环境钙对中华大蟾蜍肾脏斯钙素-1基因表达与质膜Ca^(2+)-ATP酶活性的影响

Influence of the ambient calcium level on stanniocalcin-1 gene expression and plasma membrane Ca^(2+)-ATPase activity in kidney of Bufo bufo gargarizans

目的探讨血浆钙水平对肾组织斯钙素-1(STC1)基因表达与质膜Ca2+-ATP酶(PMCA)活性的影响。方法将72只成体中华大蟾蜍随机分为3组,分别暴露于添加0.1mol/L CaCl2的自来水、添加0.03mol/L乙二胺四乙酸(EDTA)的自来水及自来水中,于暴露前及暴露后12、24、48、72、96、120和144h取血浆和肾脏,利用比色法和半定量RT-PCR法分别检测血浆钙水平、PMCA活性和STC1 mRNA水平,利用免疫组织化学染色确定STC1免疫反应。结果 0.1mol/L CaCl2暴露致中华大蟾蜍血浆钙水平在12和24h时升高,48h时降至正常水平,48h后持续升高;12～48h内肾组织PMCA活性增强,STC1 mRNA水平上调,72h后回复至正常水平。而0.03mol/L EDTA暴露则引起血浆钙水平下降,但肾组织PMCA活性和STC1 mRNA水平与对照组相比未出现明显变化。免疫组织化学技术显示,肾脏远曲小管和集合管上皮细胞出现STC1免疫反应,且0.1mol/L CaCl2暴露可致STC1免疫反应增强。结论血浆钙水平升高致肾小管和集合管上皮细胞PMCA活性增强,STC1表达上调,而STC1表达上调又使血浆钙水平下降;但血浆钙水平持续升高则使PMCA活性下降,STC1基因表达下调。

Objective To investigate the influence of the plasma calcium level on stanniocalcin-1 （STC1） gene expression and on plasma membrane Ca2＋ -ATPase （PMCA） activity in kidney. Methods Seventy-two adult toads, Bufo bufo gargarizans, were randomly divided into 3 groups, which were exposed to the tap water only, the tap water with 0. lmol/L CaCI2 , and the tap water with 0.03mol/L ethylene diamine tetraacetic acid（EDTA） , respectively. The toad＇ s plasma was prepared. Kidneys were excised at pre-exposure and post-exposure 12, 24, 48, 72, 96, 120, and 144 hours, respectively. Both of plasma Ca2＋ level and PMCA activity in kidney were detected with colorimetry. STCI gene expression in kidney were analyzed with semi-quantitative RT-PCR. STC1 immunohistochemical staining were showed by using rabbit antibody for mouse STC1 in conjunction with streptavidin/HRP-conjugated second antibodies. Results Compared with the un-treated group, 0. lmol/L CaC12 exposure induced the plasma calcium level to increase at the 12 th and 24 th hour, and to restore to the normal level at the 48 th hour, and to increase after 72 hours. Furthermore, 0. 1mol/L CaC12 exposure enhanced PMCA activity, and up-regulated STC1 gene expression during 12-24 hours, and restored them to the normal level at the 72 th hour. Compared with un-treated group, the plasma calcium level decreased, but both of PMCA activity and STCI gene expression did not change after 0. 03mol/L EDTA exposure. The immunohistochemistry staining showed that STC1 immunoreactive cells were found in distal convoluted tubules, and in medullary collecting ducts. Moreover, 0. lmol/ L CaC12 exposur enhanced STC1 immunoreactivity. Conclusion High plasma calcium level stimulates PMCA activity and up-regulation of STC1 gene expression in kidney. The up-regulation of STC1 decreases the plasma calcium level. However, substained high plasma calcium level results in inhibition of PMCA activity and down-regulation of STC1 level.