Lead Induced Apoptosis In Human Renal Proximal Tubular Epithelial Cells (HK-2) And Its Mechanism

Objective:To observe the injure of human proximal renal tubular epithelial ceils (HK-2) by lead, and study the apoptotic pathways that are initiated by lead.Methods:HK-2 were cultured under 5% CO₂ and 95% air in an incubator set at 37℃, and exposed to various concentrations of lead acetate (0, 50, 100, 200, 400μmol/L)for 48 hours; HK-2were treated with 400μmol/L lead acetate for 0, 12, 24, 48 hours. The viability of cells were mediated by colony formation assay. The cell apoptosis was determined by nuclear staining with Hoechst33258, flow cytometric analysis with Annexin-V(FITC) and Propidium Iodide(PI). Activity of caspase-3, -8, -9 was determined by colodmetric assay. Cytochrome c(Cty c) releasing from mitochondrion to endochylema was observed by flow cytometric analysis. Western-bolting was utilized to measure the expression of caspase- 3, caspase-9, bcl-2 and bax.Results:1. Colony formation assay results revealed that lead could inhibit the viability of cells in does and time dependent.2. DNA-binding fluorochrome Hoechst 33258 showed the typical apoptotic nuclei condensation and fragmentation of chromatin.3. The rates of apoptosis of lead-treated HK-2 cells increased significantly which were determined by Flow Cytometer with FITC-AnnexinVand PI double staining (p＜0.05, p＜0.01).4. colodmetric assay results showed that lead could stimulated the activity of caspase-3, caspase-9, but have no effect on caspase-8 activity.5. Flowcytometer analysis also revealed that C_tv c releasing from mitochondrion to endochylema, and the quantity increased in dose and timc dependent. 6. Caspase-3 inhibitor, z-DEVD-CHO protected HK-2 from apoptosis induced by lead.7. Western-bolt result showed that lead stimulated the expression of the active fragment of caspase-3, -9 as well as bax expression, but inhibited bcl-2 expression.Conclusion:1. Lead can induce injure on HK-2.2. Lead can induce apoptosis in cultured HK-2.3. Lead induces HK-2 apoptosis via caspase-3 activation and mitochondr -ial depolarization.4. bcl-2 expression decreased in HK-2 and bax expressed increased, as probably is one of the cause of HK-2 apoptosis.