**Abstract**

For decades, the Na/K-ATPase has been proposed and recognized as one of the targets for the regulation of renal salt handling. While direct inhibition of the Na/K-ATPase ion transport activity and sodium reabsorption was the focus, the underlying mechanism is not well understood since decreases in basolateral Na/K-ATPase activity alone do not appear sufficient to decrease net sodium reabsorption across the renal tubular epithelium. The newly appreciated signaling function of Na/K-ATPase, which can be regulated by Na/K-ATPase ligands (cardiotonic steroids (CTS)) and reactive oxygen species (ROS), has been widely confirmed and provides a mechanistic framework for natriuresis regulation in renal proximal tubule (RPT). The focus of this review aims to understand, in renal proximal tubule, how the activation of Na/K-ATPase signaling function, either by CTS or ROS, stimulates a coordinated reduction of cell surface Na/K-ATPase and sodium/hydrogen exchanger isoform 3 (NHE3) that leads to ultimately decreases in net transcellular sodium transport/reabsorption.

**Keywords:** cardiotonic steroids, natriuresis, renal proximal tubule, Na/K-ATPase, NHE3, signaling, ROS

#### **1. Introduction**

Since J.C. Skou's discovery in 1957 [1], the energy-transducing Na/K-ATPase has been extensively studied for its ion-pumping function and, later on, its signaling function. While the signaling function was first demonstrated in cardiac myocyte primary culture, the phenomenon has been confirmed in different cell types and animal models. The roles of Na/K-ATPase signaling in renal proximal tubule (RPT) sodium handling and oxidative modification of the Na/K-ATPase α1 subunit in Na/K-ATPase signaling were explored both in vitro and in vivo. The findings may explain certain mechanism(s) related to the Na/K-ATPase signaling-ROS amplification loop and subsequent regulation of salt sensitivity.

The RPT mediates over 60% of the filtered Na<sup>+</sup> reabsorption [2, 3]. There are two Na+ reabsorption pathways in RPTs. One is through the transcellular pathway, mainly through the apical Na+ entry mainly via NHE3 (and other apical Na+ coupled transporters like Na+ -glucose cotransporters 1 and 2, to a lesser extent) and basolateral Na+ extrusion through the Na/K-ATPase [2, 3]. A coordinated and coupled regulation of sodium/hydrogen exchanger isoform 3 (NHE3, SLC9A3) and the Na/K-ATPase is critical in maintaining intracellular Na<sup>+</sup> homeostasis and extracellular fluid volume. The other one is the paracellular Na+ reabsorption pathway through a tight junction (TJ), which depends on the transepithelial electrochemical force and tight junction permeability. Claudin-2 forms paracellular channels with other protein that are selective for small cations like Na+ and K+ , small anion like Cl<sup>−</sup>, as well as water [4–6]. Interestingly, the Na/K-ATPase signaling function is able to regulate the apical/basolateral polarity of the Na/K-ATPase as well as the tight junctions' components like claudins in distal tubule MDCK cells [7, 8].

The Na/K-ATPase belongs to the P-type ATPase family and consists of two non-covalently linked α- and β-subunits. Several α- and β-isoforms, expressed in a tissue-specific manner, have been identified and functionally characterized [9–12]. In RPTs, the γ-subunit (γa and γb, also known as FXYD2, one of the small type I single-span membrane FXYD protein families) also interacts with the α1 subunit to regulate the Na/K-ATPase activity [13–15]. There is also a fifth member of the β-subunit family, named βm coded by an ATP1B4 gene, that is predominantly expressed in skeletal muscle. Interestingly, the βm is not associated with the α1 subunit like other β-subunits, but accumulated in the nuclear membrane and associated with transcriptional coregulator Ski-interacting protein, which led to the regulation of TGF-β-responsive reporter Smad7 [16]. The α1 subunit contains multiple structural motifs that interact with soluble, membrane, and structural proteins. Binding to these proteins not only regulates the ion-pumping function of the enzyme, but it also conveys signal-transducing functions to the Na/K-ATPase [17–32]. NHE3 belongs to a family of electroneutral mammalian Na+ /H+ exchangers [33–35]. In RPT, NHE3 resides in the apical membrane of S1 and S2 segments, mediating transcellular reabsorption of Na+ and HCO3 <sup>−</sup> and fluid reabsorption [36, 37]. In the kidney, more than 85% of the filtered NaHCO3 is reabsorbed in the RPTs, and NHE3 contributes up to ∼60% of the total reabsorption of this segment [38]. RPT NHE3 secrets the largest portion of net H+ to the lumen and interacts with HCO3 <sup>−</sup> to form H2O and CO2 which can freely translocate into RPT cytosol. In cytosol, H2O and CO2 form H+ and HCO3 <sup>−</sup> through carbonic anhydrase catalyzation. Finally, the newly formed cytosolic H<sup>+</sup> will be secreted to the lumen, and HCO3 <sup>−</sup> will be moved to the blood through the basolateral-resided Na+ /HCO3 <sup>−</sup> cotransporter (NBCe1-A, SLC4A4). This cycling carbonic anhydrase-controlled CO2-HCO3 <sup>−</sup> system links the NHE3-mediated H<sup>+</sup> secretion to HCO3 <sup>−</sup> reabsorption, to achieve an acid-base equilibrium [39, 40]. Moreover, vesicular NHE3 activity also regulates endosomal pH and consequently affects receptor-mediated endocytosis as well as endocytic vesicle fusion [41, 42]. Under normal conditions, the Na/K-ATPase resides at the basolateral surface, providing the driving force for the vectorial transport of Na+ from the tubular lumen to the vascular compartment, while the NHE3 resides at the apical surface providing a rate-limiting Na<sup>+</sup> entry into cells.
