Department of Cardiovascular and Renal Research, Institute of Molecular Medicine, University of Southern Denmark, Odense C, Denmark.
Acta Physiol (Oxf). 2010 Dec;200(4):347-59. doi: 10.1111/j.1748-1716.2010.02170.x.
We hypothesized that the serine protease prostasin is necessary for differentiation of a high-resistance renal collecting duct epithelium governed by glucocorticoid.
Postnatal rat kidney and adult human kidney was used to study the expression and localization of prostasin. The murine collecting duct cell line (M-1) was cultured in Snapwell inserts to investigate the significance of prostasin for the development of transepithelial electrical resistance (TER).
In the cortex and medulla of rat kidney, prostasin mRNA and protein increased significantly between birth and weaning (day 21) and was detected in collecting ducts. Immunoreactive prostasin was associated with collecting ducts and loops of Henle in human kidney. In rat, adrenalectomy at day 10 had no effect on prostasin mRNA level in kidney at day 20. Cultured M-1 cells exhibited parallel increases in prostasin mRNA, protein and TER 5 days after seeding. Apical addition of the serine protease inhibitor aprotinin to M-1 cell cultures inhibited development of TER and led to aberrant localization of E-cadherin. This effect was mimicked by the protease inhibitor nafamostat. Apical addition of phospholipase C to cleave glycosylphosphatidylinositol (GPI) anchors released prostasin to the medium and attenuated development of TER with time of culture. Disruption of lipid rafts by methyl-β-cyclodextrin attenuated development of TER in M-1 cells. Omission of dexamethasone impaired development of TER in M-1 cells, while prostasin protein abundance and E-cadherin distribution did not change.
Apical, GPI-anchored, lipid raft-associated serine protease activity, compatible with prostasin, is necessary for the development of a high-resistance collecting duct epithelium.
我们假设丝氨酸蛋白酶原酶对于糖皮质激素调控的高电阻肾集合管上皮细胞分化是必需的。
利用新生大鼠肾和成人肾研究原酶的表达和定位。在 Snapwell 插入物中培养鼠集合管细胞系（M-1），以研究原酶对跨上皮电阻（TER）发育的意义。
在大鼠肾的皮质和髓质中，原酶 mRNA 和蛋白在出生到断奶（第 21 天）期间显著增加，并在集合管中检测到。免疫反应性原酶与人类肾中的集合管和 Henle 袢有关。在第 10 天进行肾上腺切除术对第 20 天肾中的原酶 mRNA 水平没有影响。培养的 M-1 细胞在接种后 5 天内，原酶 mRNA、蛋白和 TER 平行增加。在 M-1 细胞培养物中，向顶端添加丝氨酸蛋白酶抑制剂抑肽酶抑制了 TER 的发育，并导致 E-钙粘蛋白异常定位。蛋白酶抑制剂萘甲司坦产生了类似的作用。向顶端添加磷脂酶 C 以裂解糖基磷脂酰肌醇（GPI）锚定物将原酶释放到培养基中，并随着培养时间的延长减弱 TER 的发育。用甲基-β-环糊精破坏脂筏减弱了 M-1 细胞中 TER 的发育。在 M-1 细胞中省略地塞米松会损害 TER 的发育，而原酶蛋白丰度和 E-钙粘蛋白分布没有变化。
顶端、GPI 锚定、脂筏相关的丝氨酸蛋白酶活性，与原酶一致，是高电阻集合管上皮细胞发育所必需的。
