**8. Endocytosis and signaling transduction**

The clathrin-dependent endocytosis is the main endocytosis pathway for many membrane proteins in mammalian cells [166, 167, 173–175]. Apart from its endocytic function, the clathrin-coated pits also represent a specialized microdomain, where proteins are assembled into active signaling complexes before internalization of some or all of their components [176]. Some molecules involved in transmembrane signaling, such as β-arrestin, RGS-GAIP (a GTPase-activating protein for Gαi heterotrimeric G proteins) [177], GIPC (a PDZ domain-containing protein) [178], and Src family kinases [179], have been localized to clathrin-coated pits, suggesting that the interaction with the components of the pit machinery may facilitate some signaling functions of transmembrane receptors.

Caveolae/lipid rafts play a central role in transcytosis and endocytosis [180–184]. Many signaling molecules and membrane receptors are dynamically associated with caveolae, such as the Src family kinases, Ras, PKC, ERK, insulin receptor, platelet-derived growth factor receptor (PDGFR), EGFR, and some entire signaling modules like PDGFR-Ras-ERK, mainly through their interactions with caveolins [182, 185, 186]. Caveolins stabilize caveolae and modulate signal transduction by attracting signaling molecules to caveolae and regulating their activities [186]. There is also evidence that caveolins modulate endocytosis through their interactions with clathrin [187–190]. Interestingly, both caveolin and clathrin heavy chain are substrates of Src kinase [169, 184].

The Na/K-ATPase α-subunit, c-Src, and caveolin are present in caveolae isolated by a detergent-free method, in adult rat cardiac myocytes, human embryonic kidney (HEK)-293 cells, and LLC-PK1 cells. In adult rat cardiac myocytes, ouabain not only recruits α-subunit and c-Src to caveolae but also activates caveolar ERK1/2 [191]. Furthermore, some signaling molecules, such as EGFR and c-Src, are also concentrated in clathrin-coated pits and endosomes in response to ouabain [116], suggesting that both clathrin-coated pits and caveolae are involved in ouabainmediated Na/K-ATPase signal transduction and endocytosis.

The receptor-mediated endocytosis has been shown not only to attenuate ligand-activated signaling but also to continue the signaling on the endocytic pathway, especially from endosomes [166, 167, 192–194]. While endocytosis is important in the activation and propagation of signaling pathways [168, 195, 196], signal transduction can also regulate endocytosis [169, 197]. Endocytic receptor tyrosine kinase (RTK) receptors could control the magnitude of the original signaling responses (generated at the cell surface) or initiate distinct signaling cascades (qualitatively different from that generated at the cell surface) [170]. In polarized epithelial cells, the distribution of RTK substrates could affect cellular responses [118]. The endosomal signaling appears to be dependent on both the receptor and cell type.

In LLC-PK1 cells, ouabain not only induced compartmentalization of Na/K-ATPase, c-Src, EGF receptor, and ERK in early endosomes but also bound to Na/K-ATPase along the endocytic route [116]. Interestingly, caveolin-1 is also present in early or late endosomes. These facts make it possible that endosomal ouabain-Na/K-ATPase/c-Src might be able to propagate its original signaling or to initiate distinct signaling cascades. This is supported by the findings that ouabain-induced NHE3 regulation is mediated by the activation of the receptor function of Na/K-ATPase. Furthermore, endocytosis is required for ouabain to remove basolateral Na/K-ATPase, which induces a significant inhibition of the pumping activity. Moreover, blockade of Na/K-ATPase signaling/endocytosis appears to be sufficient to abolish ouabain-induced trafficking and transcriptional regulation of NHE3.

#### *Biomarkers and Bioanalysis Overview*

Although the mechanisms that involved ouabain-initiated endocytosis of the Na/K-ATPase and NHE3 (and expression) are not fully understood, endocytosis of the Na/K-ATPase may play an important role in renal sodium handling. This is because if ouabain induces a significant depletion of plasmalemmal Na/K-ATPase in proximal tubule type cells (rat proximal tubule primary culture, LLC-PK1) but not in distal tubule type cells (rat distal tubule primary culture, MDCK), it will make physiological "sense" in terms of allowing bulk sodium transport (primarily in the proximal tubule) to be altered and leaving fine-tuning (distal tubule) sodium handling intact.

### **9. ROS and the Na/K-ATPase signaling: the possible link from CTS-stimulated signaling to NHE3 regulation**

It is well established that both oxidative stress and high BP are a cause and consequence of each other. The increase in oxidative stress occurs in many forms of experimental models of hypertension, including Dahl salt-sensitive hypertension [198–204]*.* Increases in ROS can regulate physiological processes including renal tubular ion transport, fluid reabsorption, and sodium excretion [79, 205–210]. In particular, increases in ROS regulate the activity and cellular distribution of the basolateral Na/K-ATPase as well as the apical NHE3 and sodium/glucose cotransporter, at least under normal circumstances [79, 151, 208, 211–216]. Oxidative modification can affect the Na/K-ATPase activity through different mechanisms. For example, S-glutathionylation cysteine residue(s) of the Na/K-ATPase α-subunit can block the intracellular ATP-binding site [217], and S-glutathionylation of cysteine of the Na/K-ATPase β1-subunit can affect the Na/K-ATPase conformational poise [218, 219]. Oxidant and oxidative modification of the Na/K-ATPase can lead to degradation, functional changes, and formation of Na/K-ATPase oligomeric structure [74, 84–87, 217, 219–230]. In LLC-PK1 cells, increase in ROS generation, induced by either ouabain or glucose oxidase, is critical in the activation of Na/K-ATPase signaling which mediates trafficking of the Na/K-ATPase and NHE3 and transcellular Na+ transport [86, 87]. Pretreatment with higher doses, but not a low dose, of NAC attenuated the effect of ouabain on c-Src activation and transcellular 22Na+ flux, suggesting a role of basal physiological redox status in the initiation of ouabain-induced Na/K-ATPase signaling. While CTS stimulates ROS generation and Na/K-ATPase signaling in different in vitro and in vivo models [63, 85, 231–233], an increase in ROS alone (without the presence of ouabain) by extracellularly added glucose oxidase is also able to activate Na/K-ATPase signaling, indicating that activation of Na/K-ATPase signaling can be achieved by general stimuli like ROS, other than its specific ligands. Glucose oxidase-induced H2O2 alone also stimulates Na/K-ATPase endocytosis and inhibits active transcellular 22Na+ transport [85, 86]. The phenomenon of redox sensitivity of the Na/K-ATPase has been demonstrated in different cell types, tissues, and animal species.

In LLC-PK1 cells, both ouabain and glucose oxidase-induced H2O2 stimulate Na/K-ATPase signaling as well as direct protein carbonylation of Pro222 and Thr224 residues of the Na/K-ATPase α1 subunit (α1-carbonylation) [86]. The Pro222 and Thr224 are located in peptide 211VDNSSLTGESEPQTR225 [UniProtKB*/Swiss-Prot No P05024 (AT1A1\_PIG)*]. While the α1 subunit is highly conserved among humans, pigs, rats, and mice (the homology is over 98.5%), the identified peptide is 100% identical among these four species. This peptide is located in the actuator (A) domain of α1 subunit, and Pro222/Thr224 are highly exposed and facing the nucleotide binding (N) domain of the α1 subunit. Upon ouabain binding, Na/K-ATPase undergoes conformational changes, in which the A domain is rotated to the N

#### *The Na/K-ATPase Signaling Regulates Natriuresis in Renal Proximal Tubule DOI: http://dx.doi.org/10.5772/intechopen.92968*

domain favoring an E2-P conformation. The structure-function analysis indicates that these conformational changes may affect binding of the α1 subunit to signaling molecules such as c-Src and PI3K [234]. In addition, the peptide also contains the TGES motif that is the anchor of A domain rotation [234].

Biologically, ROS can oxidize various types of biological molecules including proteins, leading to their functional changes. Through Fenton's reaction, H2O2 is reduced to HO• by coupling oxidation of reduced ferrous ion (Fe2+) to ferric ion (Fe3+). This metal-catalyzed oxidation (MCO) process oxidizes proteins by introducing carbonyl groups (such as aldehydes, ketones, or lactams) into the side chains of certain amino acids (such as proline, arginine, lysine, and threonine) that named direct (primary) carbonylation that have been implied in various conditions like chronic renal failure [235–240]. Since Fenton's reaction involves the conversion of H2O2 to HO• , any specie of ROS with H2O2 as an intermediate and/or end product may stimulate the reaction.

Protein carbonylation is reversible (decarbonylation) and may function as a regulatory mechanism of cell signaling [241–244]. We also observed an undefined decarbonylation mechanism, which apparently reverses the carbonylation of the Na/K-ATPase α1 subunit induced by ouabain [86]. The removal of ouabain from the culture medium reverses ouabain-mediated carbonylation, as seen in the reversed Na/K-ATPase ion-pumping activity [116]. Moreover, inhibition of de novo protein synthesis as well as degradation pathway through lysosome and proteasome does not affect this decarbonylation, which is still poorly understood. It is possible that carbonylation modification might stabilize the Na/K-ATPase in a certain conformational status favoring ouabain binding to the Na/K-ATPase α1 subunit and ouabain-Na/K-ATPase signaling. Nevertheless, the underlying mechanism might be physiologically significant since the carbonylation/decarbonylation process could be an important regulator of the RPT Na/K-ATPase signaling and sodium handling.

It is reasonable to propose that carbonylation modification of RPT Na/K-ATPase α1 subunit has biphasic effects. On one hand, physiological and controllable α1-carbonylation stimulates Na/K-ATPase signaling and sodium excretion, rendering salt resistance, whereas on the other hand, prolonged exposure to oxidant stress leads to overstimulated α1-carbonylation and desensitized Na/K-ATPase signaling, increasing salt sensitivity. First, Dahl S rats show considerably higher basal levels of oxidative stress than R rats, and high-salt diets increase renal oxidative stresses that contribute to salt-sensitive hypertension [202–204]. Second, while high-salt diets increase circulating CTS, a high-salt diet (HS, 2% NaCl for 7 days) stimulates the Na/K-ATPase signaling in isolated RPT from Dahl salt-resistant (R) but not salt-sensitive (S) rats (i.e., impaired Na/K-ATPase signaling in S rats) [79]. Third, CTS- and H2O2-mediated redox-sensitive Na/K-ATPase signaling and α1-carbonylation are involved in this signaling process, in a feed-forward mechanism [86]. Fourth, high but not low concentration of NAC is able to prevent α1-carbonylation and Na/K-ATPase signaling [86]. Even though it is still not clear of the carbonylation/decarbonylation process, this could be another new regulatory mechanism of Na/K-ATPase signaling. It is reasonable to postulate that prolonged excessive α1-carbonylation (by CTS and/or other factors) might overcome the decarbonylation capacity, leading to the desensitization or termination of the Na/K-ATPase signaling function. This is reminiscent of the observations in clinical trials using antioxidant supplements. The beneficial effect of antioxidant supplements is controversial and not seen in most clinical trials with administration of antioxidant supplements [200, 245]. Low doses of antioxidant supplementation may be ineffective, but high doses may be even dangerous since excess antioxidants might become prooxidants if they cannot promptly be reduced in the antioxidant chain [246]. It appears that the balance of the redox status, within a physiological range, may be critical in order to maintain beneficial ROS signaling.
