Effect Of JNK Signal Transduction Pathway On The Apoptosis Of Human Kidney Cell Induced By Ochratoxin A In Vitro

Objective: Ochratoxin A (OA) is a natural occurrence mycotoxin produced by some species of Aspergillus and Penicillium. OA could be widely found as one of the predominant contaminating mycotoxins in the grain and foodstuffs in a wide variety of climates and geographical regions. Studies showed that OA was nephrotoxic, hepatotoxic, immunotoxic,carcinogenic and teratogenic to several species of animals. The nephrotoxicity of OA is characterized by tubular epithelial cell degeneration, interstitial fibrosis, and impaired renal function. Furthermore, OA seems to be involved in the pathogenesis of Balkan endemic nephropathy and chronic interstitial nephritis.The c-Jun NH2-terminal-kinase (JNK) signal transduction pathway is one of mitogen-activated protein kinase (MAPK) signal transduction pathways. JNK could be activated by many factors, such as, inflammatory cytokines, growth factors and stress, etc. Several studies indicated that JNK signal transduction pathway plays an important role in cell differentiation, apoptosis, stress and the pathogenesis several human of diseases. Up to now, most of the studies on the mechanism of OA induced nephrotoxicity were focused on the formation of DNA adducts and inhibition of mitochondrial respiration chain. Few studies involved in the putative effects of JNK signal transduction pathway on the apoptosis of human kidney cell induced by Ochratoxin A in vitro.The possible effects of JNK signal transduction pathway on OA induced apoptosis of HKC cells in vitro were studied with MTT, flow cytometric analyses, immunocytochemical staining and western blotting.Methods1 Cell culture and treatmentHKC cells were cultured in DMEM medium supplemented with 10% new-born calf serum (NBCS), streptomycin (100μg/ml), penicillin (100U/ml) at 37℃, 5% CO2 . HKC cells in logarithmic growth phase were randomly divided into 4 groups: control group, solvent control group, OA treatment groups. OA final concentration of OA treatment groups were 1μΜand 5μΜrespectively. The cells in control group were treated with NS and in solvent control group were treated with alcohol (1:500).2 MTT assay2.1 MTT assay was carried to evaluate the effects of OA on the growth of cells. HKC cells in logarithmic growth phase were randomly divided into 4 groups: control group, solvent control group, 1μΜOA treatment group and 5μΜOA treatment group. The relationship between the time of OA treatment and proliferation inhibition effects was determined at 4h, 8h, 16h, 24h and 48h respectively by MTT. The OA exposure time was determined on the bases of the study.2.2 HKC cells in logarithmic growth phase were randomly divided into 4 groups: control group, solvent control group, 1μΜOA treatment group and JNK inhibitor group. The HKC cells in inhibitor group were treated with SP600125 (the final concentration was 0.5μΜ) for 30min before OA treatment. Twenty-four hours after the OA treatment, the survival rate of HKC was observed with MTT method.3 Flow cytometric (FCM) analyses of the apoptosis of HKC cells in vitroThe apoptosis rates of HKC cells in different groups were determined with FCM DNA analysis.4 Evaluation of the expression of caspase-3, p-JNK and JNK by immunocytochemical staining and Western Blotting The expression of caspase-3, p-JNK and JNK at protein level of HKC cells in vitro in different groups was determined with SP immunocytochemical staining and Western Blotting method.Results1 The effect of OA on the growth of HKC cells in vitroThe result of MTT showed that OA could inhibit the growth of human kidney cells in vitro. 4h, 8h, 16h, 24h and 48h after OA treatment, the survival rates of HKC cells in vitro were all significantly decreased (P< 0.05). Twenty four hours after OA treatment, the survival rate of HKC cells in 1μM and 5μM OA treatment groups was 51.09% and 53.38% respectively, which was significantly lower than that in control cells (100%, P<0.05) . Thus, based on the results, 24h was chosen as the experimental OA treatment time in the following experiment.2 The effect of OA on apoptosis of HKC cells in vitro2.1 FCM analysis resultsFCM DNA analysis showed that 24 h after OA treatment, the apoptosis rates in 1μM and 5μM OA treatment group (3.85±0.29% and 4.86±0.51% respectively) were significantly higher than that in control cells( 1.23±0.05%, P<0.05).2.2 The expression of caspase-3 in HKC cells in vitro2.2.1 Results of immunocytochemical stainingThe positive immunoreaction of caspase-3 was located in the cytoplasm of HKC cells as brown granules. The caspase-3 positive expression rate in control group was 14.31±4.24%, which was significantly lower than that in OA treatment groups (54.03±9.21% and 60.75±9.95% for 1μM and 5μM OA group, P<0.05). OA treatment could significantly increase the positive expression of caspase-3 of HKC cells in vitro.2.2.2 Results of Western BlottingThe molecular weight of caspase-3 is 32kD. After SDS-PAGE electrophoresis and Western hybridization, 32kD bands were found in lanes of both the control and OA treatment groups. Image analysis result revealed that OA could significantly increase the expression of Caspase-3 in HKC in vitro. 3 The effect of OA on the expression of p-JNK3.1 Immunocytochemical resultsThe immunocytochemical positive staining of p-JNK was located in both cytoplasm and nucleus of HKC cells. The percentages of positive staining cells in OA treatment groups were all significantly higher than that in control group (P<0.05). The positive expression rates of p-JNK in 1μM and 5μM OA treatment group was 53.31±11.01% and 58.53±11.11% respectively, while that in control group was only 10.19±6.85%. It was confirmed that OA could increase the expression of p-JNK in HKC.3.2 Western Blotting resultsp-JNK is composed of p-JNK1, p-JNK2 and p-JNK3. The molecular weight of p-JNK1 is 54kD, and that of p-JNK2 and p-JNK3 is 46kD. SDS-PAGE electrophoresis and Western hybridization showed light brown 54kD and 46kD bands. The results of image analysis confirmed that OA could increase the expression of p-JNK in HKC.4 The effect of OA on the expression of JNK4.1 Immunocytochemical resultsThe cytoplasmic positive expression of JNK could be found in HKC cells of all groups. The percentage of positive staining cells in OA treatment group was not significantly higher or lower than that in control group (P>0.05).4.2 Western Blotting resultsJNK is composed of JNK1, JNK2 and JNK3. The molecular weight of JNK1 is 54kD, while that of JNK2 and JNK3 is 46kD. After SDS-PAGE electrophoresis and Western hybridization, the 54kD and 46kD light brown positive straps could be seen. The results of image analysis suggested that the significant change could not be found among different groups.5 The effect of SP600125 pretreatment on OA induced survival inhibition of HKC in vitroThe result of MTT showed that the survival rate of HKC in SP600125 pretreatment (82.09%) was significantly higher than that in OA treatment group( 66.83%, P<0.05), but the survival rate of HKC in SP600125 pretreatment group was still lower than that in solvent control( 98.58% , P<0.05). It was indicated that JNK inhibitor, SP600125 could partly inhibit OA induced impairment of HKC.Conclusion1 OA could increase the expression of caspase-3 and induce apoptosis of HKC in vitro.2 OA could increase the expression of p-JNK of HKC cells in vitro, suggesting that JNK signal transduction pathway activation be involved in the apoptosis of HKC cells after OA treatment.3 JNK inhibitor, SP600125 could partly inhibit OA induced impairment of HKC in vitro.4 All experiment results in this study showed that the possible mechanisms of apoptosis of HKC cells after OA treatment may be through the activation of JNK signal transduction pathway and increase of the expression of caspase-3.