Department of Internal Medicine, Division of Endocrinology, Diabetology and Nephrology, University Hospital Tübingen, Tuebingen, Germany.
Institute of Cellular and Molecular Physiology, Friedrich-Alexander University Erlangen-Nürnberg (FAU), Erlangen, Germany.
Acta Physiol (Oxf). 2021 May;232(1):e13640. doi: 10.1111/apha.13640. Epub 2021 Mar 11.
The serine protease prostasin (Prss8) is expressed in the distal tubule and stimulates proteolytic activation of the epithelial sodium channel (ENaC) in co-expression experiments in vitro. The aim of this study was to explore the role of prostasin in proteolytic ENaC activation in the kidney in vivo.
We used genetically modified knockin mice carrying a Prss8 mutation abolishing proteolytic activity (Prss8-S238A) or a mutation leading to a zymogen-locked state (Prss8-R44Q). Mice were challenged with low sodium diet and diuretics. Regulation of ENaC activity by Prss8-S238A and Prss8-R44Q was studied in vitro using the Xenopus laevis oocyte expression system.
Co-expression of murine ENaC with Prss8-wt or Prss8-S238A in oocytes caused maximal proteolytic ENaC activation, whereas ENaC was activated only partially in oocytes co-expressing Prss8-R44Q. This was paralleled by a reduced proteolytic activity at the cell surface of Prss8-R44Q expressing oocytes. Sodium conservation under low sodium diet was preserved in Prss8-S238A and Prss8-R44Q mice but with higher plasma aldosterone concentrations in Prss8-R44Q mice. Treatment with the ENaC inhibitor triamterene over four days was tolerated in Prss8-wt and Prss8-S238A mice, whereas Prss8-R44Q mice developed salt wasting and severe weight loss associated with hyperkalemia and acidosis consistent with impaired ENaC function and renal failure.
Unlike proteolytically inactive Prss8-S238A, zymogen-locked Prss8-R44Q produces incomplete proteolytic ENaC activation in vitro and causes a severe renal phenotype in mice treated with the ENaC inhibitor triamterene. This indicates that Prss8 plays a role in proteolytic ENaC activation and renal function independent of its proteolytic activity.
丝氨酸蛋白酶前激肽释放酶（Prss8）在远曲小管中表达，并在体外共表达实验中刺激上皮钠通道（ENaC）的蛋白水解激活。本研究的目的是探讨 Prss8 在体内肾脏中蛋白水解 ENaC 激活中的作用。
我们使用携带一种突变（Prss8-S238A）消除蛋白水解活性或导致酶原锁定状态（Prss8-R44Q）的基因修饰敲入小鼠。用低钠饮食和利尿剂对小鼠进行挑战。使用非洲爪蟾卵母细胞表达系统研究 Prss8-S238A 和 Prss8-R44Q 对 ENaC 活性的调节。
与 Prss8-wt 或 Prss8-S238A 共表达的小鼠 ENaC 在卵母细胞中引起最大的蛋白水解 ENaC 激活，而在共表达 Prss8-R44Q 的卵母细胞中仅部分激活 ENaC。这与 Prss8-R44Q 表达卵母细胞表面蛋白水解活性降低相平行。在 Prss8-S238A 和 Prss8-R44Q 小鼠中，低钠饮食下的钠保留得到保留，但 Prss8-R44Q 小鼠的血浆醛固酮浓度更高。用 ENaC 抑制剂阿米洛利治疗 4 天，Prss8-wt 和 Prss8-S238A 小鼠耐受，而 Prss8-R44Q 小鼠出现盐耗竭和严重体重减轻，伴有高钾血症和酸中毒，与 ENaC 功能受损和肾衰竭一致。
与无蛋白水解活性的 Prss8-S238A 不同，酶原锁定的 Prss8-R44Q 在体外产生不完全的蛋白水解 ENaC 激活，并导致用 ENaC 抑制剂阿米洛利治疗的小鼠出现严重的肾脏表型。这表明 Prss8 在 ENaC 激活和肾功能中发挥作用，独立于其蛋白水解活性。
