Mineralocorticoid Receptor Function as a Hormone-Regulated Transcription Factor: General Features and Subcellular Localization

In the presence of agonists, MR binds to specific genomic sites and alters the transcription rate of a subset of genes. Figure 12.3 shows the fundamental paradigm of MR function. All nuclear receptors shuttle in and out of the nucleus; however, in the absence of hormone, some, such as the estrogen and the vitamin D receptors, are predominantly nuclear, whereas others, like GR, are almost exclusively cytoplasmic. In the absence of hormone, MR is distributed relatively evenly between nuclear and cytoplasmic compartments, but in the presence of hormone, it is highly concentrated in the nucleus ( Figure 12.4 ). It is also notable that in addition to this marked change in MR cellular distribution, its subnuclear organization and protein-protein interactions are also changed. Like its close cousin the GR, the unliganded MR (in the absence of hormone) is complexed with a set of chaperone proteins, which include the heat shock proteins hsp90, hsp70, and hsp56, and immunophilins. This chaperone complex is essential for several aspects of MR function, notably high-affinity hormone binding and trafficking to the nucleus. It was thought for many years that after binding hormone, the hsp90-containing chaperone complex is jettisoned. However, it has become clear that this complex remains associated with the receptor and plays an important role in nuclear trafficking. Several members of the immunophilin family, including FKBP52, FKBP51, and CyP40, are present in the chaperone complex and provide a bridge between hsp90 and the cytoplasmic motor protein dynein, which moves the receptor-hsp90 complex retrogradely along microtubules to the nuclear envelope. Here, the receptor is handed off to the nuclear pore protein, importin-α, and translocated into the nucleus, where it functions as a transcription factor, stimulating transcription of certain genes and repressing the transcriptional activity of others. In the regulation of ion transport, stimulation of key target genes is paramount. Transcriptional repression may be essential for effects in nonepithelial cells, including neurons, cardiomyocytes, smooth muscle cells, and macrophages.

General mechanism of aldosterone action through the mineralocorticoid receptor (MR).

This simple schematic shows the general features of MR regulation of a “simple” hormone response element (HRE), common to a large subset (but not all) of aldosterone-stimulated genes. Note that in the absence of hormone, MR is found in both nucleus and cytoplasm (see Figure 12.4 ). Aldosterone triggers nuclear translocation of cytoplasmic MR, binding as a dimer to HREs, and stimulation of transcription initiation complex formation ( arrow upstream of “protein coding gene” defines the site of transcription initiation). See text for further details.

Time-dependent nuclear translocation of the mineralocorticoid receptor (MR) in the presence of aldosterone.

Cultured cells expressing green fluorescent protein–MR fusion protein (GFP-MR) were grown in a steroid-free medium and treated with 1 nmol/L aldos­terone. Translocation of GFP-MR was followed in real time, and images were captured at indicated times. It is notable that nuclear accumulation of GFP-MR started within 30 sec, was half-maximal at 7.5 min, and was complete by 10 min. Control: MR distribution prior to addition of aldosterone.

(From Fejes-Toth G, Pearce D, Naray-Fejes-Toth A: Subcellular localization of mineralocorticoid receptors in living cells: effects of receptor agonists and antagonists, Proc Natl Acad Sci U S A 95[6]:2973-2978, 1998, Figure 3.)

Domain Structure of Mineralocorticoid Receptors

The MRs of all vertebrates are highly conserved. There are only minor differences between MRs in rodents and MRs in humans. In general, the steroid and nuclear receptors have been divided into three major domains ( Figure 12.5 ): (1) an N-terminal transcriptional regulatory domain, (2) a central DNA-binding domain (DBD), and (3) a C-terminal ligand/hormone-binding domain (LBD). Each of these broadly defined domains has more than one function, and not all of the functions can be neatly assigned to separate distinct domains; however, much of what MRs do can be understood from this point of view. In the following sections, the domains are described roughly in the historical order in which they were characterized, which also parallels the clarity of functional and structural knowledge about them.

Domain structure of the mineralocorticoid receptor (MR).

Three major domains have been defined, which are common to all steroid/nuclear receptors. Further refinements have led some to use a six-letter system, which is shown; however, this adds little to the understanding of receptor structure or function, and the authors prefer the three global domain system. These large receptor sections should not be confused with the many small functional domains that have been identified, as discussed in the text. The size and amino acid designations used here are for rat MR (981 amino acids total); they apply with minor variations to human MR (984 amino acids total). A, Strip diagram of MR. N terminus is to the left, C terminus to the right. B, Schematic of MR DNA-binding domain (DBD), showing the two zinc fingers, and the positions of the coordinating Zn ions. Boxed region is the α-helix, which intercalates into the major groove of the DNA and provides the major protein-DNA interaction contacts. The dimerization interface comprises amino acids within the second zinc finger, which form van der Waals and salt bridge interactions. LBD, Ligand/hormone-binding domain.

Ligand/Hormone-Binding Domain

The MR LBD comprises amino acids 689 to 981 (see Figure 12.5 A ). Like the DBD, the LBD has multiple functions: in addition to binding with high affinity to various MR agonists and antagonists, it also harbors interaction surfaces for coactivators, dimerization, and N-C interactions. MR is distinct from GR in that it binds with equal, high affinity to cortisol, corticosterone (the physiologic glucocorticoid in rats and mice), and aldosterone. Indeed, as discussed later, MR appears to function as a high-affinity glucocorticoid receptor in some tissues, including the brain and the heart.

High-resolution representations of the crystal structures of wild-type and mutant MR have identified the structural features of the LBD and specific amino acid contacts involved in binding to the mineralocorticoid desoxycorticosterone ( Figure 12.6 ). Key features include the following: (1) The LBD of MR, like that of other nuclear receptors, is arranged into 11 α-helices and four small β-strands. (2) The C-terminal α-helix, H12, contains the activation function AF2. (3) Ligand is deeply embedded into a pocket comprising α-helices H3, H4, H5, H7, and H10, and two β-strands; numerous contacts are made between amino acids of the pocket and the hormone. This accounts for the slow off rate and high affinity of aldosterone, corticosterone, and cortisol for MR. The crystal structure of the mutant (S810L) MR, in which progesterone acts as an agonist rather than an antagonist, reveals that H12 is stabilized with AF2 in the active conformation. The crystal structure of wild-type MR LBD also provides insight into the mechanisms underlying some forms of pseudohypoaldosteronism type I. Notably, MR/S810L has an LBD mutation in helix 5, which is predicted to disrupt interaction with the steroid ring structure, whereas Q776R and L979P have been demonstrated to have markedly reduced aldosterone binding. Structural analysis reveals that Q776 is located in helix H3 at the extremity of the hydrophobic ligand-binding pocket and anchors the steroid C3-ketone group.

Mineralocorticoid receptor (MR) ligand/hormone-binding domain (LBD) crystal structure.

Structure of the MR LBD bound to corticosterone and coactivator peptides (SRC1-4). A and B, Two views of the complex (rotated by 90 degrees about the vertical axis) are shown in ribbon representation. MR is colored in gold and SRC1-4 peptide in yellow. Corticosterone, which binds MR with an affinity comparable to that of aldosterone, is shown in ball and stick representation. Note that hormone is located in a deep pocket formed by helices 3, 5, and 7 (H3, H5, and H7), which explains the slow off rate and high affinity. C, Sequence alignment of the human MR LBD with other steroid hormone receptors (GR, glucocorticoid; AR, androgen; PR, progesterone; and ER, estrogen). Residues that form the steroid-binding pockets are shaded in gray. Key structural features for the binding of SRC peptides are noted with stars, and the residues that determine MR/GR hormone specificity are labeled by arrowheads. See Li and others for further details.

(From Li Y, Suino K, Daugherty J, et al: Structural and biochemical mechanisms for the specificity of hormone binding and coactivator assembly by mineralocorticoid receptor. Mol Cell 19:367-380, 2005.)

MR binds cortisol and corticosterone with an affinity similar to that of aldosterone. 11β-HSD2 is an essential determinant of aldosterone specificity, through its effect in metabolizing glucocorticoids to their receptor-inactive keto-congeners in collecting duct principal cells, as discussed later. In tissues that do not coexpress 11β-HSD2, the physiologic ligand for MR is cortisol (corticosterone in rats and mice). The extent to which such “unprotected” MR can be pathophysiologically activated in aldosterone excess states is discussed in the “Primary Aldosteronism” section under “Disease States” and in the “Nonepithelial Actions of Aldosterone” section.

Mineralocorticoid Receptor Regulation of Transcription Initiation: Coactivators and Corepressors

The major mechanism of MR action is its effects on transcription initiation; however, there may also be effects on transcript elongation. Much has been learned about the generation of an initiation complex and the particular roles that steroid receptors play in this process. Several review articles and book chapters provide in-depth examinations of the biochemistry of the general transcription machinery, transcription initiation, promoter escape, and processive elongation. Most of the coactivators identified so far interact with the C-terminal AF2 domain and include the prototypical GRIP1/TIF2 and SRC, which sequentially recruit a series of different components of the transcriptional machinery and result in the formation of a preinitiation complex (PIC). This PIC includes all of the key components of the transcription machinery, including RNA polymerase II. A detailed picture of MR-dependent PIC formation has not been obtained. However, the general features are likely similar to those for ER and involve the sequential recruitment by the receptor of (1) chromatin-remodeling SWI/SNF and CARM1/PRTM1 proteins, which promote chromatin remodeling and initiation of complex formation; (2) histone acetylase CBP/P300 (cyclic adenosine monophosphate [cAMP]–responsive element–binding protein), which promotes an active chromatin conformation ; and (3) direct or indirect recruitment of the TATA-binding protein and other components of the general transcription machinery.

The aforementioned mechanisms are generic and are used by many transcription factors, including all steroid receptors, through interactions with the C-terminal AF2 domain. The N-terminal region of MR, which harbors the AF1 domain, diverges from the other steroid receptors, and recent evidence has identified coregulators that interact selectively with this receptor domain. ELL (eleven-nineteen lysine-rich leukemia factor) is a coactivator for MR that specifically interacts with AF1b and assists in PIC formation. It was originally identified as an elongation factor, and it may also affect transcript elongation. Other specific coregulators include the synergy inhibitory protein PIAS1, Ubc9, and p68 RNA helicase.

General Model of Aldosterone Action

Aldosterone enters the cell passively, binds to MR, triggers changes in gene transcription (as addressed in the “Mechanisms of MR Function and Gene Regulation” section), and potentially has nongenomic effects. Aldosterone effects in the ASDN have been divided into three major phases: latent, early, and late. This designation goes back to the early observations by Ganong and Mulrow that after aldosterone infusion into experimental animals, no effect was observed for at least 15 to 20 minutes. A similar delay was observed in isolated epithelia. The early phase, which is now known to involve primarily MR-dependent regulation of signaling mediators such as SGK1, culminates in increased apical localization—and possibly increased probability of the open state—of ENaC. In the late phase, aldosterone stimulates transcription of a variety of effector genes, including those that encode components of the ion transport machinery, notably the ENaC and Na ⁺ -K ⁺ –adenosine triphosphatase (Na ⁺ -K ⁺ -ATPase) subunits. The major direct effect is to increase Na ⁺ reabsorption, which is accompanied variably by Cl ⁻ reabsorption and/or K ⁺ secretion, and ultimately water reabsorption. Aldosterone’s actions in the principal cells of the connecting segment and collecting duct ( Figure 12.7 ) are of primary significance; however, it also has been shown to influence fluid and electrolyte transport in other tubule segments, as well as in other organs. These actions of aldosterone can be surmised from the clinical features of individuals with aldosterone-secreting tumors; they have volume expansion with high blood pressure and are commonly (≈50%) hypokalemic. In general, the effects of aldosterone on Na ⁺ absorption and K ⁺ secretion work together. However, there are ways that these actions can be separated, as discussed later.

Schematic of principal cells in the aldosterone-sensitive distal nephron (ASDN).

The ASDN includes the distal third of the distal convoluted tubule (DCT), the connecting tubule (CNT), and the collecting duct. The Na ⁺ -K ⁺ –adenosine triphosphatase (Na ⁺ -K ⁺ -ATPase) establishes the gradients for passive apical entry of sodium through the epithelial sodium channel (ENaC). Transport of sodium through the ENaC creates a negative lumen potential that drives potassium secretion into the lumen. Potassium is also recycled at the basolateral surface, which facilitates potassium exchange across the Na ⁺ -K ⁺ -ATPase. Chloride (Cl ⁻ ) moves via paracellular and transcellular pathways. There is evidence to support aldosterone actions in other segments, particularly the sodium-chloride cotransporter (NCC)—expressing portions of the DCT (DCT1 and DCT2).

The two basic cell processes that aldosterone regulates—Na ⁺ absorption and K ⁺ secretion—are depicted in Figure 12.7 . Most aspects of this mechanism are relevant to the various aldosterone target tissues.

Na ⁺ -K ⁺ -ATPase, located on the basolateral membrane (blood side), establishes the essential electrochemical gradients that drive ion transport (see Chapters 5 and 6 ). The most important, rate-limiting step is that of Na ⁺ entry into the cell via the epithelial Na ⁺ channel, ENaC. The discovery of the molecular composition of ENaC in 1993 opened the door to understanding how aldosterone functions to regulate this critically important ion channel. Most Na ⁺ transporters are encoded by a single gene product. In contrast, ENaC is composed of three similar but distinct subunits, each encoded by a unique gene. All three subunits come together (the stoichiometry is controversial) to form an ion channel with unique biophysical characteristics, the most striking of which is the relatively long time it stays open or closed. The complete loss of any one of these subunits in mice is incompatible with life.

The entry of Na ⁺ into the cell via ENaC in the apical membrane is the rate-limiting step in both Na ⁺ absorption and K ⁺ secretion. Na ⁺ enters the cell down a steep electrochemical gradient; intracellular [Na ⁺ ] is approximately 10 mmol/L, and the membrane voltage is strongly negative inside the cell. The Na ⁺ inside the cell is pumped out across the basolateral membrane by Na ⁺ -K ⁺ -ATPase, as addressed in detail in Chapter 6 . Most epithelial cells have a greater density of K ⁺ channels on the basolateral membrane and thus recycle K ⁺ back into the blood. The distal nephron is unique in that it has an unusually high density of K channels on the apical membrane (primarily Kir 1.1 [renal outer medullary potassium (ROMK)] and BK channels). This distribution of K ⁺ channels permits a large amount of K ⁺ that enters the cell via Na ⁺ -K ⁺ -ATPase to exit the cell into the lumen and be excreted into the urine. The vast majority of K ⁺ that appears in the urine is secreted by the distal nephron.

Much attention has been focused on the early phase of aldosterone action because it appears to be more tractable to dissection and the majority of change in Na ⁺ current occurs during this phase. This separation is probably somewhat artificial, however, and there is considerable overlap in events that define the early and late phases. Moreover, many efforts to manipulate mediators of the early phase (through overexpression and knockdown experiments) have been evaluated after prolonged alteration. Nevertheless, there is some heuristic value in considering early and late phases of aldosterone action separately.

In cultured collecting duct cells deprived of corticosteroids and then exposed to high concentrations of aldosterone, an increase in ENaC-mediated Na ⁺ transport can be observed in well under an hour, which is consistent with animal studies. Na ⁺ transport continues to increase for 2 to 3 hours, then plateaus for a few hours, and then gradually increases over the next several hours. After 12 hours of exposure to saturating aldosterone concentrations, the increase in ENaC activity is near maximal. The molecular basis for this increase in ENaC activity has been intensively investigated, and several key events are now apparent.

For aldosterone to increase ENaC activity, a change in gene transcription must occur. One of the earliest response genes is SGK1 . This discovery has had a major impact on the direction of research on aldosterone action, and this gene is addressed separately—together with its major target, Nedd4-2—later in this chapter. SGK1 is particularly important for the early actions of aldosterone. An important consideration in evaluating our understanding of this molecular pathway is that the genetic disease Liddle’s syndrome provided the first clues that the C-terminal portions of the ENaC subunits were essential in regulating ENaC surface expression (see Chapter 45 ).

Aldosterone and Epithelial Sodium Channel Trafficking

The major action of aldosterone is to increase the number of functional ENaC units on the apical membrane. This process can involve an increase in the number of channel complexes on the surface, or activation of existing complexes, or both. There is evidence to support both, although the bulk of evidence favors the idea that change in the number of ENaC complexes predominates. The redistribution of ENaC to the apical membrane can be detected in less than 2 hours after aldosterone exposure.

It is less well established whether the number of ENaC complexes is increased through increased insertion, decreased removal, or both. Aldosterone probably contributes to both processes. Rapid insertion of ENaC complexes is best understood with regard to the actions of cAMP. The extent to which the molecules involved in cAMP-mediated insertion are also involved in aldosterone action is uncertain, but some common mechanisms probably are used. Trafficking to the apical membrane appears to involve hsp70, SNARE (soluble NEM-sensitive factor attachment protein receptor) proteins, and the aldosterone-induced protein melanophilin. The mitogen-activated protein kinase pathway may also be involved, because interruption of ERK phosphorylation by GILZ increases ENaC surface expression.

Considerably more is known about how ENaC complexes are retrieved from the apical membrane. This understanding is the direct result of dissecting the molecular consequences of Liddle’s syndrome, in which mutations in the C terminus of ENaC lead to increased residence time in the apical membrane. The missing or mutated domains in the β- or γ-subunit of ENaC in this syndrome normally bind to Nedd4-2, a ubiquitin ligase, which ultimately is responsible for initiating endocytosis and degradation. The interaction of Sgk1 and Nedd4-2 in the actions of aldosterone is discussed later. ENaC is internalized via clathrin-coated vesicles and processed into early endosomes, then further processed into recycling endosomes and late endosomes. Degradation is via lysosomes or proteasomes. The processing of ENaC by vesicular trafficking and its regulation by aldosterone has been reviewed.

Phosphatidylinositol-3-kinase (PI3K)–dependent signaling is essential for epithelial Na ⁺ transport. It controls SGK1 activity (addressed later) and also appears to have independent effects on ENaC open probability through direct actions of 3-phosphorylated phosphoinositides, particularly phosphatidylinositol (3,4,5) trisphosphate. Ras-dependent signaling may also regulate ENaC and the pump in complex ways that depend on downstream signaling through Raf, MEK and ERK, as well as through PI3K.

The late phase of ENaC activation by aldosterone is less well understood than the early phase. A simple evaluation of the late phase is that aldosterone increases the transcription and protein abundance of the ENaC subunits. This idea comes from the fact that aldosterone increases the mRNA and protein abundance of α-ENaC in the kidney after a lag of several hours. However, whereas aldosterone produces an increase in α-subunit expression in the kidney, it produces an increase of β- and γ-subunits in the colon. Dietary Na ⁺ restriction, a physiologically relevant maneuver that increases aldosterone secretion, clearly increases ENaC surface expression in the renal distal nephron. However, there appear to be some important differences between chronic aldosterone administration to an Na ⁺ -replete animal and chronic dietary Na ⁺ restriction. The role of increased expression of α-ENaC in the long-term actions of aldosterone has been questioned, because overexpression of this subunit does not increase ENaC activity in models of collecting duct and lung epithelia. It appears that increased expression of α-ENaC may be important for the consolidation of the increase, but it is not sufficient to reproduce the steroid-mediated increase in ENaC activity.

Basolateral Membrane Effects of Aldosterone

Over the years, research on aldosterone action has focused with varying degrees of intensity on apical effects, basolateral effects, and effects on metabolism. There is general agreement now that the early effects of aldosterone are on apical events, primarily on ENaC, and that basolateral and metabolic effects come later. In addition, although it is somewhat less settled whether the basolateral effects are direct or result indirectly from the enhanced entry of Na ⁺ into cells, the bulk of evidence favors the latter view. Notably, increased Na ⁺ entry has been found to control more than 80% of increased Na ⁺ -K ⁺ -ATPase activity and basolateral membrane density in the rat and rabbit cortical collecting tubule. Furthermore, striking increases in basolateral membrane folding and surface area occur in aldosterone-treated animals, an effect that is markedly attenuated in animals fed a low-Na ⁺ diet. This result strongly suggests that apical Na ⁺ entry is required for basolateral changes to occur. However, good evidence exists for direct transcriptional stimulation of Na ⁺ -K ⁺ -ATPase subunit expression, as well as reports supporting some direct effects of aldosterone in increasing basolateral pump activity or at least in constituting the pool of latent pumps, which are then recruited to the basolateral membrane in response to a rise in intracellular [Na ⁺ ].

Activation of the Epithelial Sodium Channel by Proteolytic Cleavage

There is now clear evidence that when ENaC is delivered to the apical membrane, it can be activated by proteolytic cleavage. The first hint of this process was the demonstration that rats fed a low-Na diet or given aldosterone over the long term showed the appearance of a proteolytic fragment of the γ-ENaC subunit. Subsequently, investigators have shown that both the α- and the γ- but not the β-ENaC subunits can be cleaved. Furthermore, cleavage at each site apparently initiates a degree of activation of the channel complex. ENaC complexes are activated by cleavage because specific regions of the large extracellular domain (26 residues in the α-ENaC and 46 residues in the γ-ENaC) are excised. These regions contain inhibitory sequences that, when introduced exogenously, can inhibit ENaC function. Removing these regions by proteolytic cleavage releases this inhibition.

Several proteases can cleave either the α- or γ-ENaC subunits. Among them are furin, prostasin, CAP2, kallikrein, elastase, matriptase, plasmin, and trypsin. It is not clear whether activation of ENaC by proteolytic cleavage can be regulated by aldosterone, but the idea certainly has attractive features. Were aldosterone able to regulate expression of one or more rate-limiting proteases, it would be able to regulate both the number of complexes in the apical membrane and the ability of the channel complex to be active. It appears that aldosterone may regulate the expression of prostasin. Aldosterone may also regulate the expression of the protease, nexin-1 (an inhibitor of prostasin), and other proteases.

The discovery of ENaC activation by cleavage helps to explain how aldosterone might increase ENaC activity by increasing both surface expression and the activity of a single ENaC complex. By phosphorylating Nedd4-2 via SGK1 and reducing its ability to bind to the PY domains of the ENaC subunits, aldosterone increases ENaC residence time on the apical membrane. This additional time permits proteolytic activation by one or more endogenous proteases.

Potassium Secretion and Aldosterone

One of the major effects of aldosterone is to increase K ⁺ secretion (and thus excretion). This phenomenon has been demonstrated in countless patients with aldosterone-secreting tumors and in hundreds of studies in animals given excess amounts of aldosterone. The general mechanism whereby aldosterone increases K ⁺ secretion is depicted in Figure 12.7 . The key feature of this process involves the increased absorption of Na ⁺ via ENaC. The dependence of K ⁺ secretion on Na ⁺ absorption is the basis of the action of the “K-sparing” diuretics, amiloride and triamterene, both of which inhibit ENaC. These drugs have no direct effect on the apical K ⁺ channels.

Increasing ENaC activity produces two major secondary effects that, in turn, enhance K ⁺ secretion. First, the enhanced Na ⁺ conductance of the apical membrane produces depolarization of that membrane and hence a more favorable electrical driving force for K ⁺ efflux into the lumen. The second effect relates to the activity of the Na ⁺ -K ⁺ pump on the basolateral membrane. The more Na ⁺ that enters across the apical membrane, the more that must be extruded by the pump. Since the stoichiometry of the pump is constant (3Na ⁺ and 2K ⁺ ), more K ⁺ enters the cell. In isolated, perfused cortical collecting ducts, the amount of secreted K ⁺ is highly related to the amount of absorbed Na ⁺ when the stimulus for Na ⁺ absorption is mineralocorticoid hormone.

Two kinds of K ⁺ channels are found in the apical membrane of the ASDN: small conductance (SK, 30 to 40 picosiemens) channels encoded by the ROMK gene, and large conductance (BK, 100 to 200 picosiemens) channels found in many kinds of cells, including the apical membrane of the colon. The majority of the K ⁺ channels on the apical membrane of the principal cells appear to be SK, at least as far as can be assessed by patch clamp analysis. The activity of either channel is not increased by aldosterone. ^.

A feature of K ⁺ secretion is that although apical K ⁺ channels are abundant in the proximal portion of the ASDN (connecting tubule and cortical collecting duct), they are strikingly less abundant in the medullary collecting duct. Because apical K channels are not regulated by aldosterone, their absence in the medullary collecting duct might uncouple aldosterone-regulated Na ⁺ reabsorption from K ⁺ secretion in this segment.

Recent advances in understanding genetic forms of hypertension have uncovered an important role for a family of kinases that lack an otherwise conserved lysine residue in the catalytic domain. These kinases, called WNK ( w ith n o lysine; K is the one-letter code for lysine), have potent effects on pathways regulating Na ⁺ and K ⁺ transport in the distal nephron. An important feature of one member of this family, WNK4, is its ability to modulate the activity of ROMK. The activity of ROMK can be regulated via WNK4 by signaling events activated by aldosterone but modulated by serum [K ⁺ ], Na balance via angiotensin II, and SGK1 activity. The importance of this synthesis is that it provides a molecular mechanism for the collecting duct separately to regulate Na ⁺ absorption via ENaC and K ⁺ secretion via ROMK.

Separation of Sodium Absorption and Potassium Secretion by the Aldosterone-Sensitive Distal Nephron

The preceding sections establish a picture that parsimoniously accounts for the effect of aldosterone to stimulate, pari passu, Na ⁺ reabsorption and K ⁺ secretion. The simple stimulation of electrogenic Na ⁺ reabsorption (via ENaC) is sufficient to stimulate K ⁺ secretion, which fits well for organisms faced with a combined low-Na ⁺ , high-K ⁺ diet, which was maintained most of the time through millions of years of vertebrate evolution. However, organisms do not ingest a fixed amount of Na ⁺ and K ⁺ , so inexorable linkage between Na ⁺ absorption and K ⁺ secretion by the ASDN cannot possibly occur all the time. Investigators have proposed several possibilities to explain how these processes can be separated.

Differential Regulation of Intercalated Cell Mineralocorticoid Receptor

A recent study has highlighted a sixth possible mechanism allowing distinct responses to volume depletion versus hyperkalemia. The central feature of this proposed regulation is differential phosphorylation of the MR LBD in intercalated cells, as shown schematically in Figure 12.8 . When phosphorylated at S843 in the LBD, MRs cannot bind aldosterone or cortisol and thus cannot be activated. This phosphorylation, which is stimulated by hyperkalemia, occurs selectively in intercalated cells but not principal cells. Angiotensin II, on the other hand, induces S843 dephosphorylation in intercalated cells, markedly increasing ligand binding and, therefore, activation. Intercalated cells are known predominantly to mediate H ⁺ transport; however, recent studies have implicated them in electroneutral NaCl transport via the combined actions of Na ⁺ -dependent Cl ⁻ -HCO ₃ ⁻ exchanger (NDCBE) and the apical Cl ⁻ -HCO ₃ ⁻ exchanger, pendrin. Thus, when MR is active in these cells (S843 dephosphorylated), electroneutral NaCl transport occurs, without enhancing the driving force for K ⁺ secretion. Since intercalated cells lack 11β-HSD2, under these conditions, it is cortisol that binds to and activates MR. When intercalated cell MR is inactive (S843 phosphorylated), aldosterone acts in principal cells to stimulate ENaC-dependent electrogenic Na ⁺ transport, which enhances K ⁺ secretion.

The role of mineralocorticoid receptor (MR) ligand/hormone-binding domain (LBD) phosphorylation in controlling chloride reabsorption by intercalated cells.

When phosphorylated at Ser-843 in the LBD, MR cannot bind ligand and hence cannot be activated. This phosphorylated state of MR is found only in intercalated cells, not in neighboring principal cells. In states of volume depletion, elevated angiotensin II decreases MR phosphorylation at Ser-843, allowing activation. In intercalated cells, MR mediates stimulation of both the proton pump and Cl ⁻ -HCO ₃ ⁻ exchangers, thereby increasing Cl ⁻ reabsorption and promoting increased plasma volume while inhibiting K ⁺ secretion. In contrast, in states of hyperkalemia, phosphorylation of Ser-843 is increased, and hence Cl ⁻ reabsorption by intercalated cells is decreased, and principal cell-dependent K ⁺ secretion is increased.

(Reprinted with permission from Shibata S, Rinehart J, Zhang J, et al: Regulated mineralocorticoid receptor phosphorylation controls ligand binding and renal response to volume depletion and hyperkalemia. Cell Metab 18[November 5]:660-671, 2013.)

Aldosterone-Independent ENAC-Mediated Sodium Reabsorption in the Distal Nephron

The term aldosterone-sensitive distal nephron emphasizes the primacy of this key steroid in the control of ion transport in this region of the nephron. However, ENaC activity and aldosterone sensitivity exhibit axial heterogeneity from the late distal convoluted tubule (DCT2) through the connecting tubule to the cortical collecting duct and, finally, to the medullary collecting duct. In mice on a standard salt diet, total ENaC expression increases with progression from the DCT2 to the connecting tubule, although ENaC apical localization and activity are higher in the DCT2. Only under conditions of low-sodium diet or aldosterone administration does the primacy of the connecting tubule in particular, and to a lesser extent the cortical collecting duct, emerge. The total luminal surface area in the DCT2 and connecting tubule is several-fold higher than in the cortical collecting duct, and together these two segments appear to be sufficient to maintain sodium balance, even in the absence of detectable ENaC along the collecting duct. Mice lacking ENaC selectively in the collecting duct come into balance, even on a low-sodium diet. Congruent with these findings, deletion of α-ENaC from the DCT2, connecting tubule and collecting duct results in severe sodium wasting. Notably, it is the connecting tubule that appears to be most important in the response to aldosterone, while DCT2 has the highest baseline transport in the absence of MR activation. The cortical collecting duct is not as critical as was originally thought for either baseline or aldosterone-stimulated sodium reabsorption, probably due to its smaller surface area compared with the DCT2 and connecting tubule.

As we continue to traverse the nephron, further sodium reabsorption is minimal in the medullary collecting duct, under standard sodium diet conditions, and not significantly stimulated by aldosterone.

Sites of Mineralocorticoid Receptor Expression and Locus of Action Along the Nephron

Aldosterone-Sensitive Distal Nephron

In the kidney, MR is expressed at the highest levels in distal nephron cells extending from the last third of the DCT through the medullary collecting duct, which is frequently referred to as the aldosterone-sensitive distal nephron (ASDN) ( Figure 12.9 ). This pattern of expression was first demonstrated using labeled hormone–binding studies performed before the cloning of MR and has been confirmed since by several methods, including polymerase chain reaction, in situ hybridization, and immunohistochemical analysis. Effects of aldosterone on electrogenic Na ⁺ and K ⁺ transport in principal cells have been found consistently in these nephron segments, which also express ENaC, and 11β-HSD2, as addressed in detail earlier. Collecting duct intercalated cells also express MR and respond specifically to aldosterone and alter proton secretion. Aldosterone directly increases the activity of the H ⁺ -ATPase in the collecting duct, and its absence results in decreased proton secretion. Interestingly, nongenomic stimulation of H ⁺ -ATPase activity in type A intercalated cells has been demonstrated in isolated murine collecting ducts. Consistent with these effects, aldosterone deficiency results in distal renal tubular acidosis type 4, and excess aldosterone results in metabolic alkalosis. It should be noted that aldosterone also stimulates H ⁺ secretion due to effects on principal cell Na ⁺ transport, which alter the electrical gradient. These older studies must also now be interpreted in the context of more recent data, which, as noted earlier, demonstrate that the effect of aldosterone on intercalated cells depends on the genesis of the signal.

Expression and/or activity of the mineralocorticoid-dependent transport machinery in principal cells along the mature aldosterone-sensitive distal nephron (ASDN).

Mineralocorticoid specificity is conferred by the presence of the mineralocorticoid receptor (MR) and 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) beginning primarily from the latter part of the distal convoluted tubule (DCT). The thiazide-sensitive sodium-chloride cotransporter (NCC) is expressed exclusively in the DCT, but after the transition from the DCT to the connecting tubule (CNT), sodium reabsorption is distinctly determined by amiloride-sensitive sodium channel (ENaC) activity. ENaC activity is strongest in the CNT and decreases down to the inner medulla collecting duct. Variation in gene expression or activity along the nephron is indicated by the intensity of shading. Note that there is some variation in gene expression from mouse to human. However, the machinery for sodium reabsorption in the ASDN is predominantly conserved across species. Each nephron segment is drawn to scale, but expression of channels/transporters in intercalated cells is omitted. Expression and/or activity is based on messenger RNA, protein, and biochemical studies. G, Glomerulus; PCT, proximal convoluted tubule; ROMK channel, renal outer medullary potassium; SGK1, serum- and glucocorticoid-regulated kinase 1.

(Adapted from Loffing J, Korbmacher C: Regulated sodium transport in the renal connecting tubule [CNT] via the epithelial sodium channel [ENaC]. Pflugers Archiv 458[1]:111-135, 2009.)

Colon

Under physiologic conditions, approximately 1.3 to 1.8 L of electrolyte-rich fluid is reabsorbed per day from the colonic epithelium, which accounts for about 90% of the salt and water that enter the proximal colon from the terminal ileum. In nonmammalian vertebrates, sodium conservation by the colon plays an even more significant role. This transport is regulated by several transporters and channels, including ENaC. Like the nephron, the proximal colon reabsorbs sodium via an electroneutral, ENaC-independent process. In the distal colon, electrogenic Na ⁺ absorption via ENaC channels is the predominant mode of sodium transport. In disease states such as inflammatory bowel disease, ENaC-mediated sodium reabsorption can be reduced, although in diarrheal states, elevated aldosterone levels may attenuate sodium and water loss from the colon. It should be noted that in the colon, as in the distal nephron, MR signaling is aldosterone selective, reflecting the activity of 11β-HSD2. Aldosterone increases electrogenic sodium absorption and potassium secretion and inhibits electroneutral absorption. This is in contrast to glucocorticoids, which, at higher concentrations, activate GR to stimulate electroneutral absorption in the proximal and distal colon. As in the distal nephron, the aldosterone response can be characterized by an early and late response. The early response gene, SGK1, is upregulated by aldosterone via MR. However, in contrast with the kidney, aldosterone and a low-salt diet have been shown to stimulate transcription of β- but not α-ENaC in rat models.

Aldosterone stimulates electrogenic potassium secretion from colonic epithelia. The significance of this secretion is evident in anuric patients. Potassium secretion from the colon is much higher in patients undergoing long-term hemodialysis than in patients not undergoing dialysis. Indeed, administration of fludrocortisone, a mineralocorticoid agonist, to dialysis patients has been shown to reduce hyperkalemia in small clinical trials. Low doses of the common MR antagonist spironolactone do not result in significant hyperkalemia.

Lung

Vectorial transport of salt and water across the distal airway epithelium (ciliated Clara cells, nonciliated cuboidal cells) and alveoli (type I and type II alveoli) primarily determines fluid clearance from the lung. ENaC is the rate-limiting step in sodium transport in the lung and plays a primary role in several physiologic and pathophysiologic conditions determined by fluid clearance. At birth, the lung assumes a resorptive phenotype, and lack of functional ENaC channels leads to neonatal respiratory distress syndrome in mouse knockout models. In children, lack of functional ENaC (e.g., autosomal recessive pseudohypoaldosteronism type I) results in increased rates of recurrent infection due to increased airway liquid. In the mature lung, defective ENaC channels can lead to pulmonary edema and pathologic conditions (e.g., acute respiratory distress syndrome and high-altitude pulmonary edema ). Conversely, hyperab­sorption through ENaC is emerging as an important mechanism of decreased mucus clearance in cystic fibrosis.

The molecular apparatus for mineralocorticoid-stimulated liquid reabsorption via ENaC (concomitant MR and 11β-HSD2) is present in late gestational and mature adult lung in humans and rats, and there is some evidence for a significant physiologic role of aldosterone in ENaC-mediated sodium transport, although glucocorticoids acting via GR are likely to play the predominant role in lung. Importantly, glucocorticoids, but not mineralocorticoids, play a critical role in lung maturation in humans, and GR knockout mice, like α-ENaC knockout mice, die of respiratory insufficiency within hours of birth. In contrast, MR knockout mice demonstrate a severe salt-wasting phenotype but no significant lung phenotype.

Exocrine Glands and Sensors

ENaC-mediated sodium reabsorption is also measurable in salivary and sweat glands. The importance of these tissues for sodium and water homeostasis is underscored by rare genetic mutations that confer elevated plasma aldosterone levels and pseudohypoaldosteronism with normal renal tubular function but significant sodium loss from salivary or sweat glands. ENaC channels also play an important role in transduction of sodium salt taste in the anterior papillae of the tongue. The appropriate molecular machinery for mineralocorticoid-responsive sodium reabsorption is expressed in these organs, and these epithelia are model systems for the study of ENaC regulation. As in colonic epithelia, aldosterone stimulates expression of β- and γ-ENaC and sodium transport in glands and taste buds in animal models. Moreover, in humans, changes in dietary sodium are inversely proportional to sodium transport across salivary epithelia. As in the aldosterone-responsive distal nephron and distal colon, sodium uptake is coupled with potassium secretion in salivary epithelia. This effect is evident in humans with hyperaldosteronism. Such patients have a salivary [Na ⁺ ]/[K ⁺ ] ratio significantly lower than that of subjects without the disorder, although this has not been accepted as a valid means to screen for hyperaldosteronism.

Induction of SGK1 by Aldosterone

In the early to mid-1960s, primarily from the work of Isidore Edelman and colleagues, it became clear that aldosterone, like cortisol, exerted most, if not all, of its key physiologic effects by altering transcription rates of a specific subset of genes. In particular, hormone-induced changes in gene transcription were shown to be essential for its effects on epithelial Na ⁺ transport. Attention focused first on enzymes of intermediary metabolism, particularly citrate synthase, and after the cloning of the transporters involved in Na ⁺ translocation (Na ⁺ -K ⁺ -ATPase and ENaC), these were also found to be regulated by aldosterone at the transcriptional level. However, these effects are manifest several hours after most of the change in Na ⁺ transport has already occurred and hence could not explain the early and greatest proportion of effects of aldosterone. Considerable effort by many groups went into unbiased screening for aldosterone-regulated proteins and later aldosterone-regulated mRNAs (reviewed in Verrey ). In 1999, SGK1 was identified as the first early-onset aldosterone-induced gene product, which clearly stimulates ENaC-mediated sodium reabsorption in the distal nephron without pleiotropic effects on other cellular processes. The physiologic relevance of SGK1 is now firmly established, and investigations by numerous laboratories into its mechanism of action have revealed critical general features of the mechanism underlying hormone-regulated ion transport: it is therefore addressed in some detail here. SGK1 mRNA levels are increased within 15 minutes, and protein levels within 30 minutes, in cultured cells upon stimulation by aldosterone and in the collecting duct by aldosterone or a low-salt diet (a physiologic stimulus for aldosterone secretion) in vivo. Notably, SGK1 is increased more abundantly in kidney cortex (the connecting tubule and cortical collecting duct) than in medulla, which corresponds to the potency of ENaC activation in these nephron segments. SGK1 is expressed in other nephron segments, including glomeruli, proximal tubule, and papillae ; however, its rapid induction specifically in the ASDN appears to provide most of the basis for its role in aldosterone-regulated sodium and potassium transport. It is of interest that SGK1 is highly expressed in glomeruli and inner medulla and papillae in the absence of aldosterone; recent data are consistent with the idea that its inner medullary induction is related to its role in osmotic responses.

Molecular Mechanisms of SGK1 Action in the Aldosterone-Sensitive Distal Nephron

SGK1 is a serine/threonine kinase of the AGC protein kinase superfamily, and its kinase activity appears to be essential for Na ⁺ transport regulation. Although it has effects on proliferation and apoptosis in kidney cells, these effects appear to be minor, and the control of ENaC and other transporters predominates. SGK1 transcription is induced by a variety of stimuli in addition to aldosterone. As its name implies, these include serum and glucocorticoids, but also follicle-stimulating hormone, transforming growth factor–β, and hypotonic and hypertonic stress. Of these, osmotic regulation is of the clearest physiologic relevance.

SGK1 is interesting as a signaling kinase in that both its expression level and its activity are highly regulated. Regulation of the former occurs primarily through effects on gene transcription, although protein stability is also regulated, whereas regulation of the latter occurs through phosphorylation. Like that of its close relative Akt, SGK1 phosphorylation is stimulated by a variety of growth factors including insulin and insulin-like growth factor-1, which act through PI3K to trigger phosphorylation at two key residues, an activation loop (residue T256) and a hydrophobic motif (S422). Both of these phosphorylation events appear to be PI3K dependent. Specifically, the α-isoform of the p110 subunit of PI3K stimulates PI3K-dependent kinase 1 and mTORC2 (PDK2), respectively. Recent evidence has established that PDK2 is in fact the mammalian target of rapamycin [mTOR] in its complex 2 variant. mTORC2 also uses a co-factor, SIN1, to specify activation of SGK1 rather than related family members such as Akt. In turn, the upstream kinases, PI3K-dependent kinase 1 and mTORC2, phosphorylate and thereby activate SGK1 kinase, which is required for its stimulation of ENaC. Thus, SGK1 serves as a convergence point for different classes of stimuli, which act on the one hand to control its expression (aldosterone) and on the other to control its activity (insulin), which results in the coordinate regulation of ENaC.

In the study of the physiologic and pathophysiologic role of SGK1 in the ASDN, mice lacking SGK1 have provided considerable insight. Unlike MR knockout mice, mice lacking SGK1 survive the neonatal period and appear normal when consuming a diet with normal salt levels, although their aldosterone levels are markedly elevated. When subjected to a low-salt diet, these mice have a profound sodium-wasting phenotype (pseudohypoaldosteronism type I). Notably, this is a significantly milder phenotype than in the MR knockout. This comparison suggests that disruption of SGK1 signaling may be insufficient to eliminate aldosterone-mediated sodium transport due to additional aldosterone-induced and aldosterone-repressed proteins that could compensate for the lack of SGK1. Other early aldosterone-induced genes, including K-ras, GILZ, kidney-specific WNK1, Usp45, melanophilin, and promyelocytic leukemia zinc finger, have also been implicated in the stimulation of ENaC. Their distinct mechanisms of action have not been studied in vivo and are beyond the scope of this chapter.

Alternatively, SGK1 may play a more significant role in states of aldosterone excess or upregulation of hormonal activators of SGK1 (e.g., insulin). Indeed, SGK1 knockout mice are protected from the development of salt-sensitive hypertension, which accompanies the hyperinsulinemia of the metabolic syndrome. Despite its accepted role as a mediator of aldosterone-stimulated sodium reabsorption, the mechanisms by which SGK1 stimulates ENaC are not fully characterized. Several mechanistic studies have demonstrated that SGK1 is rapidly induced but also rapidly degraded. The N terminus of the kinase, which distinguishes SGK1 from other kinase family members (e.g., Akt), is important for stimulation of sodium transport but is also the target for rapid degradation of the kinase via the ubiquitin-proteasome system. In addition, several naturally occurring variants of SGK1 possess distinct N termini that modify the ability to stimulate ENaC and to be degraded. The pathophysiologic implications of the N terminus for sodium transport are unclear, but they may involve a negative feedback loop to limit sodium reabsorption in states of hypertension.

The molecular mechanisms of ENaC stimulation by SGK1 can be divided into three known categories ( Figure 12.10 ): (1) posttranslational effects on the E3 ubiquitin ligase Nedd4-2, (2) posttranslational Nedd4-2–independent effects, and (3) transcription of gene products such as α-ENaC.

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