高原低氧环境下大鼠肾脏水通道蛋白2的表达及意义

Expression of aquaporin 2 in rat kidney under hypoxia at an altitude of 4600 m

目的研究高原低氧环境下大鼠肾脏内髓水通道蛋白2（AQP2）mRNA及蛋白的表达。方法将40只雄性SD大鼠随机分为24h、48h、72h及1周组，同时设立对照组（西宁地区）。将4个实验组由西宁地区（2260m）带至青海大学可可西里高原医学研究基地（4600m），在不同时间点处死取材。用放射免疫法检测血浆抗利尿激素（AHD）浓度，Western印迹、实时定量PCR、免疫荧光等方法检测AQP2蛋白及mRNA的表达。结果暴露于高原低氧环境后，大鼠AHD水平首先呈下降趋势，24h为（142．46±10．57）；48h时达到最低水平，仅为对照组的28．5％[（86．94±6．49）μg／L比（302．53±10．48）μg／L]；后逐渐上升，72h组为（169．65±11．15）μg／L，与对照组差异均有统计学意义（均P〈0．01）；1周时为（306．46±11．14）μg／L达到对照组水平。AQP2 mRNA的表达与AHD水平的变化相一致，初入高原后呈下降趋势，48h时达到最低水平，后逐渐上升，24h、48h、72h组与对照组差异均有统计学意义（分别为0．04±0．005，0．03±0．002，0．04±0．003比0．09±0．008，均P〈0．01），1周时为（0．09±0．01），达到对照组水平。AQP2蛋白表达与mRNA的表达变化一致。免疫荧光结果显示，对照组及1周组大鼠肾脏荧光强度表达一致，其余各组较低，48h组荧光强度最低。结论在高原低氧环境下大鼠肾脏内髓AQP2 mRNA和蛋白的表达在缺氧早期呈下降趋势，后逐渐上升到正常水平，其改变可能和机体对缺氧的程度和高原低氧的习服过程有关。

Objective To investigate the change of aquaporin 2 （AQP2） mRNA and protein levels in renal collecting duct of SD rats after hypoxia caused by rising of the altitude to 4600 m. Methods Forty male SD rats were randomly divided into 4 groups （24 h, 48 h, 72 h and 1 week group）, and 10 rats in Xining city were used as control group. All the 40 SD rats were transported to Kekexili Natural Reservation areas （4600 m） in Qinghai province. Rats of four experimental groups were sacrificed and renal tissue samples were harvested at different time point respectively, the control group rats were treated in Xining city （2260 m） as well. The concentration of plasma antidiuretic hormone （ADH） was measured by radioimmunity method. The expression of AQP2 mRNA and proteins was evaluated by real-time fluorescent quantitative-PCR, Western blot and immunofluorescence assay. Results The concentration of plasma ADH was decreased at 24 h and was only 28.5% of that of control group, reaching the lowest concentration at 48 h [（86.94±6.49） μg/L vs （302.53±10.48） μg/L], then it increased gradually and was similar to the control group at 7 d [（306.46±11.14） μg/L vs （302.53±10.48） μg/L, P〉 0.05]. There were significant differences of the control group with 24 h, 48 h and 72 h groups, respectively[（302.53±10.48） μg/L vs （142.46±10.57） μg/L, （86.94±6.49） μg/L, （169.65±11.15） μg/L respectively, P〈0.01]. The change of AQP2 gene expression level was consistent with the change of ADH. It was decreased at the begining when exposure to altitude and it reached its lowest level at 48 h. It was then returned to high level similarly to that of the control group at 7 d （0.09±0.01 vs 0.09±0.008, P〉0.05 ）. There were significant differences of the control group with 24 h, 48 h and 72 h group, respectively （0.09±0.008 vs 0.04±0.005, 0.03±0.002, 0.04±0.003 respectively, P〈0.01）. Conclusions AQP2 expression in the renal collecting duct of SD rats is altered over the period exposed to altitude. It is decreased in the early hypoxia period, and is increased in later period. This change may be related to the intensity of hypoxia, which is mediated by a potential adaptation mechanisms against hypoxia caused by high altitude.