前列腺素E2在肾脏球旁器调节肾素分泌中的作用

目的 观察前列腺素E2（PGE2）对肾脏球旁器颗粒细胞（JGG）肾素分泌的调节作用。 方法 将小鼠肾脏致密斑细胞株（MMDD1）种植在特殊滤器上，滤器上下（细胞顶侧和基底侧）分别用不同培养基培养细胞，改变细胞顶侧培养基中钠离子、氯离子和血管紧张素Ⅱ（AngⅡ）浓度，分别测定不同时间点滤器上、下PGE2浓度。腹腔注射卡托普利（30 mg/kg）前后，放射免疫法测定野生型和环氧化酶基因敲除（COX-2-/-）小鼠血浆肾素活性(PRA)变化；原代分离COX-2-/-小鼠JGG，测定细胞上清肾素活性及其对PGE2刺激的反应。实时定量PCR测定低肾素（JGG细胞特异性Gsα基因缺失）小鼠肾脏皮质COX-2 mRNA表达。免疫组化法检测肾皮质COX-2蛋白表达。代谢笼中留取24 h尿，ELISA方法测定PGE2水平。 结果 (1)低氯刺激能使致密斑细胞分泌PGE2，不论基底侧还是细胞顶端PGE2浓度均显著增加（均P < 0.05）；但AngⅡ对致密斑分泌PGE2没有显著影响；(2)COX-2-/-小鼠基础PRA（378.3±96.4）比（1115.0±210.0） ng AngI&#8226;ml-1&#8226;h-1，P ＝ 0.0051，n＝10]和JGG细胞肾素分泌（153.7±14.7）比（672.4±129.0） ng AngI&#8226;ml-1&#8226;h-1，P ＝ 0.0162，n＝3]显著低于相同遗传背景的野生型小鼠；卡托普利能刺激COX-2-/-小鼠PRA增加32.8倍；PGE2能部分恢复COX-2-/-小鼠原代JGG细胞分泌肾素功能；(3)PGE2受体EP4耦联的Gsα基因敲除的低肾素小鼠，肾脏皮质COX-2 mRNA表达增加（8.07±1.08)倍（n=6，P = 0.0022)，免疫组化显示致密斑和远端小管COX-2蛋白表达增加，24 h尿PGE2分泌增加（1235±152） pg/24 h 比（385±140） pg/24 h，P = 0.0065]。 结论 致密斑PGE2直接受低氯调节，COX-2-/-小鼠基础肾素减少；下游的AngⅡ能直接作用于JGG细胞而不是致密斑负反馈调节肾素分泌。阻断JGG细胞自身的肾素产生（Gsα基因敲除）后，能负反馈上调致密斑的COX-2表达，故COX-2-PGE2-肾素在球旁器存在近距离精确调控机制。

Role of prostaglandin E2 in regulation of renin secretion at juxtaglomerular apparatus

Objective To investigate the effect and mechanism of prostaglandin E2 (PGE2) in renin regulation at the juxtaglomerular apparatus (JGA). Methods Macula densa cell line (MMDD1) was cultured on the special filter. In the medium on the apical lateral of the cells, low concentration of sodium chloride, chloride and different doses of angiotensin Ⅱ (Ang Ⅱ) were used to stimulate the PGE2 secretion. The PGE2 concentration was tested by ELISA. In the animal experiment, the response of plasma renin activity (PRA) to acute intraperitoneal administration of captopril (30 mg/kg) was determined, in conscious wild-type (WT) and cyclooxygenase COX-2-/- mice on C57BL/6 genetic backgrounds. PRA was measured in plasma obtained by tail vein puncture. Different concentrations of PGE2 were used to stimulate the renin secretion of primary cultured JGA cells from COX-2-/- mice and wild type mice. In specific Gsα gene delete mice (low renin producing mice), 24 h urine was collected to test the concentration of PGE2. The COX-2 mRNA and protein of the kidney cortex were observed by real-time PCR and immunohistochemicul staining. Results Low chloride could stimulate the PGE2 secretion both at the apical and basement of the macula densa cells. In COX-2-/- mice, the base PRA and were obviously lower than wild type mice. Captopril could stimulate the PRA of (COX)-2-/- mice increasing 32.8 times. But Ang Ⅱ had no effect on PGE2 secretion in macula densa cells. In primary cultured JGA cells, the decreasing renin seretion was partly recovered by PGE2 in cells from COX-2-/- mice. In low renin producing mice, the expression of COX-2 mRNA in the kidney cortex increased by (8.07±1.08) times (n=6, P=0.0022). The COX-2 protein of the kidney cortex and the urine PGE2 increased by several times. Conclusions Low chloride is the primary stimulation messenger of PGE2 secretion in macula densa cells. The PRA in COX-2-/- mice can be stimulated by angiotensin converting enzyme inhibitor, but the Ang Ⅱ has no direct effect on macula densa cells. When renin production is abolished in JGA cells (Gsα delete mice), COX-2 mRNA and protein up-regulation is observed in kidney cortex and macula densa. PGE2 plays an important role in regulation of renin secretion and renin release in JGA by precise feedback mechanism.