 pathway to inhibit oxidative stress.

USP19 Stabilizes TAK1 to Regulate High Glucose/Free Fatty Acid-induced Dysfunction in HK-2 Cells.

OBJECTIVE: Obesity-induced kidney injury contributes to the development of diabetic nephropathy (DN). Here, we identified the functions of ubiquitin-specific peptidase 19 (USP19) in HK-2 cells exposed to a combination of high glucose (HG) and free fatty acid (FFA) and determined its association with TGF-beta-activated kinase 1 (TAK1). METHODS: HK-2 cells were exposed to a combination of HG and FFA. USP19 mRNA expression was detected by quantitative RT-PCR (qRT-PCR), and protein analysis was performed by immunoblotting (IB). Cell growth was assessed by Cell Counting Kit-8 (CCK-8) viability and 5-ethynyl-2'-deoxyuridine (EdU) proliferation assays. Cell cycle distribution and apoptosis were detected by flow cytometry. The USP19/TAK1 interaction and ubiquitinated TAK1 levels were assayed by coimmunoprecipitation (Co-IP) assays and IB. RESULTS: In HG+FFA-challenged HK-2 cells, USP19 was highly expressed. USP19 knockdown attenuated HG+FFA-triggered growth inhibition and apoptosis promotion in HK-2 cells. Moreover, USP19 knockdown alleviated HG+FFA-mediated PTEN-induced putative kinase 1 (PINK1)/Parkin pathway inactivation and increased mitochondrial reactive oxygen species (ROS) generation in HK-2 cells. Mechanistically, USP19 stabilized the TAK1 protein through deubiquitination. Importantly, increased TAK1 expression reversed the USP19 knockdown-mediated phenotypic changes and PINK1/Parkin pathway activation in HG+FFA-challenged HK-2 cells. CONCLUSION: The findings revealed that USP19 plays a crucial role in promoting HK-2 cell dysfunction induced by combined stimulation with HG and FFAs by stabilizing TAK1, providing a potential therapeutic strategy for combating DN.

Selenoprotein-P1 (SEPP1) Expression in Human Proximal Tubule Cells after Ischemia-Reperfusion Injury: An In Vitro Model.

Background and Objectives: Selenium deficiency represents a risk factor for the occurrence of severe diseases, such as acute kidney injury (AKI). Recently, selenoprotein-p1 (SEPP1), a selenium transporter, mainly released by the liver, has emerged as a promising plasmatic biomarker of AKI as a consequence of cardio-surgery operations. The aim of the present study was to investigate, on an in vitro model of hypoxia induced in renal tubular cells, HK-2, the effects of sodium selenite (Na2SeO3) and to evaluate the expression of SEPP1 as a marker of injury. Materials and Methods: HK-2 cells were pre-incubated with 100 nM Na2SeO3 for 24 h, and then, treated for 24 h with CoCl2 (500 µM), a chemical hypoxia inducer. The results were derived from an ROS assay, MTT, and Western blot analysis. Results: The pre-treatment determined an increase in cells' viability and a reduction in reactive oxygen species (ROS), as shown by MTT and the ROS assay. Moreover, by Western blot an increase in SEPP1 expression was observed after hypoxic injury as after adding sodium selenite. Conclusions: Our preliminary results shed light on the possible role of selenium supplementation as a means to prevent oxidative damage and to increase SEPP1 after acute kidney injury. In our in vitro model, SEPP1 emerges as a promising biomarker of kidney injury, although further studies in vivo are necessary to validate our findings.

MiRNA-133a-3p Attenuates Renal Tubular Epithelial Cell Injury via Targeting MALM1 and Suppressing the Notch Signaling Pathway in Diabetic Nephropathy.

Diabetic nephropathy (DN) is a serious microvascular complication of diabetes characterized by structural and functional changes of kidneys. Human renal tubular epithelial (HK-2) cells are important for kidney recovery post injury and usually used for establishment of DN cell models. The study explored the role of microRNA (miR)-133a-3p in DN cell model and animal model. A cell model for DN was established via high glucose (HG) stimulation to HK-2 cells. Cell viability and apoptotic rate were measured by cell counting kit 8 and flow cytometry. Polymerase chain reaction was performed to quantify levels of miR-133a-3p and targets. Luciferase reporter assay was conducted to verify the binding of miR-133a-3p and MAML1. After establishment of a mouse model of DN, levels of renal function indicators were measured by biochemical analysis. Hematoxylin-eosin and periodic acid-schiff staining of kidney samples were performed to analyze histological changes. Western blotting was conducted to quantify levels of apoptotic markers, MAML1, and factors related to Notch signaling. Results showed that HG induced HK-2 cell apoptosis and the reduction of cell viability. MiR-133a-3p was lowly expressed in HG-stimulated HK-2 cells. Overexpressed miR-133a-3p improved HK-2 cell injury by increasing cell viability and hampering apoptosis under HG condition. In addition, miR-133a-3p directly targets MAML1 3'-untranslated region. MAML1 overexpression countervailed the repressive impact of miR-133a-3p on cell apoptosis in the context of HG. Moreover, miR-133a-3p inhibited the activity of Notch pathway by downregulating MAML1. MiR-133a-3p inhibits DN progression in mice, as evidenced by reduced fasting blood glucose level, improved levels of renal function parameters, and alleviation of kidney atrophy. In conclusion, miR-133a-3p improves HG-induced HK-2 cell injury and inhibits DN progression by targeting MAML1 and inactivating Notch signaling.

Astragaloside IV attenuates renal tubule injury in DKD rats via suppression of CD36-mediated NLRP3 inflammasome activation.

Background: In recent years, diabetic kidney disease (DKD) has emerged as a prominent factor contributing to end-stage renal disease. Tubulointerstitial inflammation and lipid accumulation have been identified as key factors in the development of DKD. Earlier research indicated that Astragaloside IV (AS-IV) reduces inflammation and oxidative stress, controls lipid accumulation, and provides protection to the kidneys. Nevertheless, the mechanisms responsible for its protective effects against DKD have not yet been completely elucidated. Purpose: The primary objective of this research was to examine the protective properties of AS-IV against DKD and investigate the underlying mechanism, which involves CD36, reactive oxygen species (ROS), NLR family pyrin domain containing 3 (NLRP3), and interleukin-1ß (IL-1ß). Methods: The DKD rat model was created by administering streptozotocin along with a high-fat diet. Subsequently, the DKD rats and palmitic acid (PA)-induced HK-2 cells were treated with AS-IV. Atorvastatin was used as the positive control. To assess the therapeutic effects of AS-IV on DKD, various tests including blood sugar levels, the lipid profile, renal function, and histopathological examinations were conducted. The levels of CD36, ROS, NLRP3, Caspase-1, and IL-1ß were detected using western blot analysis, PCR, and flow cytometry. Furthermore, adenovirus-mediated CD36 overexpression was applied to explore the underlying mechanisms through in vitro experiments. Results: In vivo experiments demonstrated that AS-IV significantly reduced hyperglycemia, dyslipidemia, urinary albumin excretion, and serum creatinine levels in DKD rats. Additionally, it improved renal structural abnormalities and suppressed the expression of CD36, NLRP3, IL-1ß, TNF-α, and MCP-1. In vitro experiments showed that AS-IV decreased CD36 expression, lipid accumulation, and lipid ROS production while inhibiting NLRP3 activation and IL-1ß secretion in PA-induced HK-2 cells. Conclusion: AS-IV alleviated renal tubule interstitial inflammation and tubule epithelial cell apoptosis in DKD rats by inhibiting CD36-mediated lipid accumulation and NLRP3 inflammasome activation.

The safety evaluation of Shenze Shugan capsule and mechanism of apoptosis induced by five potentially nephrotoxic components.

ETHNOPHARMACOLOGICAL RELEVANCE: Shenze Shugan capsule is a prescription of traditional Chinese medicine for nonalcoholic steatohepatitis treatment. It includes Rhei Radix et Rhizoma (RR), Cassiae Semen (CS) and Alismatis Rhizoma(AR), which widely contains rhein, emodin, aurantio-obtusin, alisol A and alisol B 23-monoacetate. AIM OF THE STUDY: In this study, we aimed to explore the safety of the medicine, and further elucidate the mechanism of apoptosis induction in HK-2 cells by five components, including rhein, emodin, aurantio-obtusin, alisol A and alisol B 23-monoacetate. MATERIALS AND METHODS: We investigated the nephrotoxicity of Shenze Shugan capsule, including RR, CS, AR and mixed herbs given for two months in rats. Superoxide dismutase (SOD) in kidney tissues, urea nitrogen (BUN) and creatinine (CRE) in serum were detected, and renal pathology analysis was performed. In cell experiments, the apoptotic rate and cell cycle distribution of HK-2 cells were tested by flow cytometry. The levels of mitochondrial membrane potential (ΔΨm) and related protein expression in mitochondrial pathway were measured as well. RESULTS: We confirmed that two months of administering high doses(60 times the dose for clinical use in adults) of RR, CS or mixed herbs upregulated the levels of CRE and RUN, inhibited SOD activity, and increased the degree of tubular degeneration and glomerular dilatation, but Shenze Shugan capsule has no significant differences in renal structure or renal function. In addition, we found that five components all concentration-dependently inhibited HK-2 cells proliferation and induced apoptosis, especially aurantio-obtusin as the novel nephrotoxic component. Rhein and emodin significantly induced S/M accumulation, but aurantio-obtusin, alisol A and alisol B 23-monoacetate significantly induced G1/M accumulation in HK-2 cells. Similarly, they could induce Caspase3 activation, loss of mitochondrial membrane potential (ΔΨm), and down-regulation of Bcl-2 and up-regulation of Bax. CONCLUSIONS: Through a two-month subchronic toxicity study in rats, our preliminary determination is that this formulation is safe and reliable for long-term use. Interestingly, the potentially toxic herbs such as RR, CS, AR can reduce toxicity by drug compatibility. When further exploring the mechanism of action of toxic herbs, we found that mitochondrial pathway is involved in the apoptosis of HK -2 cells induced by rhein, emodin, aurantio-obtusin, alisol A and alisol B 23-monoacetate. Our findings provide new ideas for safety studies of Shenze Shugan capsule.

Role of APE1/Ref-1 in hydrogen peroxide-induced apoptosis in human renal HK-2 cells.

BACKGROUND: Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a multipotent protein that plays essential roles in cellular responses to oxidative stress. METHODS: To examine the role of APE1/Ref-1 in ischemia-reperfusion (I/R) injuries and hydrogen peroxide (H2O2)-induced renal tubular apoptosis, we studied male C57BL6 mice and human proximal tubular epithelial (HK-2) cells treated with H2O2 at different concentrations. The colocalization of APE1/Ref-1 in the proximal tubule, distal tubule, thick ascending limb, and collecting duct was observed with confocal microscopy. The overexpression of APE1/Ref-1 with knockdown cell lines using an APE1/Ref-1-specific DNA or small interfering RNA (siRNA) was used for the apoptosis assay. The promotor activity of nuclear factor kappa B (NF-κB) was assessed and electrophoretic mobility shift assay was conducted. RESULTS: APE1/Ref-1 was predominantly localized to the renal tubule nucleus. In renal I/R injuries, the levels of APE1/Ref-1 protein were increased compared with those in kidneys subjected to sham operations. The overexpression of APE1/Ref-1 in HK-2 cells enhanced the Bax/Bcl-2 ratio as a marker of apoptosis. Conversely, the suppression of APE1/Ref-1 expression by siRNA in 1-mM H2O2-treated HK-2 cells decreased the Bax/Bcl-2 ratio, the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, p38, c-Jun N-terminal kinase (JNK) 1/2, and NF-κB. In HK-2 cells, the promoter activity of NF-κB increased following H2O2 exposure, and this effect was further enhanced by APE1/Ref-1 transfection. CONCLUSION: The inhibition of APE1/Ref-1 with siRNA attenuated H2O2-induced apoptosis through the modulation of mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 and the nuclear activation of NF-κB and proapoptotic factors.

Ginsenoside Rg1 Suppresses Ferroptosis of Renal Tubular Epithelial Cells in Sepsis-induced Acute Kidney Injury via the FSP1-CoQ10- NAD(P)H Pathway.

INTRODUCTION: Sepsis-induced acute kidney injury is related to an increased mortality rate by modulating ferroptosis through ginsenoside Rg1. In this study, we explored the specific mechanism of it. METHODS: Human renal tubular epithelial cells (HK-2) were transfected with oe-ferroptosis suppressor protein 1 and treated with lipopolysaccharide for ferroptosis induction, and they were then treated with ginsenoside Rg1 and ferroptosis suppressor protein 1 inhibitor. Ferroptosis suppressor protein 1, CoQ10, CoQ10H2, and intracellular NADH levels in HK-2 cells were assessed by Western blot, ELISA kit, and NAD/NADH kit. NAD+/NADH ratio was also calculated, and 4-Hydroxynonal fluorescence intensity was assessed by immunofluorescence. HK-2 cell viability and death were assessed by CCK-8 and propidium iodide staining. Ferroptosis, lipid peroxidation, and reactive oxygen species accumulation were assessed by Western blot, kits, flow cytometry, and C11 BODIPY 581/591 molecular probe. Sepsis rat models were established by cecal ligation and perforation to investigate whether ginsenoside Rg1 regulated the ferroptosis suppressor protein 1-CoQ10-NAD(P)H pathway in vivo. RESULTS: LPS treatment diminished ferroptosis suppressor protein 1, CoQ10, CoQ10H2, and NADH contents in HK-2 cells, while facilitating NAD+/NADH ratio and relative 4- Hydroxynonal fluorescence intensity. FSP1 overexpression inhibited lipopolysaccharideinduced lipid peroxidation in HK-2 cells via the ferroptosis suppressor protein 1-CoQ10- NAD(P)H pathway. The ferroptosis suppressor protein 1-CoQ10-NAD(P)H pathway suppressed lipopolysaccharide-induced ferroptosis in HK-2 cells. Ginsenoside Rg1 alleviated ferroptosis in HK-2 cells by regulating the ferroptosis suppressor protein 1-CoQ10- NAD(P)H pathway. Moreover, ginsenoside Rg1 regulated the ferroptosis suppressor protein 1-CoQ10-NAD(P)H pathway in vivo. CONCLUSION: Ginsenoside Rg1 alleviated sepsis-induced acute kidney injury by blocking renal tubular epithelial cell ferroptosis via the ferroptosis suppressor protein 1-CoQ10- NAD(P)H pathway.

Angiotensin 1-7 exerts antioxidant effects, suppresses Mammalian Target of Rapamycin (mTOR) signaling, and inhibits apoptosis in renal proximal tubular cells.

Oxidative stress is one of the crucial pathogenic factors involved in the progression of renal injury. Angiotensin (ANG) 1-7, a bioactive heptapeptide of the renin-angiotensin-aldosterone system is known to exert antioxidant and nephroprotective effects. However, the cellular mechanism involved in the beneficial effect of ANG 1-7 is not clear. Here, we assessed ANG 1-7's effect on H2O2-mediated oxidative damage in the human proximal tubular (HK2) cells and the underlying mechanisms. HK2 cells were incubated with H2O2 (500 µM, 4 h) pre-treated with and without ANG 1-7 (100 nM, 24 h), and reactive oxygen species (ROS) generation, mitochondrial dysfunction, endoplasmic reticulum (ER) stress, apoptosis and mammalian target of rapamycin (mTOR) signaling were determined H2O2 induced an increase in oxidative and ER stress together with loss of mitochondrial membrane potential, decreased ATP levels, and induced apoptosis in HK2 cells. Moreover, H2O2 treatment resulted in the activation of mTOR complexes (mTORC1 and mTORC2) in these cells. ANG 1-7 significantly attenuated H2O2-induced ROS generation, ER stress and apoptosis, and also improved mitochondrial function. Additionally, pre-treatment of ANG 1-7 inhibited the H2O2-mediated mTOR activation. These effects of ANG 1-7 were blocked by co-treatment with the Mas receptor (MasR) inhibitor, A779. Furthermore, transfection of HK2 cells with Mas receptor siRNA also abolished the inhibitory effect of ANG 1-7 on mTOR activities. In conclusion, ANG 1-7 via MasR mitigates oxidative stress, suppresses mTOR signaling, and protects HK2 cells from ER stress, mitochondrial dysfunction, and apoptosis, suggesting ANG 1-7-MasR renoprotective effects.

[Role of SPP1 in acute kidney injury induced by renal ischemia-reperfusion in rats].

OBJECTIVE: To investigate the role of SPP1 gene in acute kidney injury induced by renal ischemia-reperfusion injury (IRI). METHODS: Twelve Sprague-Dawley rats were randomly divided into sham group and IRI group (n=6) and subjected to sham operation and renal ischemia for 30 min induced by penal pedicle clamping using non-traumatic microvascular clamps, respectively.Serum creatinine and blood urea nitrogen levels were detected, and PAS staining was used for pathological examination of the kidneys in the two groups.The renal expressions of SPP1, α-SMA and caspase-3 were detected using immunohistochemistry and immunofluorescent staining.In cultured renal tubular epithelial cells (HK-2 cells), Western blotting was performed to detect the changes in expressions of SPP1, caspase-3, and Kim-1 proteins following hypoxiareoxygenation (H/R) and transfection with si-NC or si-SPP1;flow cytometry was employed to analyze apoptosis of the treated cells. RESULTS: Renal IRI caused significant elevations of serum creatinine and blood urea nitrogen levels (P<0.05) and induced severe shedding and necrosis of the renal tubular epithelial cells in the rats, resulting also in significantly up-regulated renal expressions of SPP1, α-SMA and caspase-3(P<0.05).In HK-2 cells, H/R significantly increased the protein expression levels of SPP1, caspase-3, and Kim-1(P<0.05), and compared si-NC transfection, transfection with SPP1 obviously reduced caspase-3 and Kim-1 expressions and lowered apoptosis rate of the cells with H/R exposure (P<0.05). CONCLUSION: SPP1 is up-regulated in the kidneys of rats with renal IRI, and down-regulation of SPP1 expression can inhibit H/R-induced apoptosis of renal tubular epithelial cells.

Analysis of Human microRNA Expression Profiling During Diquat-Induced Renal Proximal Tubular Epithelial Cell Injury.

Background: We established a diquat-induced human kidney-2 cells (HK-2 cells) apoptosis model in this study to identify differentially expressed microRNAs (miRNAs) and signaling pathways involved in diquat poisoning via gene sequencing and bioinformatics analysis and explored the related therapeutic benefits. Methods: The effects of diquat on the viability and apoptosis of HK-2 cells were explored using the CCK-8 and Annexin V-FITC/PI double staining methods. Total RNAs were extracted using the TRizol method and detected by Illumina HiSeq 2500. Bioinformatics analysis was performed to explore differentially expressed (DE) miRNAs, their enriched biological processes, pathways, and potential target genes. The RT-qPCR method was used to verify the reliability of the results. Results: Diquat led to HK-2 cell injury and apoptosis played an important role, hence an HK-2 cell apoptosis model in diquat poisoning was established. Thirty-six DE miRNAs were screened in diquat-treated HK-2 cells. The enriched biological process terms were mainly cell growth, regulation of apoptotic signaling pathway, extrinsic apoptotic signaling pathway, and Ras protein signal transduction. The enriched cellular components were mainly cell-cell junction, cell-substrate junction, ubiquitin ligase complex, and protein kinase complex. The enriched molecular functions were mainly Ras GTPase binding, ubiquitin-like protein transferase activity, DNA-binding transcription factor binding, ubiquitin-protein transferase activity, nucleoside-triphosphatase regulator activity, transcription coactivator activity, and ubiquitin-like protein ligase binding. Signaling pathways such as MAPK, FoxO, Ras, PIK3-Akt, and Wnt were also enriched. Conclusion: These findings aid in understanding the mechanisms of diquat poisoning and the related pathways, where DE miRNAs serve as targets for gene therapy.

Delivery and Transcriptome Assessment of an In Vitro Three-Dimensional Proximal Tubule Model Established by Human Kidney 2 Cells in Clinical Gelatin Sponges.

The high prevalence of kidney diseases and the low identification rate of drug nephrotoxicity in preclinical studies reinforce the need for representative yet feasible renal models. Although in vitro cell-based models utilizing renal proximal tubules are widely used for kidney research, many proximal tubule cell (PTC) lines have been indicated to be less sensitive to nephrotoxins, mainly due to altered expression of transporters under a two-dimensional culture (2D) environment. Here, we selected HK-2 cells to establish a simplified three-dimensional (3D) model using gelatin sponges as scaffolds. In addition to cell viability and morphology, we conducted a comprehensive transcriptome comparison and correlation analysis of 2D and 3D cultured HK-2 cells to native human PTCs. Our 3D model displayed stable and long-term growth with a tubule-like morphology and demonstrated a more comparable gene expression profile to native human PTCs compared to the 2D model. Many missing or low expressions of major genes involved in PTC transport and metabolic processes were restored, which is crucial for successful nephrotoxicity prediction. Consequently, we established a cost-effective yet more representative model for in vivo PTC studies and presented a comprehensive transcriptome analysis for the systematic characterization of PTC lines.

Sweroside plays a role in mitigating high glucose-induced damage in human renal tubular epithelial HK-2 cells by regulating the SIRT1/NF-κB signaling pathway.

Sweroside is a natural monoterpene derived from Swertia pseudochinensis Hara. Recently, studies have shown that sweroside exhibits a variety of biological activities, such as anti-inflammatory, antioxidant, and hypoglycemic effects. However, its role and mechanisms in high glucose (HG)-induced renal injury remain unclear. Herein, we established a renal injury model in vitro by inducing human renal tubular epithelial cell (HK-2 cells) injury by HG. Then, the effects of sweroside on HK-2 cell activity, inflammation, reactive oxygen species (ROS) production, and epithelial mesenchymal transition (EMT) were observed. As a result, sweroside treatment ameliorated the viability, inhibited the secretion of inflammatory cytokines (TNF-α, IL-1ß, and VCAM-1), reduced the generation of ROS, and inhibited EMT in HK-2 cells. Moreover, the protein expression of SIRT1 was increased and the acetylation of p65 NF-kB was decreased in HK-2 cells with sweroside treatment. More importantly, EX527, an inhibitor of SIRT1, that inactivated SIRT1, abolished the improvement effects of sweroside on HK-2 cells. Our findings suggested that sweroside may mitigate HG-caused injury in HK-2 cells by promoting SIRT1-mediated deacetylation of p65 NF-kB.

Polystyrene nanoplastics induce apoptosis of human kidney proximal tubular epithelial cells via oxidative stress and MAPK signaling pathways.

Polystyrene nanoplastics (PS-NPs) have recently been found to be present in human blood and kidney. However, the renal toxicity of PS-NPs and the underlying mechanisms have not been fully elucidated. Here, we found that exposure of PS-NPs induced apoptosis of human renal proximal tubular epithelial cells (HK-2) in a size- and dose-dependent manner as revealed by AnnexinV-FITC assay. In addition, PS-NPs promoted ROS production and caused structure changes of mitochondrial and endoplasmic reticulum. Mechanistically, transcriptional sequencing indicated the involvement of MAPK pathway in apoptosis, which was further confirmed by the upregulation of p-p38, p-ERK, CHOP, BAX, cytochrome C, and caspase 3 expression. This study clarified the molecular mechanism underlying PS-NP-induced apoptosis in HK-2 cells and contributed to our risk estimation of PS-NPs in human kidney.

CircTLK1 alleviates oxygen-glucose deprivation/reperfusion induced apoptosis in HK-2 cells through miR-136-5p/Bcl2 signal axis.

The biological functions of circTLK1 in acute kidney injury (AKI), which mainly results from renal ischemia-reperfusion (IR), remain largely unknown. HK-2 cell treatment with oxygen and glucose deprivation, reoxygenation, and glucose (OGD/R) was used to simulate an AKI model that was mainly caused by renal IR. Then, the circTLK1 expression level in HK-2 cells treated with OGD/R was assessed by quantitative reverse transcription polymerase chain reaction (RT-qPCR). Functional experiments were performed with circTLK1 knockdown of HK-2 cells via Cell Counting Kit-8 (CCK8), flow cytometry (FCM), RT-qPCR, and western blotting. The circTLK1-miRNAs-mRNAs network was constructed following the ceRNA mechanism and visualized by Cytoscape software to investigate the mechanism of circTLK1 in AKI. RT-qPCR was performed to verify the relationship between circTLK1, miR-136-5p, and Bcl2. The level of miR-136-5p was knocked down to ensure its function in OGD/R-triggered apoptosis through experiments, including CCK8, FCM, RT-qPCR, and western blotting. CircTLK1 was downregulated in HK-2 cells subjected to OGD/R treatment and in mouse kidney tissues after renal IR, but the expression of miR-136-5p was the opposite. Interference with circTLK1 expression accelerated HK-2 cell apoptosis, which was overturned by miR-136-5p inhibitors. CircTLK1 targets miR-136-5p to upregulate Bcl2 expression and attenuate apoptosis in HK-2 cells. These data revealed the possible role of circTLK1 as a new biomarker for diagnosis as well as a target in AKI through the miR-136-5p/Bcl2 signaling axis.

LncRNA CASC15 inhibition relieves renal fibrosis in diabetic nephropathy through down-regulating SP-A by sponging to miR-424.

Study has demonstrated the abnormal expression and role of lncRNA CASC15 in diabetes patients with chronic renal failure. However, its role in diabetes nephropathy (DN) is still unclear. This study aimed to investigate the potential mechanism and role of lncRNA CASC15 in DN. The relationship between miR-424 and CASC15/SP-A was predicted by Starbase software and verified by luciferase reporter assay. HK-2 cells were treated with 25 mM glucose (HG) for 24 h to establish DN cell model. MTT and flow cytometry analysis were carried out to test cell proliferation and apoptosis. Epithelial-to-mesenchymal transition (EMT) markers were analyzed by RT-qPCR and western blot assay. We proved that CASC15 could interact with miR-424, and SP-A was a target of miR-424. HG-treatment significantly enhanced lncRNA CASC15 level and decreased miR-424 level in HK-2 cells. LncRNA CASC15-siRNA significantly improved cell viability, repressed apoptosis, promoted E-cadherin expression, and inhibited N-cadherin expression in HG-treated HK-2 cells, and these effects were reversed by miR-424 inhibitor. SP-A was highly expressed in HG-treated HK-2 cells. The biological effects of miR-424 mimic on HG-treated HK-2 cells were reversed by SP-A-plasmid. In conclusion, lncRNA CASC15 inhibition relieved HG-induced HK-2 cell injury and EMT through miR-424/SP-A axis.

Fisetin treatment alleviates kidney injury in mice with diabetes-exacerbated atherosclerosis through inhibiting CD36/fibrosis pathway.

Diabetes-related vascular complications include diabetic cardiovascular diseases (CVD), diabetic nephropathy (DN) and diabetic retinopathy, etc. DN can promote the process of end-stage renal disease. On the other hand, atherosclerosis accelerates kidney damage. It is really an urge to explore the mechanisms of diabetes-exacerbated atherosclerosis as well as new agents for treatment of diabetes-exacerbated atherosclerosis and the complications. In this study we investigated the therapeutic effects of fisetin, a natural flavonoid from fruits and vegetables, on kidney injury caused by streptozotocin (STZ)-induced diabetic atherosclerosis in low density lipoprotein receptor deficient (LDLR-/-) mice. Diabetes was induced in LDLR-/- mice by injecting STZ, and the mice were fed high-fat diet (HFD) containing fisetin for 12 weeks. We found that fisetin treatment effectively attenuated diabetes-exacerbated atherosclerosis. Furthermore, we showed that fisetin treatment significantly ameliorated atherosclerosis-enhanced diabetic kidney injury, evidenced by regulating uric acid, urea and creatinine levels in urine and serum, and ameliorating morphological damages and fibrosis in the kidney. In addition, we found that the improvement of glomerular function by fisetin was mediated by reducing the production of reactive oxygen species (ROS), advanced glycosylation end products (AGEs) and inflammatory cytokines. Furthermore, fisetin treatment reduced accumulation of extracellular matrix (ECM) in the kidney by inhibiting the expression of vascular endothelial growth factor A (VEGFA), fibronectin and collagens, while enhancing matrix metalloproteinases 2 (MMP2) and MMP9, which was mainly mediated by inactivating transforming growth factor ß (TGFß)/SMAD family member 2/3 (Smad2/3) pathways. In both in vivo and in vitro experiments, we demonstrated that the therapeutic effects of fisetin on kidney fibrosis resulted from inhibiting CD36 expression. In conclusion, our results suggest that fisetin is a promising natural agent for the treatment of renal injury caused by diabetes and atherosclerosis. We reveal that fisetin is an inhibitor of CD36 for reducing the progression of kidney fibrosis, and fisetin-regulated CD36 may be a therapeutic target for the treatment of renal fibrosis.

Exploring the Metabolic Differences between Cisplatin- and UV Light-Induced Apoptotic Bodies in HK-2 Cells by an Untargeted Metabolomics Approach.

Among the extracellular vesicles, apoptotic bodies (ABs) are only formed during the apoptosis and perform a relevant role in the pathogenesis of different diseases. Recently, it has been demonstrated that ABs from human renal proximal tubular HK-2 cells, either induced by cisplatin or by UV light, can lead to further apoptotic death in naïve HK-2 cells. Thus, the aim of this work was to carry out a non-targeted metabolomic approach to study if the apoptotic stimulus (cisplatin or UV light) affects in a different way the metabolites involved in the propagation of apoptosis. Both ABs and their extracellular fluid were analyzed using a reverse-phase liquid chromatography-mass spectrometry setup. Principal components analysis showed a tight clustering of each experimental group and partial least square discriminant analysis was used to assess the metabolic differences existing between these groups. Considering the variable importance in the projection values, molecular features were selected and some of them could be identified either unequivocally or tentatively. The resulting pathways indicated that there are significant, stimulus-specific differences in metabolites abundancies that may propagate apoptosis to healthy proximal tubular cells; thus, we hypothesize that the share in apoptosis of these metabolites might vary depending on the apoptotic stimulus.

Panax Notoginseng Saponins Alleviate LPS-induced Fibrosis of HK-2 Cells by Inhibiting the Activation of NLRP3 Inflammasome and Pyroptosis.

BACKGROUND: Renal fibrosis is related to impaired kidney function and can eventually lead to end-stage renal disease, for which no effective treatment is available. Panax notoginseng saponins (PNS), as a commonly used traditional Chinese medicine, is considered a possible alternative for the treatment of fibrosis. OBJECTIVE: The purpose of the present study was to investigate the effects and possible mechanisms of PNS on renal fibrosis. METHODS: HK-2 cells were used to induce renal fibrosis cell model by lipopolysaccharide (LPS), and the cytotoxicity of PNS on HK-2 cells was investigated. Cell damage, pyroptosis, and fibrosis were analyzed to investigate the effects of PNS on LPS-induced HK-2 cells. NLRP3 agonist Nigericin was used further to explore the inhibitory effect of PNS on LPS-induced pyroptosis so as to clarify the possible mechanism of PNS on renal fibrosis. RESULTS: PNS had no cytotoxicity on HK-2 cells, and could reduce the apoptosis and the release of lactate dehydrogenase (LDH) and inflammatory cytokines of LPS-induced HK-2 cells, showing an alleviating effect on cell damage. PNS also reduced the expression of pyroptosis proteins NLRP3, IL-1ß, IL-18, and Caspase-1, as well as fibrosis proteins α-SMA, collagen Ⅰ and p-Smad3/Smad3, which showed an inhibitory effect on LPS-induced pyroptosis and fibrosis. In addition, LPS-induced cell damage, pyroptosis, and fibrosis were aggravated after Nigericin treatment, while PNS alleviated the aggravation caused by Nigericin. CONCLUSION: PNS inhibits pyroptosis by inhibiting the activation of NLRP3 inflammasome in LPS-induced HK-2 cells, which ultimately alleviates renal fibrosis and plays a good role in the treatment of kidney diseases.

ß-Cryptoxanthin Maintains Mitochondrial Function by Promoting NRF2 Nuclear Translocation to Inhibit Oxidative Stress-Induced Senescence in HK-2 Cells.

The mechanisms of acute kidney injury and chronic kidney disease remain incompletely revealed, and drug development is a pressing clinical challenge. Oxidative stress-induced cellular senescence and mitochondrial damage are important biological events in a variety of kidney diseases. As a type of carotenoid, ß-Cryptoxanthin (BCX) has various biological functions, which means it is a potential therapeutic candidate for the treatment of kidney disease. However, the role of BCX in the kidney is unclear, and the effect of BCX on oxidative stress and cellular senescence in renal cells is also unknown. Therefore, we conducted a series of studies on human renal tubular epithelial (HK-2) cells in vitro. In the present study, we investigated the effect of BCX pretreatment on H2O2-induced oxidative stress and cellular senescence and explored the potential mechanism of BCX action. The results showed that BCX attenuated H2O2-induced oxidative stress and cellular senescence in HK-2 cells. Moreover, BCX promoted NRF2 nuclear expression, maintained mitochondrial function, and reduced mitochondrial damage in HK-2 cells. In addition, silencing NRF2 altered the protective effect of BCX on mitochondria and significantly reversed the anti-oxidative stress and anti-senescence effects of BCX in HK-2 cells. We concluded that BCX maintained mitochondrial function by promoting NRF2 nuclear translocation to inhibit oxidative stress-induced senescence in HK-2 cells. In light of these findings, the application of BCX might be a promising strategy for the prevention and treatment of kidney diseases.