PURPOSE: Diabetic nephropathy (DN) is a prevalent chronic microvascular complication of diabetes mellitus involving disturbances in electrolytes and the acid-base balance caused by a disorder of glucose metabolism. NHE1 is a Na+/H+ exchanger responsible for keeping intracellular pH (pHi) balance and cell growth. Our study aimed to investigate roles of NHE1 in high glucose (HG)-induced apoptosis in renal tubular epithelial cells. MATERIALS AND METHODS: Renal epithelial tubular cell line HK-2 was cultured in medium containing 5 mM or 30 mM glucose. Then, cell apoptosis, oxidative stress, NHE1 expression, and pHi were evaluated. NHE1 siRNA and inhibitor were used to evaluate its role in cell apoptosis. RESULTS: HG significantly increased cell apoptosis and the production of reactive oxygen species (ROS) and 8-OHdG (p<0.05). Meanwhile, we found that HG induced the expression of NHE1 and increased the pHi from 7.0 to 7.6 after 48 h of incubation. However, inhibiting NHE1 using its specific siRNA or antagonist DMA markedly reduced cell apoptosis stimulated by HG. In addition, suppressing cellular oxidative stress using antioxidants, such as glutathione and N-acetyl cysteine, significantly reduced the production of ROS, accompanied by a decrease in NHE1. We also found that activated cyclic GMP-Dependent Protein Kinase Type I (PKG) signaling promoted the production of ROS, which contributed to the regulation of NHE1 functions. CONCLUSION: Our study indicated that HG activates PKG signaling and elevates the production of ROS, which was responsible for the induction of NHE1 expression and dysfunction, as well as subsequent cell apoptosis, in renal tubular epithelial cells.
Antioxidants/metabolism ; Apoptosis/*drug effects ; Cation Transport Proteins/*metabolism ; Cell Cycle/drug effects ; Cell Line ; Dose-Response Relationship, Drug ; Epithelial Cells/*cytology/drug effects/*metabolism ; Glucose/*pharmacology ; Glutathione/metabolism ; Humans ; Kidney Tubules/*cytology ; Oxidative Stress/*drug effects ; Reactive Oxygen Species/metabolism ; Signal Transduction/drug effects ; Sodium-Hydrogen Antiporter/*metabolism

Carbon monoxide (CO), as a vital small molecule in signaling pathways, is found to be involved in ischemia-reperfusion injury (IRI) in renal transplantation. CO-releasing molecule-2 (CORM-2), a CO-releasing molecule, is a type of metal carbonyl complexes which can quickly release CO in vivo. In this study, an in vitro oxidative stress injury model was established to examine the effect of CORM-2 pretreatment on the nuclear-cytoplasmic translocation of high mobility group box 1 protein (HMGB1) in mouse primary renal proximal tubular epithelial cells (RPTECs). Immunofluorescence staining showed that HMGB1 in the medium- and CORM-2-treated groups was predominantly localized in the nucleus of the cells, whereas higher amounts of HMGB1 translocated to the cytoplasm in the HO- and inactive CORM-2 (iCORM-2)-treated groups. Western blotting of HMGB1 showed that the total amounts of cytoplasmic HMGB1 in the HO-treated (0.59±0.27) and iCORM-2-treated (0.57±0.22) groups were markedly higher than those in the medium-treated (0.19±0.05) and CORM-2-treated (0.21±0.10) groups (P<0.05). Co-immunoprecipitation showed that the levels of acetylated HMGB1 in the HO-treated (642.98±57.25) and iCORM-2-treated (342.11±131.25) groups were markedly increased as compared with the medium-treated (78.72±74.17) and CORM-2-treated (71.42±53.35) groups (P<0.05), and no significant difference was observed between the medium-treated and CORM-2-treated groups (P>0.05). In conclusion, our study demonstrated that in the in vitro oxidative stress injury model of primary RPTECs, CORM-2 can significantly inhibit the nuclear-cytoplasmic translocation of HMGB1, which is probably associated with the prevention of HMGB1 acetylation.
Active Transport, Cell Nucleus ; drug effects ; Animals ; Carbon Monoxide ; pharmacology ; Cell Nucleus ; metabolism ; Cells, Cultured ; Epithelial Cells ; drug effects ; metabolism ; HMGB1 Protein ; metabolism ; Kidney Tubules ; cytology ; Mice ; Organometallic Compounds ; pharmacology ; Oxidative Stress

Biliverdin (BV) has long been thought to be a cytotoxic metabolic waste product. It has also been demonstrated to have important cytoprotective functions during oxidative stress. The present study aimed to examine the cytoprotective effect of BV on NRK-52E cells, a proximal tubular cell line derived from rat kidney. Cells were treated with 50 µmol/L cisplatin for 24 h (cisplatin group) or pre-treated with BV for 30 min, then with 50 µmol/L cisplatin for 24 h (cisplatin+BV group). Those given no treatment served as a control. Cell apoptosis was evaluated by flow cytometry and cell viability by Cell Counting Kit-8 (CCK-8). The protein expressions of cleaved caspase3, Bax and Bcl-2 were assessed by Western blotting. Reactive oxygen species (ROS) levels were measured using carboxydichlorodihydrofluorescein diacetate (H2DCF). The results showed that cisplatin induced the apoptosis of NRK-52E cells, decreased cell viability, and increased the formation of ROS by upregulating the expression of cleaved caspase3 and Bax and decreasing Bcl-2 protein expression. These effects could be significantly reversed by pretreatment with BV. It was concluded that BV can protect against cisplatin-induced cell apoptosis through the anti-oxidative effects.
Animals ; Antioxidants ; pharmacology ; Apoptosis ; Biliverdine ; pharmacology ; Cell Line ; Cisplatin ; toxicity ; Epithelial Cells ; drug effects ; metabolism ; Kidney Tubules ; cytology ; Rats ; Reactive Oxygen Species ; metabolism

OBJECTIVETo investigate the effect of arctiin on advanced oxidation protein product (AOPP)-induced epithelial-to-mesenchymal transition (EMT) in tubular cells and explore the mechanisms underlying this effect.

METHODSHuman proximal tubular cells (HK-2 cells) were treated with bovine serum albumin (BSA) or AOPPs in the presence or absence of arctiin. The expressions of E-cadherin, vimentin, and GRP78 at the protein and mRNA levels in the cells were examined using Western blotting and quantitative real-time PCR. The level of reactive oxygen species (ROS) was measured by flow cytometry with DCFH-DA as the fluorescent probe.

RESULTSCompared with BSA-treated cells, the cells treated with AOPPs showed decreased expression of epithelial cell marker E-cadherin and overexpression of mesenchymal marker vimentin and endoplasmic reticulum stress marker GRP78 with an increased ROS level. These changes induced by AOPPs were partly inhibited by arctiin.

CONCLUSIONArctiin can ameliorate AOPP-induced EMT in tubular cells by inhibiting endoplasmic reticulum stress, and oxidative stress response may participate in this process.

Advanced Oxidation Protein Products ; adverse effects ; Cadherins ; metabolism ; Cell Line ; Endoplasmic Reticulum Stress ; Epithelial Cells ; cytology ; drug effects ; Epithelial-Mesenchymal Transition ; Furans ; pharmacology ; Glucosides ; pharmacology ; Heat-Shock Proteins ; metabolism ; Humans ; Kidney Tubules ; cytology ; drug effects ; Oxidative Stress ; Reactive Oxygen Species ; metabolism ; Vimentin ; metabolism

OBJECTIVETo explore the regulative effects and possible mechanisms of emodin on autophagy induced by starvation in rat's renal tubular epithelial cells (NRK-52E).

METHODFirstly, Hank's balanced salt solution (HBSS) was used to induce starvation and the protein expression of microtubule-associated protein 1 light chain 3 (LC3) I/II, an autophagic marker of mammalian congener, was detected by Western blot with or without the treatment of emodin. Secondly, the changes of red fluorescent protein-microtubule associated protein light chain3 (RFP-LC3) fluorescent particles, treated by HBSS (1 mL) and bafilomycin A1 (10 nmol x L(-1)) with or without emodin, were observed through fluorescence microscopy in NRK-52E cells transient transfected by RFP-LC3 plasmid. With the intervention of mammalian target of rapamycin mTOR inhibitor rapamycin (100 nmol x L(-1)) , the effect of blocking mTOR signaling pathway on autophagic inhibition of emodin was observed. Finally, the effect of mTOR signaling pathway on autophagic inhibition of emodin was further evaluated through the over-expression of endogenous mTOR inhibitory protein DEP domain-containing mTOR-interacting protein-(DEPTOR).

RESULTHBSS hunger could induce high protein expression of LC3 II in NRK-52E cells, and the intervention of emodin could reverse the unregulated protein expression of LC3 II induced by HBSS. The number of RFP-LC3 fluorescent particles was increased after the co-treatment of HBSS and bafilomycin A1, and this increase was inhibited by emodin. After the co-treatment of rapamycin, emodin and HBSS, the LC3 II protein expression restored in NRK-52E cells, compared with the treatment of HBSS. Over-expression of DEPTOR could also block the inhibitive effect of emodin on LC3 II protein expression.

CONCLUSIONEmodin could inhibit HBSS-induced LC3 II protein expression and the activation of autophagy in NRK-52E cells, and the effect of blocking autophagy may be mediated through mTOR signaling pathway.

Animals ; Autophagy ; drug effects ; Cell Line ; Down-Regulation ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Emodin ; pharmacology ; Isotonic Solutions ; adverse effects ; Kidney Tubules ; cytology ; drug effects ; metabolism ; Microtubule-Associated Proteins ; genetics ; metabolism ; Rats ; Signal Transduction ; drug effects ; TOR Serine-Threonine Kinases ; genetics ; metabolism

OBJECTIVETo investigate the differentiation capability of kidney stem cells (KSCs) into renal tubular epithelial cells (RTECs).

METHODSKSCs isolated from the renal papilla of 4-week-old SD rats were co-cultured with hypoxia-exposed RTEC in induced medium (containing activin A, BMP-7, and retinoic acid) and renal epithelial cell growth medium (REGM) alternately. The KSCs cultured in MSC medium served as the control. The KSC differentiation rates in both groups were determined using flow cytometry, immunofluorescence assay and qRT-PCR.

RESULTSFlow cytometry showed a CK-18 positive rate of 6.5Percnt; in the control KSC group and of 44.2% in the induced group. Immunofluorescence assay detected the positivity for mature epithelial cell markers CK-18, E-cadherin, and ZO-1 in the induced cells. The results of qRT-PCR showed significantly increased expression of E-cadherin and AQP-1 mRNAs in the induced cells compared with the control cells (P<0.01).

CONCLUSIONRat KSCs can be induced to differentiate into RTECs in vitro.

Activins ; chemistry ; Animals ; Aquaporin 1 ; metabolism ; Bone Morphogenetic Protein 7 ; chemistry ; Cadherins ; metabolism ; Cell Differentiation ; Coculture Techniques ; Culture Media ; chemistry ; Epithelial Cells ; cytology ; Keratin-18 ; metabolism ; Kidney Tubules ; cytology ; Rats ; Rats, Sprague-Dawley ; Stem Cells ; cytology ; Tretinoin ; chemistry ; Zonula Occludens-1 Protein ; metabolism

We investigated the protective effects of electromagnetic field (EMF) on the survival of the human renal proximal tubular cell line, HK-2, using an in vitro hypoxia/reoxygenation (H/R) injury model. The survival rate of cells cultured under H/R condition declined significantly, while the intracellular reactive oxygen species (ROS) levels markedly increased. The 10 Hz/1 mT EMF exposure reversed the H/R induced reduction in cell survival and induction of intracellular ROS. Our results suggest that 10 Hz/1 mT EMF exposure could inhibit H/R-induced cell death of HK-2 via suppression of intracellular ROS production and that this treatment might be clinically useful for the amelioration of renal ischemia/reperfusion injury.
Cell Hypoxia ; Cell Line ; Electromagnetic Fields ; Humans ; Kidney Tubules, Proximal ; cytology ; metabolism ; radiation effects ; Reactive Oxygen Species ; metabolism ; Reperfusion Injury ; prevention & control

The expression of hypoxia-inducible factor (HIF) is influenced by reactive oxygen species (ROS). Effect of bilirubin on HIF-1 expression in proximal tubular cells was investigated under physiological oxygen concentration, which is relative hypoxic condition mimicking oxygen content in the medulla of renal tissue. The human kidney (HK2) cells were cultured in 5% oxygen with or without bilirubin. HIF-1alpha protein expression was increased by bilirubin treatment at 0.01-0.2 mg/dL concentration. The messenger RNA expression of HIF-1alpha was increased by 1.69+/-0.05 folds in the cells cultured with 0.1 mg/dL bilirubin, compared to the control cells. The inhibitors of PI3K/mTOR, PI3K/AKT, and ERK 1/2 pathways did not attenuate increased HIF-1alpha expression by bilirubin. HIF-1alpha expression decreased by 10 microM exogenous hydrogen peroxide (H2O2); scavenger of ROS with or without bilirubin in the HK2 cells increased HIF-1alpha concentration more than that in the cells without bilirubin. Exogenous H2O2 decreased the phosphorylation of P70S6 kinase, which was completely reversed by bilirubin treatment. Knockdown of NOX4 gene by small interfering RNA (siRNA) increased HIF-1alpha mRNA expression. In coonclusion, bilirubin enhances HIF-1alpha transcription as well as the up-regulation of HIF-1alpha protein translation through the attenuation of ROS and subunits of NADPH oxidase.
Bilirubin/*pharmacology ; Cell Line ; Epithelial Cells/cytology/metabolism ; Humans ; Hydrogen Peroxide/toxicity ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics/*metabolism ; Kidney Tubules, Proximal/cytology ; Mitogen-Activated Protein Kinase 1/metabolism ; Mitogen-Activated Protein Kinase 3/metabolism ; NADPH Oxidase/antagonists & inhibitors/genetics/metabolism ; Oxygen/*pharmacology ; Phosphatidylinositol 3-Kinases/metabolism ; Phosphorylation/drug effects ; Proto-Oncogene Proteins c-akt/metabolism ; RNA Interference ; Ribosomal Protein S6 Kinases, 70-kDa/metabolism ; Signal Transduction/drug effects ; TOR Serine-Threonine Kinases/metabolism ; Transcriptional Activation/*drug effects ; Up-Regulation/drug effects

We evaluated the effect of cobalt chloride (CoCl2) on TNF-alpha and IFN-gamma-induced-inflammation and reactive oxygen species (ROS) in renal tubular epithelial cells (HK-2 cells). We treated HK-2 cells with CoCl2 before the administration of TNF-alpha/IFN-gamma. To regulate hemeoxygenase-1 (HO-1) expression, the cells were treated CoCl2 or HO-1 siRNA. CoCl2 reduced the generation of ROS induced by TNF-alpha/IFN-gamma. TNF-alpha/IFN-gamma-treated-cells showed an increase in the nuclear translocation of phosphorylated NF-kappaBp65 protein, the DNA-binding activity of NF-kappaBp50 and NF-kappaB transcriptional activity and a decrease in IkappaBalpha protein expression. These changes were restored by CoCl2. We noted an intense increase in monocyte chemoattractant protein-1 (MCP-1) and regulated on activation normal T cell expressed and secreted (RANTES) production in TNF-alpha/IFN-gamma-treated cells. We demonstrated that this effect was mediated through NF-kappaB signaling because an NF-kappaB inhibitor significantly reduced MCP-1 and RANTES production. CoCl2 effectively reduced MCP-1 and RANTES production. The expression of HO-1 was increased by CoCl2 and decreased by HO-1 siRNA. However, knockdown of HO-1 by RNA interference did not affect MCP-1 or RANTES production. We suggest that CoCl2 has a protective effect on TNF-alpha/IFN-gamma-induced inflammation through the inhibition of NF-kappaB and ROS in HK-2 cells. However, CoCl2 appears to act in an HO-1-independent manner.
Cell Line ; Chemokine CCL2/metabolism ; Chemokine CCL5/metabolism ; Cobalt/*pharmacology ; Epithelial Cells/cytology/metabolism ; Heme Oxygenase-1/antagonists & inhibitors/genetics/metabolism ; Humans ; *Inflammation ; Interferon-gamma/pharmacology ; Kidney Tubules, Proximal/cytology ; NF-kappa B/antagonists & inhibitors/genetics/*metabolism ; NF-kappa B p50 Subunit/genetics/metabolism ; Oxidative Stress/*drug effects ; Phosphorylation ; Protein Binding ; RNA Interference ; RNA, Small Interfering/metabolism ; Transcription Factor RelA/metabolism ; Tumor Necrosis Factor-alpha/pharmacology

BACKGROUNDSurfactant protein A (SP-A) contributes to the regulation of sepsis-induced acute lung injury. In a previous study, we demonstrated the expression and localization of SP-A in the kidneys. The present study evaluated the effect of SP-A on lipopolysaccharide (LPS)-induced tumor necrosis factor-a (TNF-α) expression and its underlying mechanisms in the human renal tubular epithelial (HK-2) cells.

METHODSIndirect immunofluorescence assay was used to detect SP-A distribution and expression in HK-2 cells. HK-2 cells were treated with various concentrations of LPS (0, 0.1, 1, 2, 5, and 10 mg/L) for 8 hours and with 5 mg/L LPS for different times (0, 2, 4, 8, 16, and 24 hours) to determine the effects of LPS on SP-A and TNF-α expression. Then, HK-2 cells were transfected with SP-A siRNA to analyze nuclear factor κB (NF-κB) P65 and TNF-α expression of HK-2 cells after LPS-treatment.

RESULTSIndirect immunofluorescence assay revealed that SP-A is localized to the membrane and cytoplasm of HK-2 cells. Interestingly, SP-A1/SP-A2 and TNF-a expression were found to be significantly increased in HK-2 cells upon LPS treatment. Transfection of LPS-treated HK-2 cells with SP-A siRNA resulted in significant increases in the levels of NF-κB P65 protein and TNF-α mRNA and protein compared to those in non-transfected LPS-treated HK-2 cells.

CONCLUSIONSP-A plays an important role in protecting cells against sepsis-induced acute kidney injury by inhibiting NF-κB activity to modulate LPS-induced increase in TNF-α expression.

Cell Line ; Epithelial Cells ; cytology ; drug effects ; metabolism ; Fluorescent Antibody Technique, Indirect ; Humans ; Kidney Tubules, Proximal ; cytology ; Lipopolysaccharides ; pharmacology ; Pulmonary Surfactant-Associated Protein A ; metabolism ; pharmacology ; Tumor Necrosis Factor-alpha ; metabolism