Wakida N, Kitamura K, Tuyen D G, Maekawa A, Miyoshi T, Adachi M, Shiraishi N, Ko T, Ha V, Nonoguchi H, Tomita K
Department of Nephrology, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan.
Kidney Int. 2006 Oct;70(8):1432-8. doi: 10.1038/sj.ki.5001787. Epub 2006 Aug 30.
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-beta1 (TGF-beta1) 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-beta1 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 20 ng/ml TGF-beta1 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-beta1 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.
前列腺素酶已被证明可调节肾脏中的钠处理。最近，一种丝氨酸蛋白酶抑制剂，即蛋白酶nexin-1（PN-1），被鉴定为前列腺素酶的内源性抑制剂。因此，我们推测PN-1可能通过降低前列腺素酶活性来调节钠重吸收，并且PN-1的表达像前列腺素酶一样受转化生长因子-β1（TGF-β1）或醛固酮调节。上皮钠通道（ENaC）、前列腺素酶和PN-1的cRNA在非洲爪蟾卵母细胞中表达，并测量了amiloride敏感的钠电流（I(Na)）。通过实时聚合酶链反应和免疫印迹法在M-1细胞中检测TGF-β1和醛固酮对PN-1和ENaC的mRNA和蛋白质丰度的影响。PN-1的表达使卵母细胞中前列腺素酶诱导的I(Na)在约68%的水平上显著降低。用20 ng/ml TGF-β1处理M-1细胞可使PN-1的蛋白质表达显著增加3.8±0.5倍，而给予10(-6) M醛固酮则使PN-1的蛋白质表达显著降低至53.7±6.7%。从基底外侧而非顶端施加TGF-β1可显著降低I(eq)。为了阐明PN-1在基础ENaC活性中的作用，我们使用短干扰RNA使PN-1的表达沉默。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酶活性发挥利钠作用，并提示醛固酮和TGF-β可能分别通过一种额外机制相互调节肾上皮细胞中PN-1的表达，从而分别导致钠潴留或利钠。PN-1可能是一种有助于调节肾脏中ENaC活性的新因子。
