急性马兜铃酸肾损伤大鼠血清分泌型磷脂酶A2的变化

Activition of serum secretory phospholipase A2 in rats with acute kidney injury induced by aristolochic acid

目的通过测定急性马兜铃酸肾损伤大鼠血清分泌型磷脂酶A2（sPLA2）活性的变化，探讨sPLA2在马兜铃酸肾病（AAN）发病机制中的作用。方法将Wistar大鼠按随机数字表法分为两组：模型组给予关木通水煎剂（相当于生药30g·kg^-1·d^-1）灌胃7d，停药后正常喂养至第14天；对照组以自来水灌胃。光镜观察肾脏病理变化并用半定量方法评价肾小管间质损伤程度；生化法测定肾小球及肾小管功能；巯基显色法分别检测血清sPLA2活性；Western印迹法测定肾皮质和肾髓质环氧化酶2（COX-2）蛋白表达；放射免疫法分别测定血清和尿液中前列腺素类（PGs）代谢产物6-keto-PGF1α与血栓烷B2（TXB2）水平并计算二者的比值。结果给药后模型组大鼠出现明显的肾小管间质损伤和肾功能下降，并在第8天达高峰，肾小管间质损伤指数（8．14±2．55）较对照组（1．50±0．71）显著升高（P〈0．05），Scr水平显著增高[（0．24±0．10）比（0．19±0．02）μmol／g，P〈0．051。给药第8天时模型组血清中sPLA2活性（μmol·min^-1·mg^-1）（133．15±17．05）较对照组（101．3±16．07）显著增强（P〈0．05），同时肾皮质COX-2蛋白表达也显著增加（1．16±0．36比0．69±0．28，P〈0．05），但肾髓质的COX-2蛋白表达无变化。尽管模型组血清中6-keto．PGF1α、TXB2及其比值无变化，但其尿液中6-keto—PGF1α和TXB2水平显著升高，分别为对照组的2倍和3倍（P〈0．05），二者的比值显著降低（207．53±17．52比296．64±51．31，P〈0．05）。上述变化除肾小管间质损伤指数外，均在第14天时恢复，与对照组差异无统计学意义。结论在急性马兜铃酸肾损伤大鼠模型中，血清sPLA2呈激活状态，伴随出现的肾皮质COX-2表达增加及尿液中缩血管类PGs代谢产物增加，提示肾脏缩血管反应增强，可能是马兜铃酸肾损害时组织缺血改变的机制之一。

Objective To investigate whether the activation of secretory prophospholipase A2 （sPLA2） plays the role in the pathophysiological mechanism of acute aristolochic acid nephropathy （AAN） in rats. Methods Wistar rats were randomly divided into two groups. Model group received decocted Aristolochia Manshuriensis Kom 30 g·kg^-1·d^-1 by gavage for 7 days following tap water in same way for additional 7 days. Control group received only tap water by gavage at parallel time. The renal pathological changes were observed at the 4th, 8th and 14th day. The injury of renal tubules and interstitium was observed under light microscope following a semiquantity grade. The level of Scr was measured to evaluate glomcrular function. Urinary N-acetylbeta-glucosaminidase （NAG） was tested as renal tubular injury marker. The activity of sPLA2 in serum was detected by manifesting the color of thiols in the substrate. The protein expression of renal cortex and medulla COX-2 was analyzed by Western blot. The metabolic products of protaglandins （PGs） including 6-keto-PGF1α and TXB2 in the plasma and urine were assayed by radioimmunoassay. The ratio of 6-keto-PGF1α/TXB2 was calculated. Results After Aristolochia administration, the tubulointerstitial injury and Scr increased in AA rats and reached the peak at the 8th day, the tubulointerstitial injury index（8.14±2.55 vs 1.50±0.71, P〈0.05 ） and Scr[（0.24±0.10） vs （0.19±0.02）μmol/g, P〈0.05] increased significantly in AA rats compared with control group. The activity of sPLA2 （μmol·min^-1·mg^-1） in AA group elevated by 1.3-fold compared to control group at 8th day （133.15±17.05 vs 101.3±16.07, P〈0.05）, while the expression of COX-2 in renal cortex increased （1.16±0.36 vs 0.69±0.28, P〈0.05） with no change in renal medulla. Even though the levels of serum 6-keto-PGF1α and TXB2 did not change obviously in both AA and control group, but urinary levels of 6-keto-PGF1α and TXB2 increased by 2-fold and 3-fold in AA group compared to control group, respectively （all P〈0.05）, while the ratio of 6-keto-PGF1α/TXB2 decreased significantly （207.53±17.52 vs 296.64±51.31, P〈0.05）. All of above changes recovered to the control level at the 14th day except the tubulointerstitial injury index. Conclusion Serum sPLA2 is activated in the rats with acute kidney injury induced by aristolochic acid, which accompanied by up-regulated expression of COX-2 in renal cotex and increased the metabolic products of vasoconstrictive PGs in urine. These changes may participate the mechanism of renal peritubular ischemia in AAN.