| An Pre-column derivation HPLC for the determination of ETM in kidney of rats was first established to lay the foundations for next step which aimed to study the relationship between etimicin nephrotoxicity, apoptosis in RTEC and histopathological changes and its accumulation in rat kidneys after repeated injection of ETM by i.p.A Shimadzu LC-10A HPLC instrument was used with a UV detector set at330nm. The separation was performed on a Platisil C18(150mm×4.6mm,5μm) with Easy Guard C18Pre-column and0.02mol· L-1sodium heptanesulfonate dissolved by a mixture consisting of20volumes of water,5volumes of acetic acid and75volumes of methanol was used as mobile phase. The flow rate was set at1.0mL· min-1. Column temperature was35℃. The rats were killed on the1st,2nd,4th day after the delivery of ETM at a single-dosage of2mg· kg-1by i.m. and the kidneys were gathered. The rats were killed and the kidneys were gathered24hours after the last dosing of ETM by i.p. for5days. Tissue protein was precipitated by isopropanol and the ETM was derivatized with o-phthalaldehyde. The rats were divided into ETM-treated group including15rats injected with ETM100mg· kg-1· d-1for5consecutive days and vehicle control group including3rats injected with equal volume of physiological saline. After cessation of ETM administration,3rats in ETM-treated group were sacrificed respectively on the1st,3rd,7th,10th and15th day, and their kidneys were gathered. Rats in control group were sacrificed on the1st day after the last dosing. Determination of ETM concentrations accumulated in rat kidneys was carried out by HPLC with pre-column derivatization. Apoptosis in RTEC was identified by TUNEL assay. Histopathological ch- anges in kidneys were evaluated by HE staining.The linear range for ETM concentration in kidney homogenate was1.25～60μg· mL-1, r=0.9973. Concentrations of ETM in kidneys on the1st,2nd,4th day after the last i.m. injection of ETM at a single-dosage of2mg· kg-1were (32.88±3.21),(37.67±4.93) and (21.63±4.91) μg· g-1tissue respectively.24h after the last i.p. injection of ETM at dose of60and100mg· kg-1· d-1for5consecutive days, the concentrations of ETM in kidneys were (241.4±158.5) and (386.4±98.3) μg· g-1tissue, respectively. Results from the present study displayed that ETM accumulated significantly in rat kidneys, especially on the1st day after cessation of ETM administration, which was (347.5±193.3) μg· g-1tissue. And then began to decrease gradually.15days after cessation of injection, ETM accumulation in renal tissue was (16.71±9.988) μg· g-1tissue. Half-life of ETM in rat kidneys was about3.05day. Simultaneously, apoptosis induced by ETM was recovered gradually from (1544±138)(n· mm2)-1on the1st day to (716±208)(n· mm2)-1on the15th day after the last dosing of ETM. Histopathological damage was also gradually recovered.The method is simple, stable and accurate, which is suitable for the determination of ETM contents in kidneys. ETM-induced nephrotoxicity can gradually restore with its accumulation decreasing in the renal cortex. |
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