OBJECTIVETo study whether aristololactam I (AL-I) induces injury in human renal proximal tubular epithelial cells.

METHODCultured human renal proximal tubular epithelial cell line HK-2 was used as the subject. Aristolochic Acid I (AA-I) was used as a positive control. Cell toxicity of AL-I was detected by LDH releasing rate. Cell apoptosis was evaluated by cellular morphology, DNA content and expression of cell membrane phosphatidylserine (PS). The secretion level of fibronectin (FN) and TGF-beta1 in HK-2 cells were assayed by ELISA.

RESULTAL-I had a direct toxicity on HK-2 in a dose dependent manner from 2.5 mg x mL(-1) to 20 mg x mL(-1); In these range of concentration, AL-I could induce cell apoptosis which was detectable by measurements of morphology, DNA content and expression of PS. AL-I could stimulate the secretion of FN and TGF-beta1. The potency of AL-I cell toxicity was higher than AA-I at the same concentration. The effects of AL-I on apoptosis, secretion of FN and TGF-beta1 were all weaker than AA-I.

CONCLUSIONAL-I as one metabolite of AA-I in vivo induces direct injury in renal proximal tubular cells. Its effects are similar to those of AA-I. AL-I may be one of toxic metabolites in Chinese herbs containing AA which participate in renal damage and fibrosis.

Apoptosis ; drug effects ; Aristolochia ; chemistry ; Aristolochic Acids ; administration & dosage ; isolation & purification ; toxicity ; Cell Line ; Cell Membrane ; metabolism ; DNA ; genetics ; Diploidy ; Dose-Response Relationship, Drug ; Epithelial Cells ; drug effects ; Humans ; Kidney Tubules, Proximal ; cytology ; metabolism ; Plants, Medicinal ; chemistry

OBJECTIVETo study the effects of core proteoglycan on the transdifferentiation of human renal tubular epithelial cell induced by transforming growth factor beta1 (TGF-beta1) in vitro.

METHODThe cultured HK-2 cells were divided into six groups: A. negative control group; B. 10 ng/ml TGF-beta1 group; C. 10 ng/ml core proteoglycan treated group; D. 100 ng/ml core proteoglycan treated group; E. 10 ng/ml TGF-beta1 + 10 ng/ml core proteoglycan group; F. 10 ng/ml TGF-beta1 + 100 ng/ml core proteoglycan group. The changes in configuration of HK-2 cells were inspected 48 hours after adding the stimulating factor. At the same time, changes in mRNA of keratin, alpha-smooth muscle actin, vimentin were analyzed.

RESULTSCompared with group A, group B showed great changes in the morphology of cells, most cells converted into spindle shape, like fibroblast; groups E and F, especially group F showed significantly reduced spindle shape cells. Compared with group A, groups C and D had no significant changes in morphology of cells Compared with 10 ng/ml TGF-beta1 group and negative control, the mRNA expression of alpha-smooth muscle actin and vimentin had significant increase, but that of keratin reduced (P < 0.05). However, after combined treatment with TGF-beta1 and core proteoglycan, alpha-smooth muscle actin and vimentin expression were reduced significantly, while expression of keratin was up-regulated. Single core proteoglycan treated group and negative control group had no dramatic differences (P > 0.05).

CONCLUSIONTGF-beta1 can induce the transdifferentiation of human renal tubular epithelial cell and core proteoglycan has some inhibitory effect on transdifferentiation of human renal tubular epithelial cell induced by TGF-beta1 in vitro.

Actins ; physiology ; Bone Morphogenetic Protein 7 ; metabolism ; Cadherins ; metabolism ; Cell Differentiation ; drug effects ; physiology ; Cell Line ; Cell Shape ; Cell Transdifferentiation ; Cells, Cultured ; Connective Tissue Growth Factor ; pharmacology ; Epithelial Cells ; cytology ; drug effects ; physiology ; Fibroblasts ; drug effects ; pathology ; Humans ; Kidney ; pathology ; Kidney Tubules ; pathology ; Kidney Tubules, Proximal ; pathology ; Proteoglycans ; chemistry ; pharmacology ; Transforming Growth Factor beta ; genetics ; pharmacology ; Transforming Growth Factor beta1 ; chemistry ; pharmacology ; Vimentin ; metabolism

To discover the nephroprotective substances from Huangkui capsule.The components of Huangkui capsule were isolated by preparative liquid chromatography, and the active components were screened by LC/MS and identified. The adriamycine-injured HK-2 cells were treated with various active components with different concentrations, and the malonaldehyde (MDA) content, adenosine triphosphate (ATP) level and mitochondrial oxygen consumption rate were measured to verify the protective activity of the compounds.Four active components in Huangkui capsule were identified to exert nephroprotective effects. Fifteen flavanoids from these four components were tentatively identified by LC/MS, and hyperin, myricetin, quercetin, rutin and isoquercetin were confirmed. Hyperin, myricetin quercetin and rutin showed dose-dependent protective effects on injured HK-2 cells. Espacially, hyperin significantly reduced MDA content, quercetin and rutin significantly increased ATP level, and myricetin significantly increased mitochondrial oxygen consumption rate.Hyperin, myricetin, querctein and rutin might be the potential nephroprotective compounds in Huangkui capsule, their effects may be related to the inhibition of lipid peroxidation and the alleviation of mitochondrial damage.
Abelmoschus ; chemistry ; drug effects ; Adenosine Triphosphate ; metabolism ; Cell Line, Transformed ; Chromatography, Liquid ; Doxorubicin ; Drugs, Chinese Herbal ; Epithelial Cells ; drug effects ; Flavonoids ; pharmacology ; Kidney Diseases ; chemically induced ; drug therapy ; prevention & control ; Kidney Tubules, Proximal ; drug effects ; Lipid Peroxidation ; drug effects ; Malondialdehyde ; metabolism ; Mass Spectrometry ; Mitochondria ; drug effects ; Oxygen Consumption ; drug effects ; Protective Agents ; chemistry ; pharmacology ; Quercetin ; analogs & derivatives ; pharmacology ; Rutin ; pharmacology

The nephrotoxicity of gentamicin (GM) has been widely recognized. Heat shock protein 72 (HSP72) has been reported to be a cytoprotectant. However, its cytoprotective effect against GM induced kidney injury has not yet been studied. In this study, we investigated the cytoprotective effect of HSP72 on GM-induced nephrotoxicity in vitro. Human Kidney tubular cell line, HK-2 cells were divided into four groups: control group, GM group (cells incubated with GM only), heat shock (HS) group (cells incubated at 43 degrees C for 30 min), and GM plus HS group, respectively. Lactate dehydrogenanse (LDH) release increased time-dependently from 24 hr to 96 hr compared to the data of cells treated with GM only. Results of NAG activities, superoxide dismutase (SOD) activities and malondialdehyde (MDA) content were similar to that of the LDH release. The amount of HSP72 positive cells increased significartly at 72 hr after cells were treated with GM only. Both HSP72 protein and gene expression increased significantly at 72 hr when cells were treated with GM. On the other hand, HS induced HSP72 expression markedly. Pretreatment of HS inhibited HK-2 cells from GM-induced injury. It could reduce LDH release and NAG activity. HS also increased SOD activity, and decreased MDA content when cells were damaged by GM. These findings suggested that HS may protect kidney cells from GM-induced injury. Pre-induction of HSP72 may provide therapeutic strategies for nephrotoxicity induced by GM.
Reactive Oxygen Species/metabolism ; RNA, Messenger/analysis ; Oxidation-Reduction ; L-Lactate Dehydrogenase/secretion ; Kidney Tubules, Proximal/chemistry/*drug effects ; Humans ; Heat ; HSP72 Heat-Shock Proteins/analysis/genetics/*physiology ; Gentamicins/*toxicity ; Cytoprotection ; Cells, Cultured

To investigate whether a series of water-soluble cross-linked chitosan derivates synthesized in the guide of imprinting technology could be used as a uranium chelating agent to protect cells exposed to depleted uranium (DU), the imprinted chitosan derivates with high UO2(2+) chelating ability were screened, and cell model of human renal proximal tubule epithelium cells (HK-2) exposed to DU (500 micromol.L-1) was built, chitosan derivates (400 mg.L-1 ) was added to test group and diethylenetriaminepentaacetic acid (DTPA, 50 mg.L-1) was added to positive control group. The results showed that three Cu2+ imprinted chitosan derivates had higher uranium chelating ability (>49 microg.mg-1) than chitosan and non-imprinted chitosan derivates. Compared to the cells exposed to DU only, survival of cells in group added chitosan derivates rose up significantly (increased from 57.3% to 88.7%, and DTPA to 72.6%), and DU intracellular accumulation decreased, membrane damage and DNA damage also eased. Among the imprinted chitosan derivates, Cu2+ imprinted penta dialdehyde cross-linked carboxymethyl chitosan (Cu-P-CMC) was the best, and better than DTPA. From ultrastructure observation, the DU precipitates of test group added Cu-P-CMC were most grouped in a big hairy clusters in a string together outside cells. It is possible that the DU-chitosan derivates precipitates are too big to enter into cells, and from this way, the DU uptake by cells decreased so as to detoxication.
Antioxidants ; metabolism ; Apoptosis ; drug effects ; Cell Line ; Cell Survival ; drug effects ; Chelating Agents ; administration & dosage ; chemistry ; pharmacology ; Chitosan ; administration & dosage ; analogs & derivatives ; chemistry ; pharmacology ; Copper ; chemistry ; pharmacology ; Cross-Linking Reagents ; administration & dosage ; chemistry ; pharmacology ; DNA Damage ; drug effects ; Epithelial Cells ; cytology ; ultrastructure ; Humans ; Inactivation, Metabolic ; Kidney Tubules, Proximal ; cytology ; ultrastructure ; Microscopy, Electron, Transmission ; Uranium ; toxicity ; Water

OBJECTIVETo observe the renal injury in rats induced by Longdan Xiegan decoction (LDXGD) containing different dosages Aristolochia manshriensis.

METHODSD rats were divided into four groups at random, and were fed with three kinds of LDXGD 13, 14.5, 17.5 g x kg(-1) (containing respectively A. manshriensis 1.5, 3, 6 g x kg(-1)) and distilled water respectively for 12 weeks. Renal functional parameters on 4,8,12 w were determined and changes of histomorphology in rats on the end of experiment were observed.

RESULTThe LDXGD containing low dose (1.5 g x kg(-1)) A. manshriensis did't induce significantly renal injury in rats during 12 weeks; the LDXGD containing midst dose(3 g x kg(-1)) A. manshriensis induced light damage of proximal convoluted tubule epithelial cells in rats during 12 weeks; the LDXGD containing high dose(6 g x kg(-1)) A. manshriensis induced significantly renal injury in rats after administed 4 weeks. Along with the lasting of administration, the degree of injury became more seriously. The main renal injury location was in proximal convoluted tubule.

CONCLUSIONThe renal toxicity of LDXGD is correlated with the dose of A. manshriensis and the time of administration. The LDXGD containing low dose A. manshriensis has relative security. However, the LDXGD containing high dose A. manshriensis can induce renal injury.

Acetylglucosaminidase ; urine ; Animals ; Aristolochia ; chemistry ; toxicity ; Blood Urea Nitrogen ; Creatinine ; blood ; Dose-Response Relationship, Drug ; Drug Combinations ; Drugs, Chinese Herbal ; administration & dosage ; isolation & purification ; toxicity ; Kidney Diseases ; chemically induced ; metabolism ; pathology ; Kidney Tubules, Proximal ; drug effects ; pathology ; Male ; Plants, Medicinal ; chemistry ; toxicity ; Proteinuria ; chemically induced ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; beta 2-Microglobulin ; urine ; gamma-Glutamyltransferase ; urine

Betaig-h3 (betaig-h3) is a secretory protein composed of fasciclin I-like repeats containing sequences that allows binding of integrins and glycosaminoglycans in vivo. Expression of betaig-h3 is responsive to TGF-beta and the protein is found to be associated with extracellular matrix (ECM) molecules, implicating betaig-h3 as an ECM adhesive protein of developmental processes. We previously observed predominant expression of betaig-h3 expression in the basement membrane of proximal tubules of kidney. In this study, the physiological relevance of such localized expression of betaig-h3 was examined in the renal proximal tubular epithelial cells (RPTEC). RPTEC constitutively expressed betaig-h3 and the expression was dramatically induced by exogenous TGF-beta1 treatment. betaig-h3 and its second and fourth FAS1 domain were able to mediate RPTEC adhesion, spreading and migration. Two known alpha3beta1 integrin-interaction motifs including aspartatic acid and isoleucine residues, NKDIL and EPDIM in betaig-h3 were responsible to mediate RPTEC adhesion, spreading, and migration. By using specific antibodies against integrins, we confirmed that alpha3beta1 integrin mediates the adhesion and migration of RPTECs on betaig-h3. In addition, it also enhanced proliferation of RPTECs through NKDIL and EPDIM. These results indicate that betaig-h3 mediates adhesion, spreading, migration and proliferation of RPTECs through the interaction with alpha3beta1 integrin and is intimately involved in the maintenance and the regeneration of renal proximal tubular epithelium.
Amino Acid Motifs ; Antibodies, Blocking/immunology ; Cell Adhesion/physiology ; Cell Movement/physiology ; Cell Proliferation ; Cells, Cultured ; Epithelial Cells/drug effects ; Extracellular Matrix Proteins/chemistry/immunology/*metabolism ; Humans ; Integrin alpha3beta1/chemistry/immunology/*metabolism ; Kidney Tubules, Proximal/cytology/metabolism/*physiology ; Peptides/chemistry/metabolism ; Protein Interaction Mapping ; Research Support, Non-U.S. Gov't ; Transforming Growth Factor beta/chemistry/immunology/*metabolism/pharmacology

A series of adefovir mono-L-amino acid esters, mono non-steroidal anti-inflammatory drugs carboxylic ester prodrugs with more potent anti-HBV activity and lower nephrotoxicity were designed and synthesized. Adefovir bis (L-amino acid) ester was used as lead compound, according to pathological and pharmacological findings that non-steroidal anti-inflammatory drugs can effectively inhibit the organic anion transporter 1 (hOAT1)-mediated adefovir phosphonic acid pairs of anion transport across tubular basement membrane thereby reducing the nephrotoxicity of adefovir. Flatten design principle was used to introducing non-steroidal anti-inflammatory drugs structural fragments to design and synthesize target adefovir mixture ester prodrugs. HepG2 2.2.15 cell line was used as in vitro anti-HBV activity evaluation model. Five compounds exhibited antiviral activity, and compound 18 showed the most potent anti-HBV activity and relatively high selective index (EC50 3.92 micromol L(-1), SI 9.97). HK-2 cell line was used as in vitro model to evaluate nephrotoxicity. Results suggested the target compounds have lower cytotoxicity than the positive control. Moreover, by analyzing the primary structure and activity relationship of these compounds, it could suggest that mono-L-amino acid ester, mono non-steroidal anti-inflammatory drugs carboxylic ester prodrugs strategy has significant potential in the acyclic nucleoside phosphonates prodrug design.
Adenine ; analogs & derivatives ; chemical synthesis ; chemistry ; pharmacology ; Amino Acids ; chemical synthesis ; chemistry ; pharmacology ; Anti-Inflammatory Agents, Non-Steroidal ; chemical synthesis ; chemistry ; pharmacology ; Antiviral Agents ; chemical synthesis ; chemistry ; pharmacology ; Carboxylic Acids ; chemistry ; pharmacology ; Cell Survival ; drug effects ; Drug Design ; Hep G2 Cells ; drug effects ; Humans ; Kidney Tubules, Proximal ; cytology ; metabolism ; L-Lactate Dehydrogenase ; metabolism ; Molecular Structure ; Organophosphonates ; chemical synthesis ; chemistry ; pharmacology ; Prodrugs ; chemical synthesis ; chemistry ; pharmacology

This study is to assess the efficacy of BPCBG on the decorporation of uranium (VI) and protecting human renal proximal tubular epithelial cells (HK-2) against uranium-induced damage. BPCBG at different doses was injected intramuscularly to male SD rats immediately after a single intraperitoneal injection of UO2(CH3COO)2. Twenty-four hours later uranium contents in urine, kidneys and femurs were measured by ICP-MS. After HK-2 cells were exposed to UO2(CH3COO)2 immediately or for 24 h followed by BPCBG treatment at different doses for another 24 or 48 h, the uranium contents in HK-2 cells were measured by ICP-MS, the cell survival was assayed by cell counting kit-8 assay, formation of micronuclei was determined by the cytokinesis-block (CB) micronucleus assay and the production of intracellular reactive oxygen species (ROS) was detected by 2',7'-dichlorofluorescin diacetate (DCFH-DA) oxidation. DTPA-CaNa3 was used as control. It was found that BPCBG at dosages of 60, 120, and 600 micromol kg(-1) resulted in 37%-61% increase in 24 h-urinary uranium excretion, and significantly decreased the amount of uranium retention in kidney and bone to 41%-31% and 86%-42% of uranium-treated group, respectively. After HK-2 cells that had been pre-treated with UO2(CH3COO)2 for 24 h were treated with the chelators for another 24 h, 55%-60% of the intracellular uranium was removed by 10-250 micromol L(-1) of BPCBG. Treatment of uranium-treated HK-2 cells with BPCBG significantly enhanced the cell survival, decreased the formation of micronuclei and inhibited the production of intracellular ROS. Although DTPA-CaNa3 markedly reduced the uranium retention in kidney of rats and HK-2 cells, its efficacy of uranium removal from body was significantly lower than that of BPCBG and it could not protect uranium-induced cell damage. It can be concluded that BPCBG effectively decorporated the uranium from UO2(CH3COO)2-treated rats and HK-2 cells, which was better than DTPA-CaNa3. It could also scavenge the uranium-induced intracellular ROS and protect against the uranium-induced cell damage. BPCBG is worth further investigation.
Animals ; Cell Line ; Cell Survival ; drug effects ; Chelating Agents ; administration & dosage ; chemistry ; pharmacology ; Dose-Response Relationship, Drug ; Epithelial Cells ; cytology ; metabolism ; Humans ; Kidney ; metabolism ; Kidney Tubules, Proximal ; cytology ; Male ; Micronucleus Tests ; Molecular Structure ; Organometallic Compounds ; toxicity ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species ; metabolism ; Uranium ; metabolism ; urine

This study is to investigate the protection effect of schisandrin B (Sch B) against oxidation stress of HK-2 cells induced by cisplatin and the mechanisms involved. HK-2 cells were cultured and divided into different groups: solvent control group, cisplatin exposure group, positive group, Sch B treatment group. Cell viability and toxicity were evaluated by MTT and LDH assay. GSH level and SOD enzymes activities were also measured. DCFH-DA as fluorescence probe was used to detect ROS level by fluorescence microplate reader. Nrf2 translocation was detected by Western blotting. Real time Q-PCR was used to detect expressions of NQO1, HO-1 and GCLC mRNA level. The results showed that Sch B could significantly inhibit the decline of cell viability induced by cisplatin treatment (P < 0.05) and the protective effect was in a dose dependent manner. Furthermore, Sch B treatment significantly inhibited the increase of ROS level induced by cisplatin and reversed the decrease of GSH level (P < 0.05). When Sch B concentration was up to 5 micromol x L(-1), SOD enzyme activities were also enhanced significantly compared with that of the cisplatin group (P < 0.05). It was shown that Sch B could cause nuclear accumulation of Nrf2 in association with downstream activation of Nrf2 mediated oxidative response genes such as GCLC, NQO1 and HO-1. These results suggested Sch B could protect against the oxidative damage of HK-2 cells induced by cisplatin via the activation of Nrf2/ARE signal pathway.
Antineoplastic Agents ; toxicity ; Antioxidants ; isolation & purification ; pharmacology ; Cell Line ; Cell Survival ; drug effects ; Cisplatin ; toxicity ; Cyclooctanes ; isolation & purification ; pharmacology ; Glutamate-Cysteine Ligase ; genetics ; metabolism ; Glutathione ; metabolism ; Heme Oxygenase-1 ; genetics ; metabolism ; Humans ; Kidney Tubules, Proximal ; cytology ; metabolism ; L-Lactate Dehydrogenase ; metabolism ; Lignans ; isolation & purification ; pharmacology ; NAD(P)H Dehydrogenase (Quinone) ; genetics ; metabolism ; NF-E2-Related Factor 2 ; genetics ; metabolism ; Polycyclic Compounds ; isolation & purification ; pharmacology ; RNA, Messenger ; metabolism ; Reactive Oxygen Species ; metabolism ; Schisandra ; chemistry ; Signal Transduction ; Superoxide Dismutase ; metabolism