FGF2 isoforms play distinct roles in tubular epithelial-to-mesenchymal transition in diabetic nephropathy.

INTRODUCTION: The role of different isoforms of Fibroblast growth factor-2 (FGF2) in tubular epithelial-to-mesenchymal transition (EMT) in diabetic nephropathy remains unknown. We aimed to evaluate the role of FGF2 isoforms in the pathogenesis of EMT. MATERIALS AND METHODS: Western blot and immunofluorescence were used to assess the expression of FGF2 isoforms in db/db mice and high glucose-stimulated HK2 cells. The effects of specific FGF2 isoforms on EMT were explored via overexpression or knockdown of the corresponding isoform in HK2 cells cultivated in high glucose. RESULTS: Expression of low molecular weight (LMW) FGF2 was up-regulated while high molecular weight (HMW) FGF2 was down-regulated in the kidney of db/db mice and HK2 cells cultured in high glucose that underwent EMT. Overexpression of the LMW FGF2 enhanced EMT changes, while overexpression of the HMW FGF2 attenuated EMT. Knockdown of HMW FGF2 in HK2 cells promoted the EMT process. CONCLUSIONS: The expression and function of LMW and HMW FGF2 differed in the process of EMT in tubular cells. LMW FGF2 contributed to EMT, while HMW FGF2 played a protective role in the EMT process.

Selective Activation of Endoplasmic Reticulum Stress by Reactive-Oxygen-Species-Mediated Ochratoxin A-Induced Apoptosis in Tubular Epithelial Cells.

Ochratoxin A (OTA), one of the major food-borne mycotoxins, impacts the health of humans and livestock by contaminating food and feed. However, the underlying mechanism of OTA nephrotoxicity remains unknown. This study demonstrated that OTA induced apoptosis through selective endoplasmic reticulum (ER) stress activation in human renal proximal tubular cells (HK-2). OTA increased ER-stress-related JNK and precursor caspase-4 cleavage apoptotic pathways. Further study revealed that OTA increased reactive oxygen species (ROS) levels, and N-acetyl cysteine (NAC) could reduce OTA-induced JNK-related apoptosis and ROS levels in HK-2 cells. Our results demonstrate that OTA induced ER stress-related apoptosis through an ROS-mediated pathway. This study provides new evidence to clarify the mechanism of OTA-induced nephrotoxicity.

[Effect of methyl eugenol on hypoxia/reoxygenation injury of human renal tubular epithelial cells and its mechanism].

This study aimed to investigate the effect of methyl eugenol(ME) on hypoxia/reoxygenation(H/R)-induced injury of human renal tubular epithelial HK-2 cells and its mechanism. The viability of HK-2 cells cultured with different concentrations of ME and exposed to H/R was detected by cell counting kit-8(CCK-8) assay. The effect of ME on the morphology of HK-2 cells was observed under an inverted microscope. The content of intracellular reactive oxygen species in different groups was detected after 2',7'-dichlorodihydrofluorescein diacetate(DCFH-DA) fluorescence staining. Cell apoptosis was determined by flow cytometry. Changes in mitochondrial membrane potential were monitored by JC-1 dye. The concentrations of nuclear factor erythroid 2 related factor 2(Nrf2), heme oxygenase-1(HO-1), and nicotinamide adenine dinucleotide phosphatase oxidase 4(Nox4) were measured by Western blot, followed by the assay of Nrf2 concentration changes in cytoplasm and nucleus by confocal fluorescence staining. The results showed that when the concentration of ME was 0-40 µmol·L~(-1), the activity of HK-2 cells was not affected. Compared with the model group, ME enhanced the activity of HK-2 cells and the cell morphology was normal. As revealed by further experiments, ME inhibited the release of reactive oxygen species and the decline in mitochondrial membrane potential of HK-2 cells after H/R injury, promoted Nrf2/HO-1 expression and Nrf2 translocation to the nucleus, and down-regulated the expression of Nox4, thereby significantly reducing apoptosis. This protective effect of ME could be reversed by the specific Nrf2 inhibitor ML385. These findings have preliminarily proved that ME effectively protected HK-2 cells against H/R injury, which might be related to its promotion of Nrf2/HO-1 signaling pathway and inhibition of Nox4. Such exploration on the possible mechanism of ME in the treatment of renal ischemia-reperfusion injury(IRI) and protection of organ function from the perspective of antioxidant stress has provided reference for related research on the treatment of acute kidney injury with traditional Chinese medicine.

IL-1ß functionally attenuates ABCG2 and PDZK1 expression in HK-2 cells partially through NF-ĸB activation.

Long-standing untreated hyperuricemia could lead to gout. Several recent studies have demonstrated a significant decrease of serum urate during acute gout attack, which is an aseptic inflammation process focusing on IL-1ß. However, how IL-1ß, by itself, alters the expression and the functional activity of urate transporters in renal tubular epithelial cells is still unclear. Herein, we revealed that IL-1ß could attenuate the mRNA and protein levels of ABCG2, a major urate efflux pump, in HK-2 cells by real-time PCR and Western-blot assays. Moreover, using an ABCG2 specific inhibitor and a new sensitive and specific detection system, it was found that IL-1ß also reduced the ABCG2 transporter activities. Incubation with specific inhibitors of the NF-κB pathway partly dampened the inhibitory effect of IL-1ß on ABCG2, indicating that IL-1ß reduced the ABCG2 expression partially through the NF-ĸB pathway. Furthermore, the decreased expression of PDZK1 induced by IL-1ß, which is dependent on the NF-κB pathway, could account for the imbalance between the functions and expressions of ABCG2 on this status. These findings demonstrated a new role for IL-1ß, whereby it leads to the inhibition of ABCG2 in renal tubular epithelial cells; this new role probably does not encompass its involvement in the process of renal urate excretion mediated by inflammation. Therefore, other regulation mechanisms of urate reabsorption in renal tubular epithelial cells deserve to be examined in further studies.

High Glucose-Induced Apoptosis in Human Kidney Cells Was Alleviated by miR-15b-5p Mimics.

MicroRNAs were involved in a wide range of biological processes of diabetic nephropathy (DN). It is reported that miR-15b-5p was downregulated in the patients with DN. However, the mechanisms underlying the regulatory effects of miR-15b-5p on patients with diabetes remain unclear. Thus, this study aimed to investigate the role of miR-15b-5p during high glucose (HG)-induced apoptosis in human kidney cells. Quantitative real-time (qRT)-PCR was used to detect the level of miR-15b-5p. CCK-8 assay, EdU staining assays and flow cytometry were used to detect cell proliferation, apoptosis respectively in vitro. In addition, Western blotting was used to determine active caspase-3, cleaved poly(ADP-ribose) polymerase (PARP), phosphorylated (p)-AKT, p-mammalian target of rapamycin (mTOR), p-S6, p-c-Jun N terminal kinase (JNK), p-p38 and p-extracellular signal-regulated kinase (ERK) proteins levels. The expression of miR-15b-5p in patients with DN were dramatically decreased compared with health persons. Similarly, HG down-regulated the expression of miR-15b-5p in HK-2 cells. In contrast, miR-15b-5p mimics alleviated HG-induced apoptosis in HK-2 cells via decreasing the expressions of active caspase 3 and cleaved PARP. EdU detection further confirmed that miR-15b-5p mimics attenuated the anti-proliferation effect of HG in HK-2 cells. Furthermore, HG-induced Akt/mTOR pathway downregulation and JNK upregulation were markedly reversed by miR-15b-5p mimics in cells. The data suggested that miR-15b-5p mimics protects HK-2 cells from HG-induced apoptosis. The anti-apoptotic effects of miR-15b-5p may due to the activation of the Akt/mTOR pathway as well as inactivation of JNK. Taken together, miR-15b-5p might be a potential therapeutic target for the treatment of patients with DN.

Salvianolic acid A attenuates kidney injury and inflammation by inhibiting NF-κB and p38 MAPK signaling pathways in 5/6 nephrectomized rats.

Salvianolic acid A (SAA) is a minor phenolic carboxylic acid extracted from Salviae miltiorrhizae Bunge (Danshen). SAA exhibits a variety of pharmacological activities, such as antioxidative, anti-thrombotic, neuroprotective, and anti-fibrotic effects, as well as protection from myocardial ischemia and prevention of diabetes and other diseases. Furthermore, SAA has shown renal-protective effects in doxorubicin-induced nephropathy. However, there has been limited research regarding the effects of SAA and underlying mechanisms in chronic kidney disease (CKD). Here, we examined the effects and molecular mechanisms of SAA in an established animal model of 5/6 nephrectomized (5/6Nx) rats. The rats were injected with SAA (2.5, 5, and 10 mg/kg per day, intraperitoneally (ip)) for 28 days. SAA dose-dependently lowered the levels of urine protein, blood urea nitrogen, serum creatinine, plasma total cholesterol, and plasma triglycerides in 5/6Nx rats. Histological examination revealed that SAA dose-dependently attenuated renal pathological lesions, evidenced by reduced renal tubulointerstitial fibrosis by decreasing the expression levels of tumor growth factor-ß1 and α-smooth muscle actin in 5/6Nx rats. Moreover, SAA dose-dependently inhibited the activation of nuclear factor-κB (NF-κB) and p38 mitogen-activated protein kinase (MAPK) signaling pathways, subsequently attenuating the secretion of tumor necrosis factor-α and interleukin-1ß and inhibiting the expression of monocyte chemotactic protein-1, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 in kidneys of 5/6Nx rats. The above results were consistent with those obtained in lipopolysaccharide-induced HK-2 cells in vitro (a recognized in vitro inflammatory model). In conclusion, our results demonstrated that SAA effectively attenuates kidney injury in 5/6Nx rats. The therapeutic effects of SAA on kidney injury can be attributed to its anti-inflammatory activities through inhibition of the activation of the NF-κB and p38 MAPK signaling pathways.

Renal tubular epithelial cells injury induced by mannitol and its potential mechanism.

Administration of mannitol with high dose could induce extensive isometric renal proximal tubular vacuolization and acute renal failure in clinic. We previously demonstrated that mannitol-induced human kidney tubular epithelial cell (HK-2) injury. The objective of our present work was to further study the cytotoxicity of mannitol in HK-2 cells and its potential mechanism. Cell viability was assessed by an MTT method. Cell morphological changes were observed. Furthermore, levels of malondialdehyde (MDA) and glutathione (GSH) were measured. Flow cytometry was performed to determine cell apoptosis by using Annexin V-FITC and PI. In addition, the F-actin of cells was labeled by FITC-Phalloidin for observation of cytoskeleton. The MTT assay displayed that the cell viability decreased significantly in a dose- and time-dependent manner. The morphological changes were observed, including cell membrane rapture and cell detachment. The GSH concentration in HK-2 cells decreased dramatically in mannitol treatment group, while MDA content increased significantly. The results of flow cytometry indicated that apoptotic percentages of HK-2 cells increased in 250 mmol/L mannitol treatment group. After treatment with 250 mmol/L mannitol for 48 h, HK-2 cells showed disorganization of cytoskeleton and even exhibited a totally destroyed cytoskeleton. Therefore, high dose of mannitol has a toxic effect on renal tubular epithelial cells, which might be attributed to oxidative stress, destroyed cellular cytoskeleton and subsequent cell apoptosis.

ATF3 attenuates cyclosporin A-induced nephrotoxicity by downregulating CHOP in HK-2 cells.

Calcineurin inhibitors such as cyclosporin A (CsA) are widely used to treat organ transplantation-associated complications. However, CsA use is limited due to renal dysfunction. This study attempts to characterize the mechanism of CsA-induced nephrotoxicity using a human embryonic kidney cell line (HK-2). We performed microarray-based whole-genome expression analysis in HK-2 cells. CsA treatment induced the expression of endoplasmic reticulum (ER) stress-related and apoptosis-inducing genes at 6 and 24h, respectively, indicating that ER-stress predisposed the cells to apoptosis. G1 phase cell-cycle arrest was also observed via ER stress in CsA-treated cells. Furthermore, we found an inverse relationship between activating transcription factor 3 (ATF3), a stress-inducible protein, and C/EBP homologous protein (CHOP), an apoptosis-inducing protein. Moreover, when ATF3 knockdown cells were exposed to CsA, a prompt induction of CHOP was observed, which stimulated ROS production and induced cell death-related genes as compared to wild type. Taken together, our data demonstrate that ATF3 plays a pivotal role in the attenuation of CsA-induced nephrotoxicity by downregulating CHOP and ROS production mediated by ER stress.

Geniposide reduced oxidative stress-induced apoptosis in HK-2 cell through PI3K/AKT3/FOXO1 by m6A modification.

Exogenous hydrogen peroxide (H2O2) may generate excessive oxidative stress, inducing renal cell apoptosis related with kidney dysfunction. Geniposide (GP) belongs to the iridoid compound with anti-inflammatory, antioxidant and anti-apoptotic effects. This study aimed to observe the intervention effect of GP on H2O2-induced apoptosis in human kidney-2 (HK-2) cells and to explore its potential mechanism in relation to N6-methyladenosine (m6A) RNA methylation. Cell viability, apotosis rate and cell cycle were tested separately after different treatments. The mRNA and protein levels of m6A related enzymes and phosphoinositide 3-kinase (PI3K)/a serine/threonine-specific protein kinase 3 (AKT3)/forkhead boxo 1 (FOXO1) and superoxide dismutase 2 (SOD2) were detected by reverse transcription-quantitative real-time PCR (RT-qPCR) and Western blot. The whole m6A methyltransferase activity and the m6A content were measured by ELISA-like colorimetric methods. The changes of m6A methylation levels of PI3K/AKT3/FOXO1 and SOD2 were determined by methylated RNA immunoprecipitation (MeRIP)-qPCR. Multiple comparisons were performed by ANOVA with Turkey's post hoc test. Exposed to 400 µmol/L H2O2, cells were arrested in G1 phase and the apoptosis rate increased, which were significantly alleviated by GP. Compared with the H2O2 apoptosis group, both the whole m6A RNA methyltransferase activity and the m6A contents were increased due to GP intervention. Besides, the SOD2 protein was increased, while PI3K and FOXO1 decreased. The m6A methylation level of AKT3 was negatively correlated with its protein level. Taken together, GP affects the global m6A methylation microenvironment and regulates the expression of PI3K/AKT3/FOXO1 signaling pathway via m6A modification, alleviating cell cycle arrest and apoptosis caused by oxidative stress in HK-2 cells with a good application prospect.

Kidney derived apolipoprotein M and its role in acute kidney injury.

Aim: Apolipoprotein M (apoM) is mainly expressed in liver and in proximal tubular epithelial cells in the kidney. In plasma, apoM associates with HDL particles via a retained signal peptide and carries sphingosine-1-phosphate (S1P), a small bioactive lipid. ApoM is undetectable in urine from healthy individuals but lack of megalin receptors in proximal tubuli cells induces loss of apoM into the urine. Besides this, very little is known about kidney-derived apoM. The aim of this study was to address the role of apoM in kidney biology and in acute kidney injury. Methods: A novel kidney-specific human apoM transgenic mouse model (RPTEC-hapoMTG) was generated and subjected to either cisplatin or ischemia/reperfusion injury. Further, a stable transfection of HK-2 cells overexpressing human apoM (HK-2-hapoMTG) was developed to study the pattern of apoM secretion in proximal tubuli cells. Results: Human apoM was present in plasma from RPTEC-hapoMTG mice (mean 0.18 µM), with a significant increase in plasma S1P levels. In vitro apoM was secreted to both the apical (urine) and basolateral (blood) compartment from proximal tubular epithelial cells. However, no differences in kidney injury score was seen between RPTEC-hapoMTG and wild type (WT) mice upon kidney injury. Further, gene expression of inflammatory markers (i.e., IL6, MCP-1) was similar upon ischemia/reperfusion injury. Conclusion: Our study suggests that kidney-derived apoM is secreted to plasma, supporting a role for apoM in sequestering molecules from excretion in urine. However, overexpression of human apoM in the kidney did not protect against acute kidney injury.

Fucoidan from Laminaria japonica protects renal tubular epithelial cells from uric acid induced NLRP3-mediated pyroptosis through inhibition of NF-κB pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Hyperuricemia is a common metabolic disease with prominent morbidity, it can lead to many adverse effects and complications, such as chronic nephrosis. Fucoidan has been used as natural drug for acute and chronic kidney disease for over 20 years in China, but the precise mechanisms underlying the renal protective function are still indefinable. PURPOSE: This study is conducted to explore alleviation of fucoidan (FPS) from Laminaria japonica on urate-induced NOD-like receptor family, pyrin domain-containing 3 (NLRP3)-mediated pyroptosis in renal tubular epithelial cells HK-2, as well as the mechanism of nuclear factor κB (NF-κB) signaling pathway involved. MATERIALS AND METHODS: HK-2 cells were treated with FPS, uric acid (UA), and inhibitor of NF-κB signaling pathway. Nitric oxide (NO) content and inducible nitric oxide synthase (iNOS) activity were determined with detection kits. Activation of intercellular NLRP3 inflammasome and NF-κB signaling pathway, gasdermin D (GSDMD) expression level were evaluated with Western blot and quantitative reverse transcription-PCR (qRT-PCR), and immunofluorescent analysis. RESULTS: Data showed that UA induced cellular inflammatory response demonstrated by elevated NO content, iNOS activity and expression level of NLRP3 inflammasome-mediated pyroptosis associated molecules including NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), Caspase-1, interleukin 18 (IL-18) and GSDMD, moreover the NF-κB signaling pathway was activated by UA. However, FPS exposure inhibited efficiently the UA induced adverse effect. CONCLUSION: It can be concluded that FPS inhibited UA-induced NLRP3-mediated pyroptosis in HK-2 cells through repressing NF-κB signaling pathway.

Dysregulation of ferroptosis may participate in the mitigating effect of CoCl2 on contrast-induced nephropathy.

BACKGROUND: Contrast agents can directly or indirectly induce renal tubular ischemia and hypoxic damage. Given that cobalt chloride (CoCl2) can protect renal tubules, the protective effect and potential mechanism of action of CoCl2 on contrast-induced nephropathy (CIN) warrant investigation. METHODS: A CIN mouse model was established to determine the protective effect of CoCl2 on renal injury in vivo. Then, TMT-based proteomics was performed to determine the differentially expressed proteins (DEPs), following which, enrichment analyses of gene ontology and the KEGG pathway were performed. In vitro, a CIN model was constructed with renal tubular epithelial cells (HK-2) to determine the effect of CoCl2 on potential targets and the role of the key protein identified from the in vivo experiments. RESULTS: CoCl2 treatment decreased the levels of BUN and serum creatinine (sCr), while increasing the levels of urea and creatinine (Cr) in the urine of mice after CIN injury. Damage to the renal tubules in the CoCl2 treatment group was significantly less than in the CIN model group. We identified 79 DEPs after treating the in vivo model with CoCl2, and frequently observed ferroptosis-related GO and KEGG pathway terms. Of these, Hp (haptoglobin) was selected and found to have a strong renoprotective effect, even though its expression level in kidney tissue decreased after CoCl2 treatment. In HK-2 cells, overexpression of Hp reduced the ferroptosis caused by erastin, while knocking down Hp negated the attenuation effect of CoCl2 on HK-2 cell ferroptosis. CONCLUSION: CoCl2 attenuated kidney damage in the CIN model, and this effect was associated with the decrease in ferroptosis mediated by Hp.

Identification of Hyperuricemia Alleviating Peptides from Yellow Tuna Thunnus albacares.

The development of food-derived antihyperuricemic substances is important for alleviating hyperuricemia (HUA) and associated inflammation. Here, novel peptides fromThunnus albacares (TAP) with strong antihyperuricemic activity were prepared. TAP was prepared by alkaline protease (molecular weight <1000 Da), with an IC50 value of xanthine oxidase inhibitory activity of 2.498 mg/mL, and 5 mg/mL TAP could reduce uric acid (UA) by 33.62% in human kidney-2 (HK-2) cells (P < 0.01). Mice were fed a high-purine diet and injected with potassium oxonate to induce HUA. Oral administration of TAP (600 mg/kg/d) reduced serum UA significantly by 42.22% and increased urine UA by 79.02% (P < 0.01) via regulating urate transporters GLUT9, organic anion transporter 1, and ATP-binding cassette subfamily G2. Meantime, TAP exhibited hepatoprotective and nephroprotective effects, according to histological analysis. Besides, HUA mice treated with TAP showed anti-inflammatory activity by decreasing the levels of toll-like receptor 4, nuclear factors-κB p65, NLRP3, ASC, and Caspase-1 in the kidneys (P < 0.01). According to serum non-targeted metabolomics, 91 differential metabolites between the MC and TAP groups were identified, and purine metabolism was considered to be the main pathway for TAP alleviating HUA. In a word, TAP exhibited strong antihyperuricemic activity both in vitro and in vivo.

Prediction of cytotoxicity of heavy metals adsorbed on nano-TiO2 with periodic table descriptors using machine learning approaches.

Nanoparticles with their unique features have attracted researchers over the past decades. Heavy metals, upon release and emission, may interact with different environmental components, which may lead to co-exposure to living organisms. Nanoscale titanium dioxide (nano-TiO2) can adsorb heavy metals. The current idea is that nanoparticles (NPs) may act as carriers and facilitate the entry of heavy metals into organisms. Thus, the present study reports nanoscale quantitative structure-activity relationship (nano-QSAR) models, which are based on an ensemble learning approach, for predicting the cytotoxicity of heavy metals adsorbed on nano-TiO2 to human renal cortex proximal tubule epithelial (HK-2) cells. The ensemble learning approach implements gradient boosting and bagging algorithms; that is, random forest, AdaBoost, Gradient Boost, and Extreme Gradient Boost were constructed and utilized to establish statistically significant relationships between the structural properties of NPs and the cause of cytotoxicity. To demonstrate the predictive ability of the developed nano-QSAR models, simple periodic table descriptors requiring low computational resources were utilized. The nano-QSAR models generated good R2 values (0.99-0.89), Q2 values (0.64-0.77), and Q2F1 values (0.99-0.71). Thus, the present work manifests that ML in conjunction with periodic table descriptors can be used to explore the features and predict unknown compounds with similar properties.

Protective effects of Polygonatum kingianum polysaccharides and aqueous extract on uranium-induced toxicity in human kidney (HK-2) cells.

The current detoxification options of uranium, a toxic radioactive heavy metal, have obvious side effects. Polygonatum kingianum (PK), a natural product with the function of antioxidant, may be effective in detoxification and prevention of uranium-induced nephrotoxicity. Here, we studied the protective effects of PK polysaccharides (PKP) and aqueous extract (PKAE) on uranium-induced toxicity in human kidney (HK-2) cells. First, the physicochemical properties of PKP and PKAE were characterized. Assays on cultured cells demonstrated that pretreatment with PKP and PKAE significantly increased metabolic activity, relieved morphological impairments, and alleviated apoptosis. The impairments caused by uranium exposure were ameliorated (mitochondrial membrane potential and ATP level increased while reactive oxygen species decreased). Molecular mechanistic studies revealed that PKP and PKAE alleviated uranium-induced cytotoxicity by regulating mitochondria-mediated apoptosis and the GSK-3ß/Fyn/Nrf2 pathway. Collectively, our data support the preventive and therapeutic applications of PKP and PKAE for uranium poisoning.

Hydroxysafflower yellow A alleviates HK-2 cells injury in cold hypoxia/reoxygenation via mitochondrial apoptosis.

Cold storage for organ preservation in kidney transplantation is a core predisposing factor for delayed graft function and the long-term outcome of transplanted kidneys. Hydroxysafflor yellow A (HSYA) is the most effective water-soluble active monomer in Safflower with a strong property of inhibiting hypoxia and reoxygenation (H/R). However, the evidence concerning the effect of HSYA on H/R in kidney transplantation is limited. To investigate whether HSYA has a protective effect on cold H/R injury，we investigated the possible protective mechanism. Here, we incubated HK-2 cells to establish a cold H/R model and observed HSYA activation in an in vitro model of cold-storage rewarming which included the cell survival rate, cell morphology and ultrastructure, protein expression of Bcl-2, Bax, CytC, Apaf-1, and caspase-3, and status of mitochondrial permeability transformation pores (MPTPs). Our data showed that HSYA pretreatment increased the survival rate of the cells, alleviated mitochondrial damage, decreased the expression of apoptosis-related proteins and inhibited the openness of mitochondrial permeability transformation pores. Our findings suggested that HSYA may be a major predisposing mediator of mitochondrial apoptosis and renal tubular injury in cold storage-associated transplantation and may be an effective therapeutic target for improving graft function and graft survival.

Effects and Mechanism of Salvianolic Acid B on the Injury of Human Renal Tubular Epithelial Cells Induced by Iopromide.

With the increasing application of medical imaging contrast materials, contrast-induced nephropathy (CIN) has become the third major cause of iatrogenic renal insufficiency. CIN is defined as an absolute increase in serum creatinine levels of at least 0.50 mg/dl or an increase >25% of serum creatinine from baseline after exposure to contrast. In this study, the protective effects of salvianolic acid B (Sal B) were detected in human renal tubular epithelial cells (HK-2) exposed to iopromide. The results showed that different concentrations of Sal B counteract the loss of cell viability induced by iopromide, and reduce cell apoptosis, the reactive oxygen species (ROS) levels, and the levels of endoplasmic reticulum stress (ERS)-related and apoptosis-related proteins such as p-IRE-1α, p-eIF-2α/eIF-2α, p-JNK, CHOP, Bax/Bcl-2, and cleaved caspase-3. In addition, Sal B at a concentration of 100 µmol/L inhibited ERS and reduced cell damage to a similar extent as the ERS inhibitor 4-PBA. Importantly, treatment with Sal B could abolish the injury induced by ERS agonist tunicamycin, increasing cell viability and the mitochondrial membrane potential, as well as significantly reducing ROS levels and the expression of Bax/Bcl-2, cleaved-caspase-3, GRP78, p-eIF2α, p-JNK, and CHOP. These results suggested that the protective effect of Sal B against HK-2 cell injury induced by iopromide may be related to the inhibition of ERS.

Ochratoxin A induces ERK1/2 phosphorylation-dependent apoptosis through NF-κB/ERK axis in human proximal tubule HK-2 cell line.

Ochratoxin A (OTA) is a food contaminant mycotoxin with hazardous effects on human and animal health, primarily affecting the kidneys. OTA's mode of action is not well understood. OTA activates both MAPK/ERK and PI3K/Akt signaling pathways, which play role in apoptosis and cell survival, respectively. OTA is also known to induce toxicity by activating the NF-κB pathway in immune cells. However, its role in determining the cell fate upon OTA exposure in a human kidney cell line (HK-2) has not been fully explored. We made use of pharmacological inhibition of NF-κB to define its role in viability of OTA-treated HK-2 cells. We show that OTA-induced p65 NF-κB subunit translocation into the nucleus in a time-dependent manner using both Western blotting and immunofluorescence (IF). We also document the DNA-binding and reporter gene expression activities of NF-κB by electrophoretic mobility shift (EMSA) and luciferase reporter assays, respectively. Our results indicate that, following 6 h of exposure, OTA fully activates NF-κB pathway and its downstream effectors in HK-2 cells. In addition, Bay11-7085 treatment causes attenuation of the relative levels of OTA-mediated ERK1/2 phosphorylation, suggesting a cross-talk between NF-κB and the MAPK/ERK pathway. Critically, co-treatment of HK-2 cells with OTA and Bay11-7085 leads to the inhibition of OTA-induced apoptosis in a time-dependent manner. Our results support a robust association between NF-κB and the MAPK/ERK pathways in the modulation of apoptotic effects of OTA in HK-2 cells.

Sika deer antler protein against acetaminophen-induced oxidative stress and apoptosis in HK-2 cells via activating Nrf2/keap1/HO-1 pathway.

Nf-E2-related transcription factor 2 (Nrf2) helps cells fight oxidative stress events in vivo and in vitro by promoting the expression of antioxidants and detoxification enzymes. The necessary factors regulating Nrf2 activity and stability during analgesic nephropathy are not fully understood. Our results suggest that acetaminophen produces nephrotoxicity in HK-2 cells by inhibiting keap1 degradation. APAP subsided Nrf2 nuclear accumulation by inhibition of keap1 degradation, thereby reducing the binding of Nrf2 to ARE, leading to the loss of expression of antioxidant proteins such as HO-1, inducing a series of oxidative stress and apoptosis events. Therefore, Nrf2/keap1/HO-1 signal transduction pathway has a poor prognosis during analgesic nephrotoxicity. Sika deer antler protein (SDAPR) significantly prevented APAP-induced HK-2 cell damage by constitutively stabilized Nrf2 nuclear retention. Excess APAP leads to a decrease in Nrf2 nuclear translocation, leading to severe oxidative stress, increasing the levels of GSH and MDA in HK-2 cells, and reducing the enzyme activities of SOD and CAT in HK-2 cells. Increased biomarker levels of acute kidney injury (AKI) in HK-2 cells, including kidney injury molecule-1, neutrophil gelatinase-associated lipocalin and cystatin C, decrease the mitochondrial membrane potential in HK-2 cells, and cause mitochondrial dysfunction, it also reduced the ratio of mitochondria-associated apoptotic protein Bax/Bcl-2, leading to cell apoptosis. SDAPR dose dependently accorded protection against acetaminophen-induced nephrotoxicity, oxidative damage, and cell apoptosis by its molecular intervention with Nrf2/keap1/HO-1 pathway via keap1 degradation. PRACTICAL APPLICATIONS: In this paper, we investigated the protective effect of SDAPR on APAP-induced AKI in HK-2 cells, and briefly explained its possible mechanism of action, providing a basis for future clinical trials and the development of anti-APAP AKI drugs.

High glucose down-regulates microRNA-181a-5p to increase pro-fibrotic gene expression by targeting early growth response factor 1 in HK-2 cells.

Tubulointerstitial fibrosis (TIF) plays an important role in the progression of renal fibrosis in diabetic nephropathy (DN). Accumulating evidence supports a crucial effect of early growth response factor 1 (Egr1) on renal fibrosis in DN, but the underlying mechanisms are not entirely clear. Here, we explored the aggravating role of Egr1 and identified microRNA-181a-5p (miR-181a-5p) as an upstream regulator of Egr1 in TIF of DN. We demonstrated that overexpression of Egr1 enhanced, whereas small interfering RNA targeting Egr1 decreased the expressions of transforming growth factor ß1 (TGF-ß1) and fibrosis-related genes including fibronectin and collagen I in human proximal tubule cell line (HK-2) cells. We then found that miR-181a-5p expression was down-regulated, accompanied by the corresponding up-regulation of Egr1, TGF-ß1, fibronectin and collagen I in renal tissues of type 2 diabetic Otsuka-Long-Evans-Tokushima-Fatty rats with DN, and that the expression of miR-181a-5p was negatively correlated with the level of Egr1 in HK-2 cells treated with high glucose. Furthermore, we identified that miR-181a-5p directly suppressed Egr1 to decrease the expressions of TGF-ß1, fibronectin and collagen I in HK-2 cells through targeting the 3' untranslated region of Egr1. The functional relevance of miR-181a-5p-induced Egr1 decrease was supported by inhibition and overexpression of miR-181a-5p in HK-2 cells. Thus, we concluded that aberrant Egr1 expression, which can be suppressed by miR-181a-5p directly, plays a crucial role in the progression of renal TIF in DN. This study indicates that targeting miR-181a-5p may be a novel therapeutic approach of DN.