Estradiol facilitates urate excretion by reducing GLUT9 expression via ERβ/TMEM106B/PI3K/AKT1 pathway in renal tubular epithelial cells.

Renal interstitial fibrosis (RIF) is the final common outcome of numerous chronic kidney diseases, contributing to end-stage renal disease. Hirudin, a thrombin inhibitor, has attracted increased attention as a potential treatment approach for renal fibrosis. The present study aimed to investigate the molecular mechanism underlying the effect of hirudin on fibrosis in renal proximal tubular epithelial cells. An in vivo mouse RIF model established using unilateral ureteral obstruction (UUO) and an in vitro of RIF using the renal tubular epithelial cell line HK-2 treated with TGF-β were used. Expressions of sphingosine-1-phosphate (S1P) receptors (S1PR)1-4 and protease-activated receptor 1 (PAR1) were measured by reverse transcription-quantitative PCR and western blotting in mice with UUO and TGF-β induced HK-2 cells. Western blotting was used to detect the expression of N-cadherin, Slug, E-cadherin, Collagen IV, fibronectin, MMP9 and monocyte chemoattractant protein-1. Immunofluorescence staining was conducted to measure α-SMA level expression. The results demonstrated that the expression levels of S1PR1, S1PR2, S1PR3, S1PR4 and PAR1 were upregulated in both TGF-β-induced HK-2 cells and renal tissues from mice with unilateral ureteral ligation. Notably, hirudin inhibited TGF-β-induced PAR1, S1PR2 and S1PR3 upregulation in both HK-2 cells and renal tissues. Additionally, the inhibition of S1PR2 and S1PR3 resulted in PAR1 downregulation. Furthermore, treatment with S1P and PAR1 agonists abolished the effect of hirudin on the expression of EMT, fibrosis-related proteins and monocyte chemoattractant protein 1. In conclusion, hirudin attenuated TGF-β-induced fibrosis in proximal renal tubular epithelial HK-2 cells by inhibiting PAR1 expression via the S1P/S1PR2/S1PR3 signaling pathway. Therefore, hirudin may be considered as a promising therapeutic agent for RIF.

EGF-recruited JunD/c- fos complexes activate CD2AP gene promoter and suppress apoptosis in renal tubular epithelial cells

Human γδ T cells recognize conserved endogenous and stress-induced antigens typically associated with autoimmune diseases. However, the role of γδ T cells in autoimmune diseases is not clear. Few autoimmune disease-related antigens recognized by T cell receptor (TCR) γδ have been defined. In this study, we compared Vδ2 TCR complementarity-determining region 3 (CDR3) between systemic lupus erythematosus (SLE) patients and healthy donors. Results show that CDR3 length distribution differed significantly and displayed oligoclonal characteristics in SLE patients when compared with healthy donors. We found no difference in the frequency of Jδ gene fragment usage between these two groups. According to the dominant CDR3δ sequences in SLE patients, synthesized SL2 peptides specifically bound to human renal proximal tubular epithelial cell line HK-2; SL2-Vm, a mutant V sequence of SL2, did not bind. We identified the putative protein ligand chaperonin-containing T-complex protein 1 subunit ζ (CCT6A) using SL2 as a probe in HK-2 cell protein extracts by affinity chromatography and liquid chromatography-electrospray ionization-tandem mass spectrometry analysis. We found CCT6A expression on the surface of HK-2 cells. Cytotoxicity of only Vδ2 γδ T cells to HK-2 cells was blocked by anti-CCT6A antibody. Finally, we note that CCT6A concentration was significantly increased in plasma of SLE and rheumatoid arthritis patients. These data suggest that CCT6A is a novel autoantigen recognized by Vδ2 γδ T cells, which deepens our understanding of mechanisms in autoimmune diseases.

Effect of Tangnaikang on TGF-β1-induced transdifferentiation of human renal tubular epithelial HK-2 cells

Ischemia-reperfusion injury is one of the major causes of acute kidney injury (AKI), which is increasingly prevalent in clinical settings. Glaucocalxin A (GLA), a biologically ent-kauranoid diterpenoid, has various pharmacological effects like antioxidation, immune regulation, and antiatherosclerosis. In this study, the effect of GLA on AKI and its mechanism were studied in vitro. HK-2 human renal tubular epithelial cells were exposed to hypoxia/reoxygenation (H/R), which were established as an in vitro AKI model. Subsequently, the mRNA expressions of inflammatory and antioxidant factors were determined by quantitative reverse transcription polymerase chain reaction (RT-qPCR). Reactive oxygen species (ROS) production and cell death were detected by fluorescence-activated cell sorting. GLA pre-treatment improved the cell viability of HK-2 cells exposed to H/R. GLA suppressed the H/R-induced ROS production in HK-2 cells. GLA also elevated the activities of superoxide dismutase of HK-2 cells exposed to H/R. Moreover, GLA prevented H/R-induced cell death in HK-2 cells. Furthermore, GLA ameliorated the activation of the protein kinase B (Akt)/nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway in HK-2 cells exposed to H/R. Our findings suggested that GLA protected HK-2 cells from H/R-induced oxidative damage, which was mediated by the Akt/Nrf2/HO-1 signaling pathway. These results indicate that GLA may serve as a promising therapeutic drug for AKI.

Objective To investigate the effect of transforming growth factor-β1 (TGF-β1) on the expression of N-myc downstream regulatory gene 2 (NDRG2) in human renal tubular epithelial cell line HK-2. Methods The control group and the experimental group were set up according to whether or not the TGF-β1 was incubated. Western blotting and real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) were applied to detect the expression of E-cadherin, α-smooth muscle actin (α-SMA), Vimentin and Snail protein in epithelial cells. The expression of NDRG2 in human renal tubular epithelial cells (HK-2) treated with different concentrations of TGF-β1 was detected by Western blotting and RT-qPCR. Results The results showed that the protein expression level expression of E-cadherin in the control group and the experimental group was (1.0±0.2) vs. (0.3±0.2) (t=4.850, P=0.008), that of α-SMA was (1.0±0.1) vs. (2.6±0.4) (t=6.721, P=0.003), that of Vimentin was (1.0±0.1) vs. (2.6±0.4) (t=6.721, P=0.003), and that of Snail was (1.0±0.1) vs. (2.5±0.2) (t=11.620, P=0.000). In the control group and the experimental group, the mRNA expression level of E-cadherin was (1.0±0.2) vs. (0.2±0.1) (t=7.686, P=0.002), that of α-SMA was (1.0±0.3) vs. (2.4±0.3) (t=5.715, P=0.005), that of Vimentin was (1.1±0.15) vs. (2.6±0.5) (t=4.977, P=0.008), and that of Snail was (0.9±0.2) vs. (1.9±0.2) (t=6.124, P=0.004). At the same time, after incubation with TGF-β1, NDRG2 in HK-2 cells was significantly down-regulated at both protein and mRNA levels. The levels of protein expression in each group were (1.00±0.04 vs. 0.80±0.03 vs. 0.50±0.05 vs. 0.30±0.03 vs. 0.20±0.02) (P=0.019), and mRNA expression in each group were (1.00±0.05 vs. 0.70±0.03 vs. 0.50±0.04 vs. 0.30±0.02 vs. 0.20±0.05) (P=0.021). Especially when incubated with TGF-β1 concentration greater than 10 ng/ml, the difference was statistically significant. Conclusion TGF-β1 can promote the process of EMT in HK-2 cells, and TGF-β1 can down-regulate the expression of NDRG2 in protein and RNA in HK-2 cells. Key words: N-myc downstream regulatory gene 2; Transforming growth factor-β1; Renal tubular epithelial cells; Epithelial-mesenchymal transition

Background: Renal ischemia reperfusion injury plays a key role in renal transplantation, in which oxidative stress is main injury. Our previous study proved baicalin, a flavonoid glycoside isolated from Scutellaria baicalensis, protects kidney from ischemia reperfusion injury. This study aimed to study the underlying mechanism in vitro. Methods: Human renal proximal tubular epithelial cell line HK-2 cells were stimulated with 500 μmol/L H2O2 for 1 hour and then culture for 6 hours. 100 μmol/L baicalin was added to the culture medium 1 hour prior to the treatment. Morphological change, cell viability, apoptotic rate, and oxidative stress level were evaluated. Western blot and real-time PCR analysis were performed to identify the expression of ER stress hallmarks such as BiP, CHOP, and GRP94. The intranuclear and extranuclear level of Nrf2 were also determined in order to assess the activation of Nrf2 signaling. Results: Hydrogen peroxide produced dramatic injury in HK-2 cells. In H2O2 group, Morphological change of apoptosis was observed. The cell viability was decreased and apoptotic rate was increased. ROS and GSH/GSSG analysis revealed increased oxidative stress. ER stress and Nrf2 signaling were also increased. Baicalin pretreatment ameliorated hydrogen peroxide induced cytotoxicity, reduced oxidative stress and ER stress, and further activated Nrf2 signaling pathway which leads to anti-oxidative response.Figure: No Caption available.Conclusion: This study revealed that baicalin pretreatment serves a protective role against hydrogen peroxide induced cytotoxicity in HK-2 cells, and the mechanism is related to the inhibition of ER stress and the activation of Nrf2 signaling.

Renal ischemia-reperfusion injury plays a key role in renal transplantation and greatly affects the outcome of allograft. Our previous study proved that Baicalin, a flavonoid glycoside isolated from Scutellaria baicalensis, protects kidney from ischemia-reperfusion injury. This study aimed to study the underlying mechanism in vitro. Human renal proximal tubular epithelial cell line HK-2 cells were stimulated by H2O2 with and without Baicalin pretreatment. The cell viability, apoptosis and oxidative stress level were measured. The expression of endoplasmic reticulum (ER) stress hallmarks, such as binding immunoglobulin protein (BiP) and C/EBP homologous protein (CHOP), were analyzed by western blot and real-time PCR. NF-E2-related factor 2 (Nrf2) expression was also measured. In the H2O2 group, cell viability decreased and cell apoptosis increased. Reactive Oxygen Species (ROS) and Glutathione/Oxidized Glutathione (GSH/GSSG) analysis revealed increased oxidative stress. ER stress and Nrf2 signaling also increased. Baicalin pretreatment ameliorated H2O2-induced cytotoxicity, reduced oxidative stress and ER stress and further activated the anti-oxidative Nrf2 signaling pathway. The inducer of ER stress and the inhibitor of Nrf2 abrogated the protective effects, while the inhibitor of ER stress and the inducer of Nrf2 did not improve the outcome. This study revealed that Baicalin pretreatment serves a protective role against H2O2-induced cytotoxicity in HK-2 cells, where the inhibition of ER stress and the activation of downstream Nrf2 signaling are involved.

Background More studies are focusing on renal tubular epithelial cells (RTECs) as a new target to restore inflammatory environment as clarifying their immune regulatory function. Here, we investigated whether histone deacetylases (HDACs) are activated in RTECs during T cell-mediated inflammation and their blockade is able to reduce the inflammatory responses. Methods Human renal proximal tubular epithelial cell line HK-2 was cultured in the presence or absence of recombinant interferon gamma (IFN-g) 200 U/ml plus tumor necrosis factor alpha(TNF-a) 5 ng/ml. The HDAC activity was determined on the expression levels of acetylated H3 and a-tubulin by immune blot assay. To determine the functional activity of HDAC inhibitor SB939, we analyzed the immune stimulatory phenotype of HK-2 cells such as class II MHC molecule, CD80, CD86, and CD40 by flow cytometry. In addition, the culture supernatants were used for measuring cytokines and chemokines by ELISA assay. Results We found that HDAC activity was markedly increased in HK-2 cells by treatment of IFN-g/TNF-a within 12 hours. Treatment of pan-HDAC inhibitor SB939 in HK-2 cells completely prevented HDAC activity increased by IFN-g treatment. SB939 treatment predominantly inhibited up-regulating CD40 expression but not MHC class II, CD80, and CD86. In addition, MCP-1 was significantly inhibited more than IL-6 and TNF-a by SB939 treatment. We found that HDAC activity was markedly increased in HK-2 cells by treatment of IFN-g/TNF-a within 12 hours. Treatment of pan-HDAC inhibitor SB939 in HK-2 cells completely prevented HDAC activity increased by IFN-g treatment. SB939 treatment predominantly inhibited up-regulating CD40 expression but not MHC class II, CD80, and CD86. In addition, MCP-1 was significantly inhibited more than IL-6 and TNF-a by SB939 treatment. Conclusion Our results demonstrate that 1) HDAC activity is increased in RTECs in response to IFN-g, 2) which further facilitates T cell-mediated inflammatory responses through CD40 and MCP-1. Therefore, our study suggests that HDAC inhibitor has a therapeutic potential for the treatment of acute renal inflammatory diseases such as allograft rejection in transplantation.

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

BACKGROUND AND AIMS Acute kidney injury (AKI) is a frequent, serious condition characterized by acute tubular necrosis involving the destruction of tubular epithelial cells. Recent studies have shown that DNA damage contributes to renal tubular cell death during kidney injury. Cyclin-dependent kinase 12 (CDK12) is a member of the cyclin-dependent kinase family that phosphorylates the carboxy-terminal domain of RNA polymerase II and selectively regulates transcription of DNA-damage response (DDR) genes. CDK12 inhibitor was reported to play an important role in a variety of cancers. So far, the functions of CDK12 deficiency in the pathogenesis of AKI have not been studied. METHOD Here, we established a cisplatin-induced AKI mouse model with CDK12 knockdown mice and created stable in vitro models with CDK12 knockdown tubular epithelial cells. Cisplatin nephrotoxicity was induced by intraperitoneal injection of cisplatin (20 mg/kg). After 2 days following cisplatin treatment, blood and kidney tissues were harvested. Renal function and histology were evaluated. DDR, tubular apoptosis, cell proliferation were evaluated by immunofluorescence assays, real-time PCR, flow cytometer and western blot analysis. RESULTS Here we found that CDK12 was downregulated in the renal tubular epithelial cells of AKI patients and murine AKI models and associated with kidney functions. RNA-sequencing of kidney cortex demonstrated increased transcripts involved in DDR and tubular apoptosis signaling pathways and decreased transcripts cell proliferation pathways, in knockout versus control mice. Moreover, more severe AKI occurred in tubular cell-specific knock-down of CDK12 mice (CDK12RTEC+/–) than age- and sex-matched wild type mice, as evidenced by increased levels of serum creatinine (Scr), blood urea nitrogen (BUN) and tubular injury score. Mechanistically, CDK12 deficiency accelerated cisplatin-induced DDR, mitochondrial apoptosis and abrogates cell proliferation of renal tubular cells in these mice. Similar results were observed after the human renal tubular epithelial cell line HK-2 cells exposure to CDK12 siRNA stimulation. In contrast, lentivirus-mediated CDK12 overexpression protected against DDR and mitochondrial apoptosis while attenuating renal dysfunction, morphological damage and renal tubular cell death in cisplatin-induced AKI models. Moreover, representative Integrative Genomics Viewer (IGV) profiles demonstrated a minimal impact of gene length-dependent elongation defects, a substantial proportion of which participate in the DDR and cell proliferation. CONCLUSION Thus, proximal tubule-specific deficiency of CDK12 exacerbates acute kidney injury and tubular apoptosis, likely due to affecting the expression of genes involved in the DDR and mRNA processing.

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

Tri-ortho-cresyl phosphate (TOCP), an organophosphate compound widely used in industry as a lubricant, plasticizer, and hydraulic fluid, could cause organophosphate-induced delayed neuropathy (OPIDN) which is a phospholipase and involved in phospholipid metabolism. However, whether TOCP could induce nephrotoxicity is not clear. In this study, we used a mouse model with subchronic exposure to TOCP, and a human renal proximal tubular epithelial cell line HK-2 cells as the in vitro model to study the TOCP-induced nephrotoxicity. We found that TOCP-exposed mice developed severe renal dysfunctions, increased serum creatinine (p < 0.01), serum urea nitrogen (p < 0.01), and urinary protein (p < 0.05) levels. Histological analyses demonstrated severe tubular degeneration, glomerular atrophy. TOCP induced oxidative stress, increased malondialdehyde (MDA) (p < 0.01) and reduced glutathione (GSH) (p < 0.01) levels in kidney and serum. The antioxidant L-ascorbic acid (Vc) cotreatment significantly decreased TOCP-induced oxidative stress, increased GSH levels (p < 0.01), and partially alleviated renal dysfunction and histological damage in vivo. In vitro, the TOCP metabolite cresyl saligenin phosphate (CBDP) caused cytotoxicity to HK-2 cells through reactive oxygen species (ROS) production, which was decreased by Vc (p < 0.05). The direct toxicity target of TOCP is neuropathy target esterase (NTE), which is highly expressed in kidney and plays a key role in kidney functions such as maintaining osmotic pressure. We found that TOCP significantly decreased the activity of NTE in kidney (p < 0.01), but TOCP treatment induced NTE gene expression (p < 0.05), possibly through negative feedback. Therefore, these findings indicate that the TOCP-induced nephrotoxicity is largely mediated by the induction of oxidative stress possibly by the inhibition of NTE. This study contributes to the understanding of the mechanisms of TOCP toxicity in kidney and provides the scientific basis for the risk assessment of TOCP exposure.

LIGHT deficiency aggravates cisplatin-induced acute kidney injury by upregulating mitochondrial apoptosis

Obstructive sleep apnea syndrome (OSAS) is a respiratory disorder and chronic intermittent hypoxia (CIH) is an important pathological characteristic of OSAS. Injuries on renal tubular epithelial cells were observed under the condition of CIH. Pyroptosis is a programmed mode of cell death following cell apoptosis and cell necrosis, which is mediated by NLRP3 signaling. The present study aims to investigate the effects of CIH on the pyroptosis of renal tubular epithelial cells and the underlying mechanism. Firstly, CIH was induced in two renal tubular epithelial cell lines, HK-2 cells and TCMK-1 cells. As the aggravation of hypoxia, an increasing trend of elevated apoptotic rate was observed in HK-2 cells and TCMK-1 cells, accompanied by the excessive release of ROS and LDH, and upregulation of NLRP3. Subsequently, the CIH model was established on rats. The pathological analysis results indicated that in CIH rats, the glomerular bottom membrane and mesangium were slightly thickened and edema was observed in the renal tubule epithelium. More serious injury was observed in the moderate intermittent hypoxia group. The expression level of IL-1β and IL-18 was promoted as the aggravation of hypoxia, accompanied by the elevated production of LDH and ROS. The expression level of cleaved Caspase-1, Caspase-1, GSDMD, TLR4, MyD88, NF-κB, p-NF-κB, and NLRP3 was found significantly upregulated as the aggravation of hypoxia. Lastly, the pathological changes in rats induced by CIH were dramatically abolished by MCC950, a specific inhibitor of NLRP3. Collectively, CIH triggered the pyroptosis of renal tubular epithelial cells by activating the NLRP3 inflammasome.