**7. Coordinated and coupled regulation of Na/K-ATPase and NHE3 by Na/K-ATPase signaling**

Although the mechanisms are still being elucidated, accumulating evidence supports the notion that the expression and activity of the basolateral Na/K-ATPase and apical NHE3 are coordinated and coupled under certain circumstances. For example, McDonough's laboratory has shown that, during pressure natriuresis and salt loading, the surface expression and activity of both NHE3 and the Na/K-ATPase are simultaneously downregulated to remove Na+ from the body [125, 127, 145, 151]. During the development of hypertension in spontaneous hypertensive rat (SHR), the expression and activity of both the Na/K-ATPase and NHE3 are elevated in comparison with the normotensive control rats [121, 152–155].

Activation of Na/K-ATPase signaling, by either ouabain or a high-salt diet, is also capable of stimulating a coordinated and coupled downregulation of apical NHE3 and basolateral Na/K-ATPase to inhibit active transepithelial Na+ transport in cultured or isolated RPTs [79, 115–117, 149]. This coordinated regulation depends on activation of the Na/K-ATPase signaling function, but not on acute inhibition

of the Na/K-ATPase activity since it requires the activation of Src and PI3K and increase in intracellular Ca2+. Moreover, MBG infusion also induced endocytosis of RPT Na/K-ATPase in rats, which could be prevented by an antibody-mediated neutralization of infused MBG [156].

A high salt intake or volume expansion increases both dopamine and CTS. It has been shown that dopamine-induced regulation of RPT Na/K-ATPase of Dahl S rats was defective because of an apparent decoupling between the binding of dopamine to its D1 receptor and activation of GPCRs [157–161]. In response to salt loading, Dahl S rats have a similar diuretic, but much less CTS-related natriuretic response than that seen in Dahl R rats [162]. Both dopamine and CTS can regulate the activity and trafficking of RPT Na/K-ATPase and NHE3. Even though the initiating steps and signaling pathways might be different, they share some signaling steps such as the activation of PLC/PKC and calcium signaling. It *will be of interest to further assess whether there is a crosstalk between CTS- and dopamine-activated signaling pathways in the regulation of renal Na<sup>+</sup> handling.*

In vivo studies suggest the essential role of CTS in modulating renal sodium excretion and BP with different approaches. First, the administration of some (e.g., ouabain) but not all CTS induces natriuresis [163, 164]. Second, in transgenic mice expressing ouabain-sensitive Na/K-ATPase α1 subunit, both acute salt load and ouabain infusion augment natriuretic responses, which were prevented by administration of an anti-digoxin antibody fragment [75, 76]. Third, immune neutralization of endogenous CTS prevents CTS-mediated natriuretic and vasoconstrictor effects [55, 59, 78, 80]. Fourth, the administration of the ouabain antagonist, rostafuroxin (also known as PST 2238), prevents not only ouabain-induced Na/K-ATPase signaling but also ouabain-induced increase in BP [64]. Finally, in humans, a high salt intake increases circulating endogenous CTS [57, 80, 165]. An increased CTS excretion is directly linked to an enhanced RPT-mediated fractional Na<sup>+</sup> excretion, but inversely related to age and to agedependent increase in salt sensitivity [165].

Although the historical focus has largely been on the direct inhibition of CTS on the Na/K-ATPase ion-pumping activity and sodium reabsorption in RPT as well as vascular tone/contractility, decreases in basolateral Na/K-ATPase activity alone do not appear to be sufficient to reduce net RPT sodium reabsorption since the apical NHE3, but not the Na/K-ATPase, is the rate-limiting step.

In contrast, the newly appreciated signaling function of Na/K-ATPase has been widely confirmed and provides a realistic, mechanistic framework that the renal Na/K-ATPase and its signaling play a key role in regulating renal sodium handling. In porcine RPT LLC-PK1 cells, ouabain activates the Na/K-ATPase signaling pathways and consequently redistributes the basolateral Na/K-ATPase and the apical NHE3 in a coordinated manner; this leads to a symmetrical reduction of cell surface Na/K-ATPase and NHE3 content and ultimately decreased net transcellular sodium transport [86, 87, 115–117]. No significant acute change in intracellular Na<sup>+</sup> concentration was observed [115], further suggesting the coordination of the downregulation of both apical and basolateral sodium transporters. This Na/K-ATPase signaling-mediated regulation of renal tubular epithelial ion transporters was further confirmed in in vivo studies [79, 156]. It has been shown that endocytosis of signaling molecules could be a way to terminate or propagate the signaling and could further regulate endocytosis itself [166–171]. In this regard, it is possible that ouabain- and ROS-induced endocytosis could be an effective way to terminate Na/K-ATPase signaling-mediated oxidant amplification loop by the degradation of carbonylated Na/K-ATPase, to maintain a certain basal level of ROS and carbonylated protein [172].
