Natriuretic effect of bufalin in isolated rat kidneys involves activation of the Na⁺-K⁺-ATPase-Src kinase pathway

Abstract

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.

Expression of Mutant α1 Na/K-ATPase Defective in Conformational Transition Attenuates Src-mediated Signal Transduction*

“…Moreover, fluorescence resonance energy transfer analyses indicated that both proteins were in close proximity, supporting a direct interaction in live cells (6). These early observations have now been confirmed by many laboratories (32)(33)(34)(35)(36)(37)(38). More recently, we have identified two potential interacting pairs between the ␣1 subunit of Na/KATPase and Src, namely the A domain/SH2 domain and the N domain/kinase domain.…”

Regulation of renal function and structure by the signaling Na/K-ATPase

“…Many laboratories have investigated the role of Src in Na/K‐ATPase‐mediated signal transduction . Moreover, the following data indicate that the Na/K‐ATPase and Src most likely interact directly to form a functional receptor complex .…”

“…Indeed, application of CTS to the basolateral but not apical side of renal epithelial cell monolayer in culture reduces the transcellular movement of Na + . Ex vivo studies provide additional evidence of the importance of CTS and Na/K‐ATPase‐mediated signal transduction in the regulation of tubular Na + movement . Finally, studies with Dahl salt‐sensitive rats have revealed the inability of CTS in the activation of Na/K‐ATPase‐mediated signal transduction in renal proximal tubular cells, resulting in decreased ability of renal salt handling and consequently an increase in blood pressure in response to salt loading .…”

Epithelial Na+,K+-ATPase — A Sticky Pump

“…A status of hormone was recommended for the endogenous CSTs as it was demonstrated that it increases during exercise [108], salty meals [109][110][111], and pathological conditions such as arterial hypertension and myocardial infarction [112]. To confirm the hormone-like function, Arnaud-Batista and colleagues [113] showed that ouabain and bufalin induce diuresis, natriuresis, and kaliuresis, mediated by signal transduction in the isolated intact rat kidney. Furthermore, at the systemic level, cardenolides and bufadienolides have been implicated in many physiological and pathophysiological mechanisms, including cell growth and cancer, body or organ weight gain, mood disorders, vascular tone homeostasis, blood pressure, hypertension, and natriuresis [114].…”