Mechanism Of Protective Effect Of L-carnitine On Human Kidney Tubular Epithelial Cell Line Injury Induced By Oxidative Stress

Increased oxidative stress have been implicated in a variety of kidney diseases, such as renal ischemia-reperfusion (I/R) injury caused by shock or during surgery or transplantation, diabetic nephropathy, and chronic tubulointerstitial injury. It is mediated by reactive oxygen species (ROS), including free radicals such as superoxide ions and hydroxyl radicals as well as non-free radical species such as hydrogen peroxide (H2O2). ROS can be generated within the nephron segments like the glomeruli and proximal tubule and mammalian cells have developed several protective mechanisms to prevent ROS formation or detoxify ROS, which include low-molecular-mass antioxidants (ascorbic acid, glutathione, tocopherols, and others), ROS-interacting enzymes (superoxide dismutase, peroxidases, and catalases), and redox regulation enzymes. I/R excessively produces reactive oxygen species (ROS) beyond this organ's scavenging capacity for ROS, simultaneously impairs antioxidant enzymes, and causes cell damage by lipid peroxidation, DNA breakdown, and protein damage. Studies have shown that ischemia/reperfusion (I/R) inevitably accompanied with renal transplantation is well characterized oxidative stress-induced tissue injury immediately after kidney transplantation. Injury initiated by the lack of oxygen during cold presentation is augumented by ROS during subsequent warm reperfusion of grafts through activation of imflammatory cascade. Reactive oxygen species (ROS) is also markedly increased after kidney transplantation and may participate in the development and/or progression of chronic renal allograft nephropathy [3]. ROS-induced mitochondrial swelling, caspase-3 activation, which contributes to both necrotic and apoptotic forms of cell death have been documented after I/R injury in the kidney and in posthypoxic isolated proximal tubules occurred. The expression of both the antiapoptotic Bcl-2 family of proteins, Bcl-2 and Bcl-XL, and the proapoptotic proteins Bax, p53, FADD, and Bak in the distal and proximal tubules during the first 24 h were increased after I/R injury in the kidney, with the net effect determining the severity of injury and dysfunction. Antioxidant strategy may reduce oxidative stress and inhibit apoptotic signaling and cell death which will allow better preservation of graft function and ameliorate the associated injury and inflammation in kidney.L-Carnitine (4-N-trimethylammonium-3-hydroxybutyric acid), an L-lysine derivative, is an endogenous mitochondrial membrane compound. The main physical function of L-carnitine in human body is facilitating the transport of long chain fatty acids into mitochondria in order to enter the P-oxidation cycle. Used as a safe and effective nutritional supplement for more than three decades, the protective effect of 1-carnitine on kidney tissue has been proved in various models involving oxidative stress, such as cisplatin-induced injury of the kidney and small intestine, gentamycin-induced nephrotoxicity, ischaemia- reperfusion injury of the kidney and chronic renal failure. It has been demonstrated that L-carnitine administration inhibits both serum and kidney tissue MDA formation in response to renal ischaemia/reperfusion injury. By using different antioxidant assays,Ⅰlhami Gulcin demonstrated that L-carnitine had an effective DPPH·scavenging, superoxide anion radical scavenging, hydrogen peroxide scavenging, total reducing power and metal chelating on ferrous ions activities compared to a-tocopherol and trolox as references antioxidants. Carnitine can also act as a chelator by decreasing the concentration of cytosolic iron, which plays a very important role in free radical chemistry. And carnitine supplementation enhances the activities of anti-oxidant enzymes, such as SOD, CAT and GPx, and GSH levels and decreases the MDA concentration in kidney tissues of 24-month-old rats.In light of the findings described above, the present study employed the human proximal tubule epithelial cell line, HK-2 cells as a cell model system and aimed to elucidate the molecular mechanisms of L-carnitine on renal oxidative stress. As the major component of ROS, H2O2 is produced during the redox process and is considered as a messenger in intracellular signaling cascades. H2O2 could cause lipid peroxidation and DNA damage. So we used H2O2 as an inducer of oxidative stress for HK-2 cells and tested whether pretreatment cells with L-carnitine resulted in the resistance of HK-2 to H2O2 challenge. Furthermore, the effect of L-carnitine on oxidative stress conditions such as ROS production, lipid peroxidation, antioxidant defensive system, mitochondrial dysfunction and DNA damage associated with cell apoptosis were also studied.Chapter one Protective effect of L-carnitine on human kidney tubular epithelial cell line damaged by oxidative stressObjective To establish the HK-2 cell oxidative model induced by H2O2, and investigate the protective effect and its mechanism of L-carnitine on human kidney tubular epithelial cell line damaged by hydrogen peroxide (H2O2)-mediated oxidative stress.Methods Human kidney tubular epithelial cell line (HK-2 cells) were exposed to H2O2 of different concentration. The exact dose of H2O2 for the oxidative model was determined according to the cell viability evaluted by MTT assays. Then the HK-2 cells were divided into 5 group:normal control group, LC alone group, H2O2 group, LC protected group(pretreated with L-carnitine for 12hs and then injuryed by H2O2), and NAC group. The cell viability evaluted by MTT assays. Enzyme activities including superoxide dismutase (SOD), glutat hione peroxidase (GSH-Px), catalase (CAT), total antioxidative capacity (T-AOC) and malondialdehyde (MDA) were determined by biochemical methods. Intracellular ROS was detected by means of an oxidation sensitive fluorescent probe (DCF-DA) and the cell apoptosis were quantified by determining DNA content of cells by propidium iodide staining by flow cytometry. The Data was shown as mean±sd, analysed by One-way ANOVA and LSD-t test, SPSS 16.0, P<0.05 was statistically significant difference.Results H2O2 500μM X 30min decreased the cell viability significantly compared to the normal control group (P＜0.001). L-carnitine(LC) 1OμM,50μM,100μM alone for 12h increased the cell viability compared to the normal control group (P＜0.001) Pretreated by LC for 12h could inhibit H2O2-induced cell viability loss(P<0.001). The activities of intracellar superoxide dismutase(SOD), glutathione peroxidase(GPx), catalase(CAT) and total anti-oxidative capacity (T-AOC) decresed 12h after the cells exposed to H2O2 500μM for 30 min(P<0.001) compared to the normal control group. Pretreated by LC for 12h could enhance the activities of these antioxidant enzymes significantly in a concentration-dependent manner compared to the H2O2 group(P<0.001). Also, L-carnitine pretreatment increased total anti-oxidative capacity (T-AOC) and inhibited MDA formation. The intracellular reactive oxygen species generation and cell apoptosis triggered by H2O2 characterized with the DNA fragment were also inhibited by L-carnitine.Conclusions These results indicated that L-carnitine exhibited protective effects on HK-2 cells injuried by oxidative stress. It may be related to its antioxidative action which included enhancing endogenous antioxiant defense components, inhibiting the ROS production, MDA formation and cell apoptosis.Chapter two Role of the mitochondrial in L-carnitine inhibiting oxidative stress-induced human kidney tubular epithelial cell apoptosisObjective To investigate the role of the mitochondrial in the inhibiting effect of L-carnitine on hydrogen peroxide (H2O2)-induced human kidney tubular epithelial cell apoptosis.Methods Human kidney tubular epithelial cell line (HK-2 cells) were pretreated with L-carnitine for 12hs and then injuryed by H2O2. The cell apoptosis were evaluted by nuclear staining assay using chromatin dye Hoechst 33258 and flow cytometric detection of caspase-3 activity. Mitochondrial membrane potential was monitored using the fluorescent dye Rh123 by flow cytometry. Expression levels of Bcl-2, Bax and the release of cytochrome c were determined by Western blot analysis. The Data was shown as mean±sd, analysed by One-way ANOVA and LSD-t test, SPSS16.0, P<0.05 was statistically significant difference.Results The apoptosis rate of H2O2 injured cells was significantly higher than that of L-carnitine pretreatment cells(P<0.001). Activities of caspase-3 in H2O2 injured cells seemed higher than normal cells, and L-cartine pretreatment could prohibit the activation of caspase-3 in a concentration-dependent manner(P<0.001). The mitochondrial trans-membrane potential (ΔψM) was rapidly reduced when HK-2 cells were exposed to H2O2 and H2O-induced dissipation ofΔψM was significantly blocked by the pretreatment with L-carnitine. There was significant decrease in the mitochondrial cytochrome c level after H2O2 injury, which was accompanied by a simultaneous increase in cytochrome c level in the cytosol. Treatment of HK-2 cells with L-carnitine reduced cytochrome c in the cytosol and increased the mitochondrial cytochrome c. In H2O2 group, the expression rate of Bcl-2 was significantly reduced and the expression of Bax was increased compared to normal group(P＜0.001). When HK-2 cells were treated with 50μM L-carnitine before H2O2 injury, the expression rate of Bcl-2 was markedly increased compared with H2O2 group.Conclusions These results suggested that L-carnitine could inhibit H2O2-induced kidney tubalar epithelial cell apoptosis through the mitochondrial pathway.