Kemp Brandon A, Howell Nancy L, Gildea John J, Padia Shetal H
Department of Medicine, Division of Endocrinology and Metabolism, University of Virginia Health System, Charlottesville, Virginia.
Department of Pathology, University of Virginia Health System, Charlottesville, Virginia.
J Endocr Soc. 2019 Sep 2;3(11):2088-2106. doi: 10.1210/js.2019-00121. eCollection 2019 Nov 1.
Intrarenal ghrelin infusion activates ghrelin receptors in the kidney collecting duct (CD) to increase epithelial sodium (Na) channel (EC)-dependent Na reabsorption , but the underlying mechanisms are unknown. Seventy-two hours following uninephrectomy, 12-week-old female Sprague-Dawley rats received the following renal interstitial (RI) infusions for 1 hour after a 1-hour control: vehicle (n = 10), ghrelin (3 μg/minute; n = 8), ghrelin + phosphatidylinositol 3-kinase (PI3K) inhibitor LY-294002 (0.1 μg/kg/minute; n = 7), ghrelin + protein kinase A (PKA) inhibitor adenosine 3'5'-cyclic monophosphorothioate, Rp-isomer (10 μg/kg/minute; n = 8), ghrelin + microtubule polymerization inhibitor nocodazole (0.3 μg/kg/minute; n = 7), or ghrelin + actin polymerization inhibitor cytochalasin D (0.3 μg/kg/minute; n = 6). Compared with vehicle infusion, RI ghrelin induced a significant anti-natriuresis (urine Na excretion was reduced by 53.7% ± 6.8%; < 0.001). This effect was abolished during concomitant PKA or microtubule inhibition (106.4% ± 9.4% and 109.7% ± 10.6% of vehicle infusion, respectively; < 0.01 from ghrelin) but not during concomitant PI3K or actin inhibition (reduced by 48.6% ± 3.9% and 52.8% ± 12.7%, respectively; < 0.001 and < 0.01 from vehicle, respectively; = not significant from ghrelin). Infusions had no effect on mean arterial pressure. Western blot analysis demonstrated that CD membrane but not total EC expression increased in response to ghrelin infusion compared with vehicle, (0.39 ± 0.05 vs 0.12 ± 0.02 arbitrary units; < 0.01). This effect was abolished during PKA or microtubule inhibition but persisted during PI3K or actin inhibition. Neural precursor cell expressed, developmentally down-regulated 4 isoform 2 (Nedd4-2) dependent internalization of EC was not affected by ghrelin, indicating that microtubule-dependent forward trafficking of EC is necessary for anti-natriuretic responses to ghrelin. Taken together, these studies highlight the importance of PKA and microtubule polymerization in ghrelin-induced EC-mediated Na reabsorption.
肾内注射胃饥饿素可激活肾集合管（CD）中的胃饥饿素受体，以增加上皮钠（Na）通道（ENaC）依赖性钠重吸收，但其潜在机制尚不清楚。单侧肾切除术后72小时，12周龄雌性Sprague-Dawley大鼠在1小时对照后接受以下肾间质（RI）注射1小时：溶剂（n = 10）、胃饥饿素（3μg/分钟；n = 8）、胃饥饿素+磷脂酰肌醇3激酶（PI3K）抑制剂LY-294002（0.1μg/kg/分钟；n = 7）、胃饥饿素+蛋白激酶A（PKA）抑制剂3',5'-环磷硫酰腺苷，Rp-异构体（10μg/kg/分钟；n = 8）、胃饥饿素+微管聚合抑制剂诺考达唑（0.3μg/kg/分钟；n = 7）或胃饥饿素+肌动蛋白聚合抑制剂细胞松弛素D（0.3μg/kg/分钟；n = 6）。与溶剂注射相比，RI胃饥饿素诱导了显著的抗利钠作用（尿钠排泄减少53.7%±6.8%；P<0.001）。在同时抑制PKA或微管时，这种作用被消除（分别为溶剂注射的106.4%±9.4%和109.7%±10.6%；与胃饥饿素相比P<0.01），但在同时抑制PI3K或肌动蛋白时未被消除（分别减少48.6%±3.9%和52.8%±12.7%；与溶剂相比P<0.001和P<0.01；与胃饥饿素相比P=不显著）。注射对平均动脉压无影响。蛋白质印迹分析表明，与溶剂相比，胃饥饿素注射后CD膜而非总ENaC表达增加（0.39±0.05对0.12±0.02任意单位；P<0.01）。在抑制PKA或微管时，这种作用被消除，但在抑制PI3K或肌动蛋白时持续存在。神经前体细胞表达的、发育性下调的4异构体2（Nedd4-2）依赖性ENaC内化不受胃饥饿素影响，表明ENaC的微管依赖性正向运输是胃饥饿素抗利钠反应所必需的。综上所述，这些研究突出了PKA和微管聚合在胃饥饿素诱导的ENaC介导的钠重吸收中的重要性。
