**2. Sodium Reabsorption in Terminal Nephrons**

Lifton et al. determined that the genes involved in the development of hereditary human hypertension are restricted to the sodium transporter and its associated proteins in the renal tubule [5]. The most important message that can be derived from this finding might be the fact that the first hit in the development of naturally occurring hereditary hypertension in human is due to the enhanced sodium reabsorption mechanism in the renal tubule. We considered that the mechanism of sodium reabsorption in tubules need to be understood to determine the genetic factors involved in salt-sensitive hypertension, i.e., "hypothesis-driven approaches", which is the purpose of this review.

The function of the kidney is to produce urine by ultrafiltration, during which the primitive urine passes through the renal tubules, and the quality and quantity of the body fluids and electrolytes are maintained through concentration by water re-absorption, appropriate reabsorption of electrolytes, and appropriate secretion of the bicarbonate ions [24,25]. Most sodium ions in the serum are filtered into primitive urine and at the rate according to the nephron segments; >99% of the sodium that is reabsorbed and ultimately filtered in each segmen<sup>t</sup> of the renal tubules is excreted into urine. Considering the mechanism that regulates sodium reabsorption, it can be divided into two segments involving the glomerulus-proximal, tubule-loop of the Henle-distal tubule to the macula densa and the tubules distal to the macula densa (connecting tubules, cortical collecting tubules).

During the first part of the process, >90% of the ultrafiltered sodium is reabsorbed. A physiological regulatory mechanism of the renal microcirculation (tubulo-glomerular-feedback; TGF) between the macula densa and the a fferent arterioles, detects the chloride ions in the renal tubular fluid flowing into the distal nephron and imports the anions, thus adjusting the diameter of the a fferent arterioles. By adjusting the diameter of the a fferent arterioles according to the di fference between the amount of filtered and reabsorbed sodium chloride (NaCl), the glomerular filtration pressure and amount were adjusted, and the glomerular filtration rate was maintained according to the oral salt intake. A myogenic reaction specific to the a fferent arterioles occurs, in which the smooth muscle around the arterioles reflexively contracts due to the blood flow brought to the a fferent arterioles, which regulates the diameter of the a fferent arterioles. Similar to TGF, this mechanism adjusts the diameter of the a fferent arterioles and maintains a constant glomerular filtration pressure and volume. Moreover, the renal tubules distal to the macula densa are collectively called aldosterone-sensitive-distal nephrons (ASDN), and sodium reabsorption is fine-tuned by the regulation of the expression of sodium ion transporters on the apical side of renal tubular cells, mainly through the action of aldosterone, an endocrine factor. Sodium reabsorption at this site comprises <5% of the total reabsorption, but because of an absent or insu fficient feedback/compensation mechanism as in TGF, abnormalities about sodium reabsorption at this site is thought to cause blood pressure abnormalities such as the Liddle syndrome or type I pseudo-hypoaldosteronism (type I PHA). Conversely, abnormalities in sodium reabsorption due to irregularities in the ion transporter from the proximal tubule to the macula densa can be canceled and corrected by powerful TGF and myogenic reactions. Even if such irregularities exist in nature, they might not be recognized due to these physiological compensations.

The onset of salt-sensitive hypertension caused by an enhanced renal tubular renin-angiotensin system (RAS) might be caused by abnormalities in the sodium reabsorption mechanism in the terminal nephron, i.e., the ASDN. Our findings in the C57Bl6/J mice showed that angiotensinogen secretion in the proximal tubular cells was enhanced as a result of the enhanced filtered sodium ion [9,11,12], due to excess oral salt intake. We then proposed a model of the onset of salt-sensitive hypertension by

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paradoxically enhancing angiotensin II stimulation on the apical side of ASDN, in which the likely ion transporter of the angiotensin II action was the epithelial sodium channels (ENaCs) [26–28].

The function of renal tubules, that comprise a highly differentiated and complex regulatory system, is difficult to describe in a few words. However, their function can be summarized to facilitate understanding, as follows [29–32]. The cellular effects of aldosterone are exerted through intracellular mineralocorticoid receptors (MR) that bind not only to aldosterone, but also to the steroid hormone, cortisol, to produce intracellular effects. As the plasma concentration of aldosterone is much lower than that of cortisol, when competing for binding with MR under the same conditions, most MR probably binds to cortisol. Thus, epithelial cells (such as renal tubule cells) that are sensitive to aldosterone express 11-βHSD2 (type 2 11β-hydroxy-steroid dehydrogenase) and cortisol is converted into cortisone, thereby losing its MR binding activity. As a result, aldosterone-specific cellular action can occur by reducing the competitive inhibitory effect of cortisol on aldosterone with regards to MR. MR is expressed in nephrons beyond the early distal convoluted tubule (DCT), but because 11-βHSD2 is expressed only in nephrons after late DCT, the effects of aldosterone are not evident in early DCT. Mineralocorticoid receptors bind cortisol, and the NaCl co-transporter (NCCT) that is sensitive to thiazide diuretics and that is expressed in early DCT, is controlled by aldosterone to a lesser degree. Regulators of NCCT were discovered in the Gordon syndrome (type II PHA) in which hereditary hypertension was thought to be due to abnormal sodium reabsorption through the NCCT as well as abnormalities in WNK1 and WNK4 [33], but details of the mechanism of hypertension onset remain unknown. Through consistent expression of MR and 11-βHSD2, because "cortisol" was inactivated into "cortisone", an aldosterone was able to exert effects by binding to the MR in the nephrons below the late DCT. Thus, aldosterone regulates the ENaC expression by binding to MR as ASDN, via the aldosterone–MR complex and through the expression of regulatory factors known as AIP (aldosterone inducible protein), such as SGK1 (serum and glucocorticoid-regulated kinase 1), in nephrons after late DCT.

The outline of sodium transport in the ASDN renal tubular epithelial cells is as shown [34] (Figure 1). Na-K-ATPase are expressed on the basolateral side of all ASDN and functions, by ATP-dependently pumping intracellular sodium out of cells and the uptake of potassium into the cell. As a result of this sodium pump and K-ATPase, a transmembrane Na gradient arises, and the sodium is reabsorbed through the ENaCs on the apical side. After intracellular uptake, potassium is secreted lumenally through the action of the potassium channel ROMK. This channel was also under the control of aldosterone, and aldosterone action in the renal tubule appears to act on potassium secretion as well as sodium reabsorption.

**Figure 1.** Schematic presentation of the ASDN epithelial cell is shown. ENaC is ubiquitinated by Nedd4-2/Nedd4L, and subsequently degraded by proteasome. Aldosterone, insulin, and vasopressin regulate ENaC gene expression via the basolateral side.

### **3. ENaC and Regulation of ENaC Expression in Renal Tubular Epithelial Cells**

As ENaC is inhibited by the diuretic amiloride, it is referred to as an "amiloride-sensitive" epithelial sodium channel and is localized in epithelial cells with polarity. That is, it is expressed only in the apical side of the distal tubule, the alveolar epithelium, and colonic mucosa. It comprises α, β, and γ subunits, at a ratio of 1 α:1β:1 γ. The expression of its function requires all three subunits [29,35]. However, the α subunit alone or the combination of αβ and αγ generates a sodium current, albeit incompletely. ENaC molecules have two transmembrane domains comprising an extracellular domain with multiple glycosylation sites, a short NH2 terminus, and COOH terminus, within cells. All three subunits have two proline-rich sequences (P1 and P2) called PY motifs, at their COOH terminal.

The endocrine factors principally regulate intracellular ENaC expression. In addition to aldosterone action through binding to MR, the action of vasopressin (ADH) secreted from the posterior pituitary gland appears to regulate the expression of ENaC via V2 receptor-adenylyl cyclase-cAMP, as well as insulin via the insulin receptor. As it acts on renal tubule cells through the blood stream, ADH acts via a receptor present on the basolateral side [26–28]. Abnormalities in the ENaC gene result in diseases such as cystic fibrosis, type I pseudohypoaldosteronism (type I PHA) [36], and Liddle syndrome [37].
