Laboratório de Farmacologia Bioquímica e Molecular, Instituto de Ciências Biomédicas, Centro de Ciências da Saúde, Av. Carlos Chagas 373, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil.
Am J Physiol Renal Physiol. 2012 Apr 15;302(8):F959-66. doi: 10.1152/ajprenal.00130.2011. Epub 2012 Jan 11.
Bufadienolides are structurally related to the clinically relevant cardenolides (e.g., digoxin) and are now considered as endogenous steroid hormones. Binding of ouabain to Na(+)-K(+)-ATPase has been associated, in kidney cells, to the activation of the Src kinase pathway and Na(+)-K(+)-ATPase internalization. Nevertheless, whether the activation of this cascade also occurs with other cardiotonic steroids and leads to diuresis and natriuresis in the isolated intact kidney is still unknown. In the present work, we perfused rat kidneys for 120 min with bufalin (1, 3, or 10 μM) and measured its vascular and tubular effects. Thereafter, we probed the effect of 10 μM 3-(4-chlorophenyl)1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-d]pyrimidin-4amine (PP2), a Src family kinase inhibitor, and 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio] butadiene (UO126), a highly selective inhibitor of both MEK1 and MEK2, on bufalin-induced renal alterations. Bufalin at 3 and 10 μM profoundly increased several parameters of renal function in a time- and/or concentration-dependent fashion. At a concentration that produced similar inhibition of the rat kidney Na(+)-K(+)-ATPase, ouabain had a much smaller diuretic and natriuretic effect. Although bufalin fully inhibited the rat kidney Na(+)-K(+)-ATPase in vitro, its IC(50) (33 ± 1 μM) was threefold higher than the concentration used ex vivo and all its renal effects were blunted by PP2 and UO126. Furthermore, the phosphorylated (activated) ERK1/2 expression was increased after bufalin perfusion and this effect was totally prevented after PP2 pretreatment. The present study shows for the first time the direct diuretic, natriuretic, and kaliuretic effects of bufalin in isolated rat kidney and the relevance of Na(+)-K(+)-ATPase-mediated signal transduction.
蟾毒配基与临床上相关的强心苷（如地高辛）在结构上有关联，现在被认为是内源性甾体激素。在肾细胞中，哇巴因与 Na(+)-K(+)-ATP 酶的结合与Src 激酶途径的激活和 Na(+)-K(+)-ATP 酶内化有关。然而，其他强心甾体是否也会激活这一级联反应，并导致在分离完整肾脏中利尿和排钠，目前尚不清楚。在本工作中，我们用蟾毒配基（1、3 或 10 μM）灌注大鼠肾脏 120 分钟，并测量其血管和管状效应。之后，我们探测了 10 μM 3-(4-氯苯基)-1-(1,1-二甲基乙基)-1H-吡唑并[3,4-d]嘧啶-4-胺（PP2），一种Src 家族激酶抑制剂，和 1,4-二氨基-2,3-二氰基-1,4-双[2-氨基苯基硫代]丁二烯（UO126），一种 MEK1 和 MEK2 的高选择性抑制剂，对蟾毒配基引起的肾脏改变的影响。蟾毒配基在 3 和 10 μM 时以时间和/或浓度依赖的方式显著增加了几个肾功能参数。在产生类似抑制大鼠肾脏 Na(+)-K(+)-ATP 酶的浓度下，哇巴因的利尿和排钠作用要小得多。尽管蟾毒配基在体外完全抑制了大鼠肾脏 Na(+)-K(+)-ATP 酶，但它的 IC(50)（33±1 μM）是在体外用的浓度的三倍，所有的肾脏作用都被 PP2 和 UO126 减弱。此外，在蟾毒配基灌注后，磷酸化（激活）的 ERK1/2 表达增加，而这种效应在 PP2 预处理后完全被阻止。本研究首次显示了蟾毒配基在分离大鼠肾脏中的直接利尿、排钠和排钾作用，以及 Na(+)-K(+)-ATP 酶介导的信号转导的相关性。
