INTRODUCTION

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BECAUSE THIS REVIEW FOCUSES on the regulation of epithelial Na⁺ channels (ENaC) in the collecting duct (CD), other nephron segments and organ systems that can be important contributors to extracellular fluid (ECF) volume and blood pressure regulation have been neglected. Thus the reader may conclude incorrectly that the kidney alone, or more specifically the cortical collecting duct (CCD) alone, is responsible for abnormalities in these processes. However, even in the case of salt-sensitive hypertension, there is a large body of evidence from animal and human studies that defects in the central control of the sympathetic nervous system may play an important role (45, 145). It is perhaps even more important to dispel any false perception that the rare genetic disorders discussed below may be important contributors to more common forms of human hypertension. While these disorders do demonstrate that single molecular defects that interfere with the regulation of renal Na⁺ excretion are sufficient to produce hypertension, there is no evidence that they are necessary components of essential hypertension. However, such single-gene defects do provide impressive (and relatively uncomplicated) links among molecules, physiological systems, and pathology, and they turn our attention to associated regulatory mechanisms that may be involved in essential hypertension.

The role of ECF volume in the control of arterial blood pressure, and hence the role of the kidneys vs. the cardiovascular system in hypertension and edematous states such as congestive heart failure, have been matters of controversy for decades. Using parameters and relationships derived from animal studies, Guyton (46, 47) proposed a dominant role for the kidneys based on a mathematical model of the interactions among the multiple organ systems that determine ECF volume and blood pressure. They showed that, because of the rapid response of baroreceptor reflexes, acute changes in ECF volume produced little change in blood pressure. In contrast, chronic changes in ECF volume could only be corrected by increased Na⁺ and water excretion, which were determined by a feedback mechanism with an apparent "infinite gain." Guyton described the relationship between blood pressure and renal Na⁺ excretion in terms of renal function curves, which predicted that when Na⁺ excretion is impaired, blood pressure rises until the resulting pressure natriuresis achieves Na⁺ balance (46, 47).

Many of the predictions of the Guyton model have been confirmed in humans (e.g., see Ref. 67). Arguably, however, the best support has come from the identification of molecular defects linked to heritable forms of hypertension and salt wasting and from the explanations of the pathophysiology of those disorders. As a consequence of previous physiological studies that provided the fundamental information about the location and regulation of ion and water transporters in the nephron, certain single-gene defects have been shown to produce renal salt retention and hypertension, whereas other defects lead to salt wasting and hypotension. Consequently, these defects support the primacy of the kidneys in ECF volume regulation predicted by the Guyton model.

For the most part, these single-gene defects affect Na⁺ reabsorption in those segments of the nephron that express the mineralocorticoid receptor (MR), i.e., the distal convoluted tubule (DCT), the connecting tubule (CNT), and CD (10, 27). The DCT and CCD respond to aldosterone with increased Na⁺ reabsorption and K⁺ secretion, and, although the evidence is sparser (see below), aldosterone may produce the same effects in the CNT and portions of the medullary CD. This review, however, concentrates on the CCD, in which Na⁺ reabsorption is mediated primarily, if not exclusively, by ENaC.

In the DCT, Na⁺ reabsorption occurs via the thiazide-sensitive NaCl cotransporter (NCCT; also known as NCC or TSC; OMIM no. 600968; for references, see Ref. 96) in the luminal membrane.¹ However, the DCT is difficult to characterize because it is composed of multiple subsegments with different cell types within which the distribution of transporters and associated enzymes varies markedly among species (for an excellent editorial comment, see Ref. 136). ENaC appears to have no functional role in the DCT, although expression of ENaC subunits is variably found in the CNT and DCT (76, 133). In the DCT, the distributions of MR and the enzyme 11-hydroxysteroid dehydrogenase type 2 (11-HSD2; see below) are also quite variable among subsegments and species (10, 27, 96, 136). Finally, vasopressin (VP) has no action in the DCT, which lacks the necessary V₂ receptors.

Some subsegments of the DCT respond to aldosterone (probably in combination with glucocorticoids; see Refs. 96 and 136) with increased K⁺ secretion and Na⁺ reabsorption due to de novo synthesis of NCCT, and increased Na⁺ delivery to the DCT enhances this effect (66, 96). Thus the DCT contributes to Na⁺ retention or wasting in those disorders involving the synthesis or actions of aldosterone and other adrenal corticosteroids. Furthermore, at least one gene defect resulting in altered Na⁺ balance, the Gitelman's syndrome (a variant of Barrter's syndrome; OMIM no. 263800), is confined to the DCT. In this disorder, various mutations in the NCCT gene result in a loss of function, and thus salt wasting, which is accompanied by hypocalciuria, and K⁺ and Mg²⁺ depletion (114).

This review also neglects the medullary CD, which, like the DCT, is quite heterogeneous (123). Although MR, 11-HSD2, and all three ENaC subunits are present in outer and inner medullary CD segments (10, 27, 48, 133), the actions of aldosterone appear to be confined primarily to the CCD (123). For these reasons, this review focuses on the CCD, but the possibility that some of the same mechanisms are involved in the medullary CD should be kept in mind.

It may at first seem quite remarkable that, despite the fact that the aldosterone-responsive segments of the nephron in combination reabsorb <10% of the filtered load of Na⁺, regulatory defects in the control of that reabsorption result in marked changes in body Na⁺ balance and blood pressure. These consequences appear to be disproportionate to the amount of Na⁺ reabsorbed; however, as argued cogently by Schnermann (110), relatively gross abnormalities in Na⁺ and water reabsorption in the more proximal regions of the nephron are offset by changes in the glomerular filtration rate due to tubuloglomerular feedback, but this mechanism does not respond to changes in salt and water delivery beyond the macula densa.

	CCD ION TRANSPORTERS

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Figure 1 illustrates the primary transporters involved in Na⁺ reabsorption and K⁺ secretion in the CCD. Microelectrode and patch-clamp studies in isolated, perfused CCD segments (e.g., 37, 38, 88, 100, 109), as well as in planar epithelia and cell cultures derived from or resembling the CCD, established the general characteristics of the apical Na⁺ channel, especially its high selectivity to Na⁺ and its inhibition by low concentrations of amiloride (42, 134). Mechanisms regulating the activity of this ENaC have been the subject of intensive study since the cloning and sequencing of its three component subunits, -, -, and -ENaC, by Canessa et al. (14, 16). All three have two transmembrane regions connected by an extracellular loop, with both NH₂- and COOH-terminal regions on the cytoplasmic side of the membrane. Although the stoichiometry of -, -, and -subunits that comprise a functional channel remains controversial, the identification of an ENaC complex as the mediator of amiloride-sensitive Na⁺ entry (42, 134) has provided a wealth of information concerning the normal and abnormal regulation of Na⁺ transport in the CCD.

View larger version (47K): [in this window] [in a new window]   Fig. 1.   Major ion transport processes in the cortical collecting duct (CCD). Shown is a schematic representation of a principal cell in the CCD epithelium, with the luminal membrane on the left and the basolateral membrane on the right. The junctional complexes are shown as crosshatched structures between the principal cell and its neighboring cells. The pathway for transepithelial Na+ transport includes passive movement into the cells across the luminal membrane by the epithelial Na+ channel (ENaC; composed of ENaC subunits) and active extrusion from the cell by Na+-K+-ATPase in the basolateral membrane. The latter transporter also actively transports K+ into the cell, where it can diffuse back to the interstitial fluid on the right via low- and intermediate-conductance K+ channels in the basolateral membrane, or into the lumen via ROMK1 channels, resulting in transepithelial secretion. Maxi-K+ channels may also mediate secretion across the luminal membrane, but only under special circumstances (see text).

As is the case throughout the nephron and similar epithelia, the motive force for Na⁺ and K⁺ transport by the CD is the ubiquitous Na⁺-K⁺-ATPase, and it is activated by aldosterone, although only in the presence of increased Na⁺ entry (8, 90). Thus it appears that the primary regulation of Na⁺ reabsorption by aldosterone occurs by changes in ENaC activity, which is the rate-limiting step in transepithelial transport, and that there is no increase in Na⁺-K⁺-ATPase activity unless there is apical Na⁺ entry (90).

Under most conditions, K⁺ transport across the apical membrane of the CCD is mediated by ROMK1 (also called SK) channels that may be regulated somewhat differently than those in the thick ascending limb (86, 87). However, as with other ROMK channels, these channels are inwardly rectifying, inhibited by ATP, and have a low unit conductance (25-40 pS). In addition to ROMK1, K⁺ secretion across the apical membrane of the CCD is also mediated by maxi-K (or BK) channels, so named for their high unit conductance (80-140 pS), which may be activated by elevated intracellular calcium concentration ([Ca²⁺]_i) and membrane stretch (36, 58, 87, 107). Under the usual experimental conditions, the maxi-K⁺ channels are inactive (81), but they may be activated by cell swelling and increased luminal flow (54, 144). The basolateral membrane conductance is dominated by both low (~30-pS)- and intermediate (~80-pS)-conductance K⁺ channels (55, 56, 81, 138).

The mechanism for Cl reabsorption in the CCD has been neglected relative to the cations, an oversight that will be addressed in more detail below. Electrophysiological studies in the rabbit CCD established that there is a substantial Cl conductance in the basolateral membrane that under some conditions may exceed the K⁺ conductance (82, 85, 100, 101). Although the CLC-K family of channels, members of which are expressed in many segments of the nephron, might seem to offer the best candidates, only CLC-K2 has been found in the CCD, and only in the basolateral membranes of A-type intercalated and not principal cells (68). In their study of the basolateral Cl channel, Sansom et al. (101) could find no evidence for an apical conductance and suggested that an electroneutral anion exchanger might mediate apical Cl transport. Although the anion exchanger AE2 was previously thought to be expressed only in intercalated cells, more recent observations in primary cultures of rabbit CCD cells also demonstrated AE2 in principal cells (30).

Figure 2A illustrates the putative transcellular route for Cl reabsorption in the rabbit CCD. However, even in the rabbit CCD, a substantial fraction of transepithelial Cl transport occurs paracellulary through the junctional complexes and is driven by the lumen negative transepithelial voltage (139), as depicted in Fig. 2B. In the rat CCD, paracellular diffusion appears to be the sole possible route for Cl reabsorption because no Cl conductance could be found in either the apical or basolateral membranes (108).

View larger version (33K): [in this window] [in a new window]   Fig. 2.   Routes of transepithelial Cl reabsorption in the CCD. The schematic cellular structures are the same as described in Fig. 1. A: transcellular Cl pathway. As demonstrated in the rabbit CCD, there is a Cl conductance on the basolateral membrane that is mediated by an unidentified Cl channel. Movement across the apical membrane is assumed to be mediated by electroneutral exchange for another unspecified anion (X). B: paracellular Cl transport through junctional complexes. Diffusion of Cl through the junctional complexes is driven by the lumen negative voltage. In the rabbit CCD, paracellular transport may occur in parallel with the transcellular transport, but in the rat CCD it appears to be the only mechanism for Cl reabsorption.

	REGULATION OF NA+ Transport by Aldosterone

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The primary hormonal regulator of Na⁺ reabsorption and K⁺ secretion in the CD and similar epithelia is aldosterone, which induces the transcription of specific genes via the MR, and hence, the synthesis of "aldosterone-induced proteins" (AIPs). The ultimate results of this change in transcription include increased ENaC-mediated Na⁺ conductance of the apical membrane, increased activity of Na⁺-K⁺-ATPase in the basolateral membrane, and increased levels of mitochondrial enzymes such as citrate synthase (41, 42, 98, 134).

Despite general agreement about the ultimate actions of aldosterone, the identity of the "proximate" AIPs, i.e., the proteins, the expression of which is directly elevated by the MR, has been elusive. The most promising candidate in the CD is a serine-threonine kinase that was initially characterized as being activated by serum and glucocorticoids (SGK; also variably referred to as SGK1 or sgk). Differential gene expression techniques in A6 cells (21) and primary cultures of rabbit CCD cells (83) indicate that SGK is a proximate AIP. Aldosterone increases SGK mRNA within 30 min in these cells as well as in the M-1 mouse CCD cell line and in the rat CCD in situ (21, 76, 83, 89, 111). Because coexpression of SGK with the three ENaC isoforms in Xenopus laevis oocytes produced much higher amiloride-sensitive currents than the injection of mRNA for the ENaC subunits alone, SGK activation appears to be coupled to increased ENaC activity independently of any changes in subunit expression (21, 83, 89, 111).

Rather than phosphorylating ENaC subunits themselves, SKG appears to phosphorylate other proteins that are involved in the trafficking of ENaC into and retrieval from the cell membrane. Alterations in the rates of insertion and retrieval are envisioned as determining the density of ENaC in the apical membrane, as depicted in Fig. 3. Attention has focused recently on the retrieval pathway in which the binding of ubiquitin to specific membrane proteins leads to their internalization and degradation by proteosomes. Several studies have shown that the WW domain of the ubiquitin ligases Nedd1-1 and Nedd4-2 bind specifically to a "PY domain" (PPPXY) that is present in the cytoplasmic COOH-terminal regions of all three ENaC subunits and that the binding of these ligases decreases ENaC activity (29, 50, 64, 65, 115, 118-120). This evidence has been reconciled with the action of SGK in the model shown in Fig. 4, which is based on the very recent studies of Debonneville et al. (24) and Snyder et al. (116). These investigators showed that SGK binds to and phosphorylates Nedd4-1 and/or Nedd4-2. This phosphorylation was found to reduce Nedd4-2 affinity for one or more of the ENaC subunits (24, 116). With diminished Nedd4 activity, less ENaC is ubiquitinylated, internalized, and degraded. Thus the model predicts that aldosterone, by inducing SGK as a proximate AIP, leads to ENaC channel accumulation in the membrane.

View larger version (67K): [in this window] [in a new window]   Fig. 3.   Regulation of luminal membrane Na+ conductance by trafficking of the ENaC. The diagram is a schematic representation of a cross section of the luminal membrane of a principal (or similar) cell, with the channels shown as cylinders. The Na+ conductance of the luminal membrane is directly proportional to the number of ENaC channels present, which is determined by the rate at which channels assembled in the trans Golgi (shown here as 3 channels in the membrane of exocytic vesicle) are inserted and the rate at which they are internalized (retrieved) by endocytosis.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Involvement of Nedd4 and serum and glucocorticoid-stimulated kinase (SGK) in the regulation of ENaC retrieval and degradation by the ubiquitin pathway. An ENaC subunit of a Na+ channel is depicted in the form of its secondary structure in the luminal membrane, i.e., 2 transmembrane segments with an extracellular loop. The cytoplasmic COOH-terminal region (shown toward the top) contains the PPPXY motif that allows specific binding of the WW domain of the ubiquitin ligases Nedd4-1 and Nedd4-2. When Nedd4 binds to an ENaC subunit, it catalyzes the binding of ubiquitin (U; ubiquitinylation), which "marks" the channel for internalization by endocytosis and degradation in proteosomes. Aldosterone stimulates the synthesis of SGK. SGK mediates the phosphorylation of a serine in Nedd4 (S-PO4), which decreases its affinity for the ENaC subunit and decreases ubiquitylation of ENaC. Thus, via SGK, aldosterone causes an increase in the number of ENaC channels in the luminal membrane by decreasing their rate of internalization and degradation (see Refs. 24 and 116).

In the late phase of aldosterone action (after several hours), there is increased synthesis of both ENaC subunits and Na⁺-K⁺-ATPase that probably represents secondary effects of the proximate AIPs. Although there appear to be cis elements that are activated by aldosterone and dexamethasone in the 5'-flanking region of the -ENaC gene (79), aldosterone produces only a modest increase in -ENaC mRNA and protein and no changes in - and -ENaC in the rat and mouse CCD (76-78, 125). There is also no association between aldosterone deficiency and the expression of ENaC subunits. Berger et al. (7) showed there was no decrease in ENaC subunit mRNA expression in mice in which the MR receptor had been knocked out despite their greatly increased fractional Na⁺ excretion. Despite the fact that aldosterone has little effect on ENaC abundance, it does enhance the migration of ENaC into the apical membrane (48, 75, 76, 78).

Methylation of ENaC may also be a component of the response to aldosterone that occurs before any increases in ENaC or Na⁺-K⁺-ATPase synthesis (121). In detached patches from A6 cells, application of a substrate for protein methyltransferase increased the open probability of single ENaC channels, and -ENaC has been shown to be directly methylated in studies with the in vitro translated protein in lipid bilayers and in intact A6 cells, and this methylation increases channel activity (59, 97). The most puzzling aspect of methylation as an early aldosterone effect has been the failure to show any increase in the levels of methyltransferase proteins despite their increased activity. It appears that these proteins are not regulated at the transcriptional or translational level but through posttranslational modification by a proximate AIP (121).

Although aldosterone activation of SGK and transmethylases occurs within 30 min and well before any synthesis of new ENaC or Na⁺-K⁺-ATPase subunits, mounting evidence suggests that aldosterone and other steroid hormones have even more rapid initial effects that involve membrane receptors coupled to the activation of the phospholipase C (PLC) signaling system and elevation of [Ca²⁺]_i (26). In patch-clamp studies of isolated rabbit CD principal cells, Zhou and Bubien (152) have shown that aldosterone increases ENaC activity within 2 min. This effect was not prevented by the MR blocker spironolactone but was completely inhibited by amiloride.

	DEFECTS IN THE REGULATION OF CCD NA+ Transport Leading to Volume Expansion

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Syndromes of abnormal Na⁺ retention by the CD that result in the expansion of ECF fall into two basic classifications of etiology, all of which are characterized by severe hypertension, hypokalemia, and, to a more variable degree, alkalosis, as depicted in Fig. 5. The first group includes those conditions in which there is an excess of mineralocorticoid activity due to either aldosterone overproduction or abnormal steroid metabolism. A primary example is the long-used experimental model of hypertension produced by exogenous DOC in combination with elevated dietary salt (43). The second group consists of mutations in the genes encoding the ENaC subunit that result in a gain of function and/or loss of their regulation, which are now collectively referred to as Liddle's syndrome.

View larger version (32K): [in this window] [in a new window]   Fig. 5.   Volume expansion and hypertension due to defects in the regulation of ENaC. These defects fall into 2 categories: 1) excessive mineralocorticoid action (MC), in which ENaC activity in the luminal membrane (left) is increased; and 2) Liddle's syndrome, in which various mutations in - or -ENaC subunits produce a gain of channel function. In both conditions, there is excessive NaCl reabsorption, resulting in extracellular fluid (ECF) volume expansion and hypertension. In both categories, increased ENaC activity increases the depolarization of the luminal membrane and thereby increases K+ secretion, which leads to hypokalemia.

In all of these conditions, excessive K⁺ secretion accompanies the accelerated Na⁺ reabsorption. As shown in Fig. 1, the lumen negative voltage in the CCD is a consequence of the depolarization of the luminal membrane produced by the ENaC channels, and it is augmented by increased ENaC activity. Microelectrode studies in the CCD have shown that this increased driving force is sufficient to account quantitatively for the stimulation of K⁺ secretion by aldosterone and VP in the rat CCD (105), and patch-clamp studies have shown no effect of short-term aldosterone administration on K⁺ channel density (87). In the rabbit CCD, however, aldosterone also increases the K⁺ conductance of the apical membrane (69, 100). Thus the K⁺ wasting with Liddle's syndrome or excess mineralocorticoid activity is a secondary consequence of the depolarization of the apical membrane, possibly accompanied by increased ROMK activity in the case of the latter etiology.

Syndromes of mineralocorticoid excess are seldom related to abnormalities of the hypothalamic-anterior pituitary axis, because ACTH exerts little or no control over aldosterone secretion. Primary aldosteronism (Conn's syndrome; see OMIM no. 103900) is the unregulated and augmented secretion of aldosterone due to bilateral hyperplasia of the adrenal cortex or an aldosterone-producing adrenal adenoma. Excessive aldosterone production can also be driven by an appropriate response to aberrant activation of the renin-angiotensin system, in which case it is referred to as secondary aldosteronism. Another cause of aldosterone overproduction is an autosomal dominant disorder produced by a chimeric gene duplication event that results in aldosterone synthase expression driven by an abnormal promoter that responds to ACTH (74). The condition can be treated by glucocorticoid therapy (which suppresses ACTH release), and thus it is referred to as glucocorticoid-remediable aldosteronism (GRA; also known as GSH or familial hyperaldosteronism type I; OMIM no. 103900).

Under some circumstances, glucocorticoids may produce mineralocorticoid actions. For example, when glucocorticoids are given therapeutically, retention of salt and water is a common side effect. A more interesting example in the present context is the autosomal recessive genetic disorder of apparent mineralocorticoid excess (AME; alternatively, AME1 or AME2; see OMIM nos. 218030 and 207765). The range of plasma cortisol concentrations in man is >100-fold higher than that of aldosterone, yet the affinities of these hormones for MR are similar, and thus glucocorticoids could cause excessive mineralocortidoid actions (27). This normally does not occur because the aldosterone-sensitive regions of the nephron express the enzyme 11-HSD2 (also called HSD11B2; OMIM no. 217030), which converts circulating cortisol to cortisone. Because cortisone does not activate the MR, the enzyme effectively "protects" these receptors from occupancy by glucocorticoids (27, 40). In AME, deficient 11-HSD2 activity allows cortisol to exert the same effects as excess aldosterone (27). Several different mutations in the 11-HSD2 gene are known to reduce or eliminate its activity, and this polymorphism may account for variability of the phenotype that has led to categorization as types I and II AME (OMIM nos. 218030 and 207765, respectively).

Since the molecular identification of the amiloride-sensitive Na⁺ channel in the CCD as a heterotrimeric assembly of ENaC subunits, there has been renewed interest in Liddle's syndrome (Fig. 5). Liddle's syndrome [also known as pseudoaldosteronism (PHA); OMIM no. 177200] is a rare autosomal dominant disorder that resembles primary aldosteronism but with no elevation of aldosterone and no amelioration by glucocorticoids as found in AME. In the original kindred, the disorder was shown to be linked to a mutation in the -ENaC gene that results in truncation of the cytoplasmic COOH-terminal region of the protein (112). These observations stimulated a worldwide search for other kindreds in which low-renin hypertension might be associated with ENaC mutations. Multiple kindreds exhibiting Liddle's syndrome have been identified with autosomal dominant linkage to a variety of mutations in the COOH-terminal region of -ENaC, including missense, nonsense, and truncation mutations (49, 62, 122, 132), and a single kindred with truncation of the cytoplasmic COOH-terminal domain of -ENaC (49).

The evidence associating Nedd4 and the ubiquitin degradation system with ENaC regulation has provided a compelling explanation for the gain of function of ENaC in Liddle's syndrome. As can be seen in Fig. 4, truncation of the COOH-terminal region or mutations that disrupt its PPPXY motif would prevent Nedd4 binding and ubiquitin-mediated internalization of the mutant ENaC. Staub et al. (119) proposed this mechanism as the basis of Liddle's syndrome; i.e., defective internalization of ENaC subunits would lead to an increase in their steady-state level in the apical membrane independently of aldosterone regulation. This hypothesis is supported by observations that the introduction of Liddle mutations in the - and -ENaC subunits expressed in X. laevis oocytes and Madin-Darby canine kidney cells increases amiloride-sensitive transport and the surface density of ENaC channels (34, 117).

The association between Na⁺ retention and hypertension is further supported by experiments in transgenic mice expressing the Liddle trait. Mice that are heterozygous or homozygous for the Liddle mutation develop normally during the first 3 mo of life but exhibit low plasma aldosterone consistent with chronic hypervolemia. When given a high-salt diet, the mice rapidly develop hypertension, hypokalemia, and metabolic alkalosis (91).

	DEFECTS IN THE REGULATION OF CCD NA+ Transport Leading to Volume Contraction

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Disorders associated with renal Na⁺ wasting and extracellular volume contraction that often lead to hypotension are effectively the mirror image of those producing volume contraction. As outlined in Fig. 6, they can be categorized as disorders in which aldosterone production is impeded or its action is prevented, or there are loss-of-function mutations in ENaC subunits. Disorders in both categories are associated with salt wasting, hyperkalemia, and variable acidosis. Hyperkalemia arises because of the diminished activity of Na⁺ channels in the luminal membrane, which reduces the depolarization of that membrane and hence reduces the driving force for K⁺ secretion across the luminal membrane.

View larger version (35K): [in this window] [in a new window]   Fig. 6.   Salt wasting and hypotension due to defects in the regulation of ENaC. These defects can also be divided in 2 categories: 1) low or absent levels of aldosterone or mineralocorticoid receptor (MR) that remove the mineralocorticoid enhancement of ENaC and Na+-K+-ATPase activity; and 2) various mutations in any of the 3 ENaC subunits that result in a loss of channel function. The resulting salt wasting leads to hypotension. Decreased ENaC activity results in less depolarization of the luminal membrane and less K+ secretion, which leads to hyperkalemia.

Addison disease (primary adrenocortical deficiency), which is due to idiopathic or autoimmune destruction of the adrenal cortex, can produce severe hypovolemia and hyperkalemia. The syndrome of PHA type I (PHA1) is also characterized by salt wasting, hypotension, and hyperkalemia but in the presence of high plasma renin and aldosterone. PHA1 is now recognized to include two different and rare heritable disorders, one being autosomal dominant and the other autosomal recessive. The autosomal dominant form of PHA1 (OMIM no. 177735) represents a loss of aldosterone effect in the nephron because of mutations in MR (44, 135). Autosomal recessive PHA1 (OMIM no. 264350) is caused by loss-of-function mutations in ENaC that have been demonstrated in all three isoforms in different pedigrees (18, 33, 126). Interestingly, these mutations occur either in the cytoplasmic NH₂-terminal region (- and -ENaC) or in the extracellular loop (- and -ENaC) but not in the COOH-terminal region. Firsov et al. (32) expressed one of the mutant variants of -ENaC in X. laevis oocytes together with wild-type - and -ENaC and observed proportional decreases in the amiloride-sensitive current and the surface expression of ENaC. They hypothesized that the mutant ENaC isoforms associated with autosomal recessive PHA1 interfered with the normal trafficking of assembled ENaC channels to the plasma membrane.

	A ROLE FOR A CL Transporter in Regulating Salt Transport in the CCD?

TOP ABSTRACT INTRODUCTION CCD ION TRANSPORTERS REGULATION OF NA+ Transport... DEFECTS IN THE REGULATION... DEFECTS IN THE REGULATION... A ROLE FOR A... OTHER HORMONAL REGULATORS OF... IS THE ANTINATURETIC EFFECT... CONCLUSIONS REFERENCES

All of the disorders of ECF volume regulation discussed above are attributable to abnormalities in the regulation of Na⁺ transport in the aldosterone-responsive segments of the nephron, especially the CCD. A common aspect of these disorders is the reciprocal relationship between Na⁺ and K⁺ transport-augmented Na⁺ reabsorption being associated with excessive K⁺ secretion and wasting (Fig. 5), whereas Na⁺ wasting is accompanied by diminished K⁺ secretion and hyperkalemia (Fig. 6). This relationship might seem to be an inescapable consequence of the electrical coupling between Na⁺ reabsorption and K⁺ secretion due to the depolarization of the luminal membrane produced by ENaC (Fig. 7). The apparent invariance of the relationship between Na⁺ reabsorption and K⁺ secretion leads one to question how the CCD can be a primary regulator of both. For example, how can the CCD increase Na⁺ reabsorption and decrease K⁺ secretion (or even produce net K⁺ reabsorption) when there is a deficit of both?

View larger version (45K): [in this window] [in a new window]   Fig. 7.   The reciprocal relationship between Na+ reabsorption and K+ secretion in the CCD. Because increased Na+ reabsorption is produced by increased ENaC activity and results in depolarization of the luminal membrane, there is an increased driving force for K+ secretion via ROMK channels. Conversely, decreased Na+ reabsorption results in decreased K+ secretion. The extent to which these countermovements of the 2 cations are linked is determined primarily by the Cl permeability of the junctional complexes. At one extreme, if there were no transepithelial Cl movement, electroneutrality would demand a 1:1 exchange of K+ for Na+. At the other extreme, if the junctional complexes were extremely permeable to Cl, no lumen negative voltage would develop and there would be no K+ secretion. In the latter case, Na+ reabsorption would be increased due to a greater electrical driving force across the luminal membrane. An abnormally high junctional Cl permeability is the putative cause of pseudohypoaldosteronism type II (106, 142).

A possible explanation is that the usual assumption that the body's K⁺ balance is achieved primarily by aldosterone-mediated regulation of K⁺ secretion in the CCD is incorrect. Rabinowitz (for references, see Ref. 93) has patiently but persistently presented evidence that aldosterone is not a kaliuretic hormone at normal plasma concentrations and that marked diurnal changes in K⁺ excretion, or changes in excretion in response to changes in dietary intake, are not correlated with changes in plasma aldosterone or K⁺ concentrations. He and a group of Russian investigators (31, 92, 93) have hypothesized that the central nervous system mediates meal-induced and diurnal variations in K⁺ excretion. Unfortunately, the postulated K⁺ receptors in the gastrointestinal tract and regulatory signaling mechanism that affects the kidney have not been identified. However, even if there are mechanisms of K⁺ regulation other than aldosterone, they must be overwhelmed by the more extreme changes in mineralocorticoid regulation or ENaC activity summarized in Figs. 5 and 6. Indeed, Rabinowitz (92) agrees that aldosterone is kaliuretic at supraphysiological concentrations. Thus the question of whether Na⁺ reabsorption in K⁺ secretion can be dissociated at the level of the CCD remains regardless of the role of aldosterone and variations in ENaC activity in the normal regulation of K⁺ balance.

Intercalated cells in the CCD present a possible pathway by which the reciprocal relationship between Na⁺ and K⁺ transport might be dissociated. It has been shown that type A intercalated cells can actively reabsorb K⁺ via the H⁺-K⁺-ATPase (143), but is it reasonable that this transporter must be activated to counteract elevated K⁺ secretion by the principal cells in Na⁺ depletion? Another solution to dissociating Na⁺ and K⁺ transport in the CCD could involve independent regulation of the basolateral and apical K⁺ channels depicted in Fig. 1. One might argue that increased K⁺ secretion coupled to increased Na⁺ reabsorption would not be obligatorily if the ratio of apical (ROMK1) to basolateral K⁺ conductances could be regulated. Alternatively, K⁺ secretion might be diminished by the increase in basolateral K⁺ conductance that accompanies increased Na⁺ reabsorption (109). In either case, however, net K⁺ secretion would be decreased only if there were a driving force for K⁺ efflux from CCD cells across the basolateral membrane. The latter is questionable, especially in situations when Na⁺-K⁺-ATPase activity is stimulated, because the net K⁺ electrochemical potential gradient across the basolateral membrane actually favors K⁺ uptake (100, 109).

Once again, a congenital abnormality, PHA type II [PHA2; also known as Gordon (or familial) hypertensive-hyperkalemia; OMIM no. 145260], provides a clue to solve this apparent dilemma. The most consistent clinical characteristics of this rare autosomal dominant disorder include hyperkalemia and hypertension with normal renal function, and the exceptional amenability of the hypertension and ion imbalances to treatment with thiazide diuretics.

By a careful clinical study of one patient with this syndrome, and from descriptions of other such patients, Schamblen et al. (106) proposed that this disorder represents a defect in the regulation of Cl reabsorption by the distal nephron, such that the lumen-negative driving force that normally favors K⁺ secretion is diminished by an augmented Cl conductance. As shown in Fig. 7, the lumen negative voltage, which develops in the CCD because of the ENaC-dependent depolarization of the luminal membrane, provides the driving force for Cl reabsorption as well as K⁺ secretion. An increase in the Cl permeability of the junctional complexes, or the introduction of a conductive pathway for transcellular Cl movement, would increase NaCl reabsorption, leading to ECF volume expansion and hypertension, but it would also decrease, or in effect "short-circuit," the lumen negative voltage that drives K⁺ secretion.

Genetic studies of different PHA2 pedigrees have shown linkage to chromosomes 1, 12, and 17. The gene involved on chromosome 1 has not yet been identified, but Wilson et al. (142) have recently shown that the genes associated with the other two encode members of a unique family of WNK ("with no lysine") serine-threonine kinases (146). A mutation in one intron of the WNK1 gene, which is located on chromosome 12, and various mutations in the coding regions of WNK4, on chromosome 17, lead to elevated levels of both kinases, i.e., a genetic gain of function (142). Wilson et al. (142) also showed that WNK4 is only expressed in the kidney, where it is localized to aldosterone-responsive segments of the nephron. WNK1 is expressed in several tissues, but in the kidney, it too is found only in the distal nephron. Finally, and most intriguingly, immunohistological studies showed that, whereas WNK1 is distributed throughout the cytoplasm of the distal nephron, WNK4 is present only in the junctional complexes where it was colocalized with ZO-1, a protein characteristic of these structures (142).

The above observations provide at least circumstantial evidence that higher WNK4 activity is in some way associated with an increased Cl permeability of the junctional complexes of the distal convoluted tubule and/or CCD in at least one pedigree of PHA2 patients. More important in the present context is the potential identification of a mechanism for Cl reabsorption that could be just as important as ENaC in regulating NaCl reabsorption by the distal nephron. The activity of such a Cl transporter would also determine the strength of coupling between Na⁺ reabsorption and K⁺ secretion. There are as yet no good candidates for Cl channels in the CCD or other regions of the aldosterone-responsive segments of the nephron; however, since the discovery that a specific protein (paracellin) mediates Mg²⁺ and Ca²⁺ reabsorption through the junctional complexes of the thick ascending limb (113), the possibility of regulated Cl movement through junctional complexes no longer seems remote.

	OTHER HORMONAL REGULATORS OF SALT TRANSPORT IN THE CCD

TOP ABSTRACT INTRODUCTION CCD ION TRANSPORTERS REGULATION OF NA+ Transport... DEFECTS IN THE REGULATION... DEFECTS IN THE REGULATION... A ROLE FOR A... OTHER HORMONAL REGULATORS OF... IS THE ANTINATURETIC EFFECT... CONCLUSIONS REFERENCES

The clear and consistent effects of ENaC subunit mutations and alterations in their regulation by abnormal mineralocorticoid metabolism have been seductive in advancing the optimistic view that a single gene defect might be the cause of essential hypertension. However, the connection between the rare genetic defects discussed above and essential hypertension is remote. In AME, glucocorticoid-remediable hypertension, and Liddle's syndrome, hypertension is usually severe and begins in childhood. Essential hypertension, on the other hand, typically begins later in life and progresses more slowly. It is only with certain forms (pedigrees) of PHA2 that a more slowly developing course is seen. This observation in no way discounts the importance of the single-gene defects described. Any one of them can cause severe hypertension, which clearly demonstrates the importance of transport regulation in the DCT and CCD. The question is only whether these particular candidate genes are likely to be involved in essential hypertension. Given the emerging picture of the polygenic nature of this and other diseases, it seems more likely that various coincidental combinations of gene alterations, diet, and environment are the cause.

The following sections discuss the possible role of VP, as well as autacoids and paracrine agents that alter the response to this hormone, in Na⁺ retention and hypertension. This focus on VP does not imply that it is potentially a more important or more likely contributor to derangements in ECF volume and blood pressure regulation but rather that the role of this regulatory system has been relatively neglected compared with others. Despite the fact that VP was the first hormone demonstrated to augment Na⁺ reabsorption in frog skin and toad bladder, the classic models of the CCD, it has traditionally been regarded to be a natriuretic hormone in mammals. Most of the studies that support this view, however, were conducted in water- or volume-loaded rats infused with VP (4, 137). More recent studies have demonstrated that VP can be antinatriuretic in euvolemic rats and humans (2, 4, 5, 22). VP was found to markedly decrease Na⁺ excretion even in isolated perfused kidney preparations, in which alternative explanations of the effect are obviated (73).

In the isolated, perfused rat CCD, VP acts synergistically with aldosterone to stimulate both Na⁺ reabsorption and K⁺ secretion. Both hormones individually increase transport, but in combination the effect is greater than with either hormone alone (20, 94, 95, 103, 105, 130, 131). VP also stimulates 11-HSD2 activity in the rat CCD, and this mechanism has been proposed to account for at least some of the synergism between VP and aldosterone (1). The stimulation of Na⁺ reabsorption by VP is mediated by cAMP, which produces a rapid and selective increase in the Na⁺ conductance of the luminal membrane (104, 109), and the resulting depolarization of the luminal membrane voltage increases K⁺ secretion (103, 105). With Madin-Darby canine kidney cells as a model of the rat CCD, we have used surface labeling to demonstrate that cAMP increases the density of ENaC subunits in the apical membrane in direct proportion to the increase in the amiloride-sensitive short-circuit current (80). Thus the acute action of VP appears to be due primarily to trafficking of ENaC into that membrane.

Chronic treatment of rats with VP also leads to a dramatic increase in the expression, primarily, of the - and -ENaC subunits at both mRNA (25) and protein (84) levels. It is interesting to note that this effect is quite different from that observed with aldosterone, which increases the expression only of -ENaC and to a far lesser extent than the effect of VP on the - and -ENaC subunits (77, 78, 84, 125).

The results in the rat CCD stand in marked contrast to earlier observations in the rabbit CCD. Although VP produced an initial hyperpolarization of the lumen negative voltage in the rabbit CCD that was suggestive of an increase in the luminal membrane Na⁺ conductance, after 15-30 min the voltage returned to control levels or below, and there was no stimulation of Na⁺ transport in the presence or absence of mineralocorticoid (20, 35, 57).

As summarized in Figs. 8 and 9, the rat and rabbit CCD also exhibit quite different responses to other agents that appear to act as autocrine or paracrine modulators of CCD transport. PGE₂ has long been known to be an inhibitor of both Na⁺ reabsorption and the water permeability increase produced by VP in the rabbit CCD (17, 19, 57, 124), possibly by elevation of [Ca²⁺]_i (53); however, neither PGE₂ nor elevation of [Ca²⁺]_i has any effect on Na⁺ transport or the water permeability response to VP in the rat CCD (17, 19, 99). It seems likely that, as proposed years ago by Holt and Lechene (57), PGE₂ produced by CCD cells in response to VP acts as a negative-feedback inhibitor to prevent stimulation of Na⁺ reabsorption and that this pathway is absent in the rat CCD. PGE₂ is now known to interact with two receptors, EP₁ and EP₃, on the basolateral membrane of the CCD (11). EP₃ inhibits adenylyl cyclase via a G_i protein, whereas EP₁ activates the PLC signaling system (11). The increase in [Ca²⁺]_i and protein kinase C (PKC) activity that results from PLC stimulation has been shown to inhibit VP-dependent Na⁺ transport and water permeability in the rabbit, but not the rat, CCD (99).

View larger version (31K): [in this window] [in a new window]   Fig. 8.   Putative model for intrinsic inhibition of the salt-retentive effect of vasopressin (VP) in the intact rabbit CCD. VP, acting through the Gs-coupled V2 receptor, activates adenylyl cyclase (AC), which elevates cAMP. This second messenger, by protein kinase A (PKA)-dependent phosphorylation of unknown intermediate proteins, results in the insertion of aquaporin-2 (AQP2) water channels and ENaC into the luminal membrane. While the resulting increase in water permeability is stable, the increase in Na+ reabsorption is very short-lived, possibly because of a regulatory autacoid feedback by PGE2. PGE2 synthesis in the CCD is driven by VP-dependent activation of phospholipase A2 (PLA2) through an unknown mechanism (?) possibly involving cAMP (28, 60). Synthesized PGE2 interacts with EP3 receptors that inhibit AC via a Gi protein and with EP1 receptors that stimulate phospholipase C (PLC) by an unknown (Gq ?) G protein (11). The resulting increase in intracellular Ca2+, and possibly activation of 1 or more PKC isoforms, inhibits ENaC insertion into the apical membrane.

View larger version (21K): [in this window] [in a new window]   Fig. 9.   Regulation of VP-dependent Na+ and water reabsorption in the intact rat CCD and rabbit CCD cells in primary culture. VP acts to increase the insertion of AQP2 and ENaC in the luminal membrane, as described in Fig. 8; however, in these systems the resulting increase in Na+ reabsorption is stable and synergistic to the effect of aldosterone because of the absence of a PGE2-regulatory feedback. In addition, the antidiuretic and antinatriuretic effects of VP can be inhibited by dopamine or norepinephrine, acting, respectively, through D4 and 2 (probably 2B)-receptors.

In the rat CCD, catecholamines appear to be the primary modulators of VP-dependent Na⁺ transport and water permeability, but they are without effect in the rabbit CCD (17, 19). In the rat CCD, dopamine inhibits VP action through a D₄ receptor (127, 128), and epinephrine or norepinephrine through an ₂-receptor (51, 52, 141), both of which are linked to G_i proteins associated with adenylyl cyclase, as shown in Fig. 9. Dopamine, epinephrine, and clonidine have been demonstrated to decrease VP-dependent cAMP production in the rat CCD (17, 71).

This discussion of the differences between the response of the rat and rabbit CCD to VP is not intended to be a thorough examination of the regulatory processes involved but rather a prelude to the argument that these differences may not just be "hardwired" in a given species but may have physiologically relevant plasticity. In support of this hypothesis, consider the following changes in the phenotype of the rabbit CCD grown in culture. When rabbit CCD cells are grown in primary culture, Na⁺ transport, measured as the amiloride-sensitive short-circuit current, is indefinitely stimulated by VP (15). This stimulation by VP is additive to the stimulatory effect of overnight incubation with aldosterone, and it is not inhibited by PGE₂ or PKC as it is in the isolated perfused rabbit CCD (15). In other words, rabbit CCD cells grown in primary culture assume the regulatory phenotype of the rat CCD with regard to the regulation of Na⁺ transport by VP. We presently have only circumstantial evidence for the mechanism producing this conversion. The atypical PKC -isoform is found in rabbit, but not rat, CCD, and its expression disappears when rabbit CCD cells are grown in culture, although other PKC isoforms are not downregulated (140). However, regardless of the mechanism, the change in the response of rabbit CCD cells to VP after growth in culture raises the possibility that the human CCD might also acquire an enhanced antinatriuretic response to VP in pathophysiological circumstances.

	IS THE ANTINATURETIC EFFECT OF VP INVOLVED IN HYPERTENSION OR EDEMATOUS STATES?

TOP ABSTRACT INTRODUCTION CCD ION TRANSPORTERS REGULATION OF NA+ Transport... DEFECTS IN THE REGULATION... DEFECTS IN THE REGULATION... A ROLE FOR A... OTHER HORMONAL REGULATORS OF... IS THE ANTINATURETIC EFFECT... CONCLUSIONS REFERENCES

It is perhaps not surprising that the antinatriuretic effects of VP differ between rat and rabbit. Like the human, in antidiuresis the rabbit can only concentrate its urine to ~1,200 mosmol/l. In contrast, rat urine samples are in the range of ~1,000-3,000 mosmol/l and in antidiuresis exceed 3,000 mosmol/l. As pointed out by Bankir (4), omnivores such as rats and mice have a higher obligatory osmolal clearance due to higher urea production, normalized to body weight, than an herbivore such as the rabbit. Omnivores and carnivores thus require a higher urine osmolality to accommodate their higher rates of urea production and yet conserve water. The ability to concentrate the urine maximally is very dependent on water reabsorption in the cortical regions of the collecting duct, which is quantitatively much larger than the water reabsorption that occurs in the medullary collecting duct in antidiuresis (4, 61). Because the water permeability of the rat CCD is very high in the presence of VP, osmotic equilibration with the cortical interstitium occurs in the initial region. Distal to the point of equilibration, continued NaCl reabsorption in combination with the high water permeability leads to isosmotic fluid absorption. The rate of this isosmotic water reabsorption is directly proportional to the rate of NaCl reabsorption, and thus enhancement of Na⁺ reabsorption by VP in the rat CCD minimizes salt and water delivery to the medulla and maximizes water conservation (102).

In light of these observations, it is important to consider the possibility that VP may contribute to Na⁺ retention in humans. Of course, we do not know whether VP stimulates Na⁺ reabsorption in the human CCD under physiological conditions, but, given the similar concentrating ability of humans and rabbits, we might suspect not. Nevertheless, the necessary ingredients for such a response appear to be available in the rabbit CCD and are assembled during growth in culture. Can the response of human CCD to VP shift to cause Na⁺ as well as water retention, or can this antinatriuretic effect of VP be augmented if it already exists? If so, elevated VP might be an important contributor to Na⁺ retention and ECF volume expansion not only in hypertension but also in the edematous states produced by congestive heart failure, cirrhosis, and the nephrotic syndrome. There are no certain answers to these questions, but there is evidence linking VP to both hypertension and to the edematous states.

In the classic DOC-salt rat model, hypertension does not develop in the absence of plasma VP but can be restored by the selective V₂-receptor agonist dDVP (6, 23, 72, 129, 150). Also, Yagil et al. (148) have shown that rats of the Sabra SBH/y strain, which provides a model of salt-sensitive hypertension, have elevated hypothalamic VP production and plasma VP levels on normal-salt intake compared with the corresponding control SBN/y line, which does not develop hypertension when placed on a high-salt diet. They concluded that elevated VP was a definite candidate gene for the salt-sensitive trait, at least in this model of hypertension (148, 149)

Clinical studies have also shown a correlation between plasma VP and hypertension. In healthy human subjects, Choukroun et al. (22) have shown that antidiuresis compromises the ability to excrete a Na⁺ load efficiently, which they attributed to the effect of VP on Na⁺ reabsorption by the CCD. These investigators further suggested that low water intake could compromise Na⁺ excretion even under conditions of normal daily intake. Bakris et al. (3) examined African-American and Caucasian patients with hypertension and found that baseline plasma VP was higher and the plasma renin lower in the former group. Furthermore, administration of a specific V₂-receptor blocker significantly reduced blood pressure in the African-American, but not the Caucasian, group. In a large study that included both black and white patients, plasma levels of VP were positively correlated with arterial blood pressure and hypertension, particularly in those patients who also had low renin levels (151). Finally, in a recent study of sibling pairs of hypertensive (and hypercholesterolemic) black patients, Kotchen et al. (70) found that the response of plasma VP to NaCl loading was one of several heritable traits that were significantly associated with the hypertension.

There is also an association of VP with Na⁺ retentiveness in animal models of edematous states. For example, in rat models of chronic heart failure (147) and cirrhosis (39), it was found that specific V₂-receptor antagonists induced diuresis and natriuresis. V₂-receptor antagonists have also been suggested to be useful in treating both hypertension and Na⁺ and water retention in heart failure (12). Localization of these palliative effects to the V₂ receptor indicates that they are a consequence of diminished antidiuretic and antinatriuretic effects of VP rather than vasodilation. Certainly, inhibition of the antidiuresis that causes the frequent development of hyponatremia in patients with congestive heart failure or cirrhosis is the most obvious and perhaps the most beneficial action of V₂-receptor antagonists (13); however, they also relieve the extreme Na⁺ retentiveness that characterizes these disorders (9, 63).

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION CCD ION TRANSPORTERS REGULATION OF NA+ Transport... DEFECTS IN THE REGULATION... DEFECTS IN THE REGULATION... A ROLE FOR A... OTHER HORMONAL REGULATORS OF... IS THE ANTINATURETIC EFFECT... CONCLUSIONS REFERENCES

The genetic disorders discussed above provide ample evidence of the importance of the aldosterone-responsive segments of the nephron for regulating Na⁺ excretion. They also reveal highly critical elements in this regulation, including the production and metabolism of aldosterone and other adrenal steroids, the synthesis and proper function of ENaC subunits, and possibly the Cl permeability of the junctional complexes in the DCT and CCD.

These genetic defects and their pathophysiological consequences also provide more general support for the close association between the renal regulation of ECF volume and blood pressure proposed by Guyton (46, 47). One must be cautious, however, in applying these observations to any understanding of the pathophysiology of essential hypertension. Nevertheless, more subtle alterations in other processes that regulate Na⁺ reabsorption by the CCD, and perhaps other aldosterone-responsive nephron segments, may be components of essential hypertension, especially the low-renin, salt-sensitive variants. One candidate regulator is VP, which can, at least in some settings, act in synergy with aldosterone to increase Na⁺ reabsorption. This antinatriuretic action of VP, as well as its effect on the water permeability of the CCD, is variably inhibited in different animal models by activation of the PLC and PLA₂ intracellular signaling systems, the autacoid actions of PGE₂, and the paracrine actions of catecholamines, including dopamine. Therefore, alterations in any of these regulatory systems are also possible contributors to excessive Na⁺ retention or loss that lead to hypertension or salt wasting.