Inhibition of prostasin-induced ENaC activities by PN-1 and regulation of PN-1 expression by TGF-β1 and aldosterone
Inhibition of prostasin-induced ENaC activities by PN-1 and regulation of PN-1 expression by TGF-β1 and aldosterone
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DOI：
10.1038/sj.ki.5001787
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发表时间：
2006-10-01
影响因子：
19.6
通讯作者：
Tomita, K.
中科院分区：
文献类型：
--
作者：
Wakida, N.;Kitamura, K.;Tomita, K.
前列腺素已被证明可以调节肾脏中的钠处理。最近，一种丝氨酸蛋白酶抑制剂蛋白酶连接蛋白-1 （PN-1）被确定为前列腺蛋白酶的内源性抑制剂。因此，我们假设PN-1可能通过降低前列腺素活性来调节钠的重吸收，PN-1的表达受转化生长因子- β 1 （tgf - β 1）或醛固酮（如前列腺素）的调节。在爪蟾卵母细胞中表达上皮钠通道（ENaC）、前列腺素（prostasin）和PN-1的crna，并测量阿米洛利敏感钠电流（I-Na）。采用实时聚合酶链反应和免疫印迹法检测tgf - β 1和醛固酮对M-1细胞PN-1和ENaC mRNA和蛋白丰度的影响。在卵母细胞中，PN-1的表达显著降低前列腺素诱导的I-Na约68%。20ng/ml tgf - β 1处理M-1细胞后，PN-1蛋白表达量显著增加3.8 +/- 0.5倍，而10(-6)M醛固酮处理后，PN-1蛋白表达量显著降低至53.7 +/- 6.7%。基底外侧（而非根尖）应用tgf - β 1可显著降低I-eq。为了阐明PN-1在ENaC基础活性中的作用，我们使用短干扰RNA沉默了PN-1的表达。这使I-eq增加了1.6 +/- 0.1倍。我们的研究表明PN-1可能通过抑制前列腺素活性而具有尿钠作用，并提示醛固酮和tgf - β可能通过另一种机制相互调节PN-1在肾上皮细胞中的表达，从而分别促进盐潴留或尿钠。PN-1可能是一种有助于调节肾脏ENaC活性的新因子。
Prostasin has been shown to regulate sodium handling in the kidney. Recently, a serine protease inhibitor, protease nexin-1 (PN-1), was identified as an endogenous inhibitor for prostasin. Therefore, we hypothesized that PN-1 may regulate sodium reabsorption by reducing prostasin activity, and that expression of PN-1 was regulated by transforming growth factor-beta 1 (TGF-beta 1) or aldosterone, like prostasin. cRNAs for epithelial sodium channel (ENaC), prostasin, and PN-1 were expressed in Xenopus oocytes, and the amiloride-sensitive sodium currents (I-Na) were measured. The effect of TGF-beta 1 and aldosterone on the mRNA and protein abundance of PN-1 and ENaC was detected by real-time polymerase chain reaction and immunoblotting in M-1 cells. Expression of PN-1 substantially decreased prostasin-induced I-Na by approximately 68% in oocytes. Treatment of M-1 cells with 20ng/ml TGF-beta 1 significantly increased protein expression of PN-1 by 3.8 +/- 0.5-fold, whereas administration of 10(-6) M aldosterone markedly decreased protein expression of PN-1 to 53.7 +/- 6.7%. Basolateral, but not apical, application of TGF-beta 1 significantly reduced I-eq. To elucidate the involvement of PN-1 in basal ENaC activity, we silenced the expression of PN-1 by using short-interfering RNA. This increased I-eq by 1.6 +/- 0.1-fold. Our study indicates that PN-1 could have a natriuretic role by inhibiting prostasin activity and suggests the possibility that aldosterone and TGF-beta reciprocally regulate the expression of PN-1 in renal epithelial cells contributing to salt retention or natriuresis, respectively by an additional mechanism. PN-1 could represent a new factor that contributes to regulation of ENaC activity in the kidney.