Mineralocorticoid Action in the Aldosterone Sensitive Distal Nephron

The Aldosterone Sensitive Distal Nephron (ASDN)

Each day, the glomeruli of the human kidney filter about 1.5 kg of NaCl out of the blood into the primary urine. However, usually less than 1% of the filtered NaCl is finally excreted. Thus, most of it is reabsorbed along the renal tubular system, which is composed by serial arrangement of the proximal tubule (PCT and PST), the thin limb (TL), the thick ascending limb (TAL), the distal convoluted tubule (DCT), the connecting tubule (CNT), and the collecting duct (CD) ( Figure 35.1 ). While the bulk of Na ⁺ reabsorption occurs in the proximal tubule (PT) and the TAL, the final regulation of Na ⁺ excretion takes place in the so-called Aldosterone-Sensitive Distal Nephron (ASDN) which extends from the late portion of the DCT (also called DCT-2) to the end of the collecting duct (IMCD) in the renal papilla. The three ASDN segments differ in their histotopographical localization and epithelial structure. The DCT and CNT are located in the cortical labyrinth, while the CD extends from the medullary rays in the renal cortex down to the renal medulla until its opening on the tip of the renal papilla. DCT cells have an apical localized cell nucleus and numerous mitochondria that are densely stuffed into the interdigitating basolateral membrane infoldings. The CNT cells are usually less tall than the DCT cells, have less mitochondria and a basal membranous labyrinth that is predominantly formed by membrane infoldings. The CCD cells are even smaller, and are characterized by a basal thin rim of mitochondria-free membrane infoldings. The medullary CD is formed by cuboidal cells that have few cell organelles ( Figure 35.1 ). Membrane infoldings and mitochondrial density progressively decrease from DCT to CCD, in parallel with a reduction in the abundance and activity of the Na ⁺ ,K ⁺ -ATPase, suggesting different ion transport rates along the ASDN. In fact, recordings of transepithelial Na ⁺ fluxes in isolated rabbit CNTs and rat CCDs, indicate at least a 10-fold higher Na ⁺ transport rate in the CNT than in the CCD (600–120 versus 0.2 to 24 pmoles Na ⁺ /min/mm). The rates of Na ⁺ transport in OMCD and IMCD are generally low, but Na ⁺ channels are also expressed in the OMCD and IMCD, and can be activated, at least to some extent, by aldosterone.

Although the sequence of tubule portions appears to be quite similar in mammals, subtle species differences in the structural and functional organization of the ASDN have to be taken into account if functional data from different nephron portions and species are compared. For example, rabbits have morphologically well-defined sharp transitions from DCT to CNT and from CNT to CD, while the segment transitions in rats, mice, and humans are gradual. These morphological differences are reflected by the distribution pattern of ion- and water-transporting proteins. In rabbits, the sharp transitions from DCT to CNT and from CNT to CCD coincide with the immediate replacement of the DCT-specific NaCl co-transporter (NCC) by the epithelial Na ⁺ channel (ENaC), and the abrupt onset of aquaporin-2 (AQP2) expression. In contrast, in rats, mice, and humans NCC and ENaC overlap in the late DCT (DCT2), and AQP2 extends into the connecting tubule.

Despite the above-mentioned segmental and species differences, all ASDN cells in common express high levels of the mineralocorticoid receptor (MR), the glucocorticoid receptor (GR), the enzyme 11-beta-hydroxysteroid-dehydrogenase type 2 (11β-HSD2), and the epithelial Na ⁺ channel (ENaC) ( Figure 35.2 ). The MR and the GR are hormone-activated transcription factors that can bind both mineralocorticoid and glucocorticoid hormones, although with different affinities. While the MR is a high-affinity receptor (K _d for aldosterone and cortisol in the range from 0.5 to 3 nM), the GR has a more than 10 times lower affinity for these steroids (K _d in the range from 20 to 65 nM). The corticosteroid levels in the plasma (cortisol in humans and corticosterone in rats) are usually far above these K _d values, and exceed the plasma concentrations of aldosterone 100–1000-fold. Under these conditions, mineralocorticoid specificity is thought to be conferred to the ASDN cells by their high expression of 11β-HSD2 that rapidly hydrolyzes the physiological corticosteroids to inactive metabolites. Upon hormone binding, the receptors are thought to homo- or heterodimerize and to translocate to the cell nucleus, where they modulate a transcriptional program that modulates the expression of ENaC, the Na ⁺ ,K ⁺ -ATPase, and of regulatory proteins that control the activity of these Na ⁺ -reabsorbing proteins.

Na ⁺ transport across the ASDN cells occurs in two steps. Na ⁺ uptake from the lumen into the cells is mediated by ENaC, while Na ⁺ exit across the basolateral plasma membrane depends on the Na ⁺ ,K ⁺ -ATPase. The activity of ENaC and the Na ⁺ ,K ⁺ -ATPase is electrogenic, and generates a transepithelial electrochemical gradient that drives paracellular Cl ⁻ reabsorption and transcellular K ⁺ secretion. The apical K ⁺ secretion is thought to occur via secretory K ⁺ channels such as the renal outer medulla K ⁺ channel (ROMK) and the flow-dependent maxi-K (BK) channel. Immunohistochemical studies revealed that ROMK is highly abundant in the DCT, CNT, and CD cells, where it becomes activated and redistributed to the apical plasma membrane in response to an increased dietary K ⁺ intake. Likewise, a high-K ⁺ diet appears to stimulate the BK channel. In rabbits, it increases the mRNA expression of the α-, β2-, and β4-subunits, and in rats it elevates the electrical activity in CCD cells. In contrast to ROMK, the BK channel is not only present in the segment-specific ASDN cells, but also in intercalated cells. Intercalated cells may not only secrete but also reabsorb K ⁺ via their apical H ⁺ ,K ⁺ -ATPase. Thereby, intercalated cells may help to modulate the interdependence of ENaC-mediated Na ⁺ reabsorption and K ⁺ secretion via apical K ⁺ channels. A close link between the function of intercalated and segment-specific ASDN cells is also indicated by the fact that, in all species investigated so far, the appearance of intercalated cells in the early ASDN coincides precisely with the onset of functional ENaC expression. Moreover, several recent studies on various rodent models further supported the idea of a functional cooperation between ASDN and intercalated cells. The ASDN has at least two types of intercalated cells. The acid-secreting type-A intercalated cells have the vacuolar H ⁺ -ATPase in the apical plasma membrane and the Cl ⁻ /HCO ₃ ⁻ anion exchanger AE1 in the basolateral plasma membrane, whereas the bicarbonate-excreting type-B intercalated cells have the H ⁺ -ATPase in the basolateral plasma membrane, and are characterized by the abundance of the Cl ⁻ /HCO ₃ ⁻ exchanger pendrin in the apical membrane, which may contribute to a combined transcellular reabsorption of Na ⁺ (via ENaC) and Cl ⁻ (via pendrin). In fact, experiments in pendrin-deficient mice indicated that bicarbonate secretion via the type-B intercalated cells is necessary for normal ENaC expression and activity. Consistent with this view, the mice are prone to develop arterial hypotension when placed on an NaCl-deficient diet, and they are protected from hypertension under mineralocorticoid treatment. Pendrin may not only cooperate with ENaC, but also with the Na ⁺ -dependent chloride-bicarbonate exchanger SLC4A8, which may account for the previously reported thiazide-sensitive NaCl reabsorption in the renal collecting system. Experiments in SLC4A8- and NCC-deficient mice indicated that about 40–70% of collecting duct Na ⁺ -absorption involve SLC4A8. Similar to ENaC, the thiazide-sensitive electroneutral Na ⁺ reabsorption, as well as SLC4A8, appear to be stimulated by aldosterone. Na ⁺ reabsorption via ENaC may not only interfere with K ⁺ , H ⁺ , and Cl ⁻ handling of the ASDN cells, but also with transepithelial water reabsorption. The ENaC activity generates an osmotic gradient that likely favors the vasopressin-dependent transepithelial water reabsorption via the water channels AQP2 in the apical and AQP3 and AQP4 in the basolateral plasma membrane of the segment-specific ASDN cells. Although there are some indications that aldosterone directly controls the expression and activity of ROMK, BK, pendrin, SLC4A8, AQP2, and AQP3, the functional significance of these regulations compared with the indirect effects mediated via enhanced activity of ENaC and Na ⁺ ,K ⁺ -ATPase are still unclear. Nevertheless, the various cell types and ion transport pathways with manifold possible interactions likely contribute significantly to the complexity of ion handling along the ASDN which may allow adaptation of urinary ion excretion precisely to homeostatic needs. Details on the regulation of K ⁺ channels, pendrin, SLC4A8, and aquaporins are given in several excellent recent reviews.

Aldosterone Action in Non-ASDN Cells

A huge variety of studies have been aimed at localizing MR and GR along the mammalian nephron. Ligand-binding studies and RNAase protection assays on microdissected nephron portions, as well as autoradiography, in situ hybridization and immunohistochemistry on kidney sections, revealed a very high abundance of MR and GR along the classical ASDN. Moreover, these studies indicated that the MR, the GR, and 11β-HSD2 are expressed in nephron segments and cell types that are considered not to be classical targets for aldosterone, including glomerular mesangial cells, podocytes, and proximal tubules (reviewed in ). Using a set of highly specific MR antibodies, Ackerman et al. recently confirmed that the MR is expressed along the entire distal tubule, including the TAL and the entire DCT. MR expression in the TAL is in line with studies describing a profound stimulatory effect of aldosterone on TAL Na ⁺ transport, but contrasts with other reports that did not reveal any effect of the mineralocorticoid on the expression and activity of the Na ⁺ ,K ⁺ -ATPase and K ⁺ transport in the TAL of adrenalectomized rabbits and rats, respectively. Likewise, there is considerable evidence that aldosterone stimulates NaCl co-transport in the DCT. Velazquez et al . demonstrated that adrenalectomy lowers the electroneutral Na ⁺ transport activity (corresponding to NCC activity) in the DCT, and that this effect can be fully restored to normal when aldosterone and/or glucocorticoids are replaced. Consistent with a stimulatory role of aldosterone on the DCT, immunoblotting and immunohistochemistry on rat kidneys showed that dietary Na ⁺ restriction, which stimulates endogenous aldosterone production, as well as exogenous aldosterone application, increases the abundance and phosphorylation of NCC in the DCT. Frindt and Palmer used in vivo cell surface biotinylation to show in the rat that dietary Na ⁺ restriction does also increase the cell surface abundance of NCC. Conversely, dietary Na ⁺ -loading removes NCC from the plasma membrane, indicating that aldosterone may affect the trafficking of NCC as well. Interestingly, the increase in NCC protein abundance and phosphorylation was significantly lower when MR was blocked by spironolactone. Although the effect of spironolactone points to an MR-mediated transcriptional response, changes in plasma aldosterone do not appear to directly affect NCC mRNA expression, indicating that mineralocorticoids elicit their effects on NCC abundance via enhanced translation and/or reduced degradation of the protein. In fact, there is increasing evidence that NCC protein abundance might be controlled by ubiquitylation, and hence by altered protein stability. Aldosterone is also known to stimulate renal H ⁺ excretion. The intercalated cells in the ASDN play an important role for renal acid–base handling. However, so far it is unclear whether aldosterone directly affects renal acid secretion via activation of the H ⁺ -ATPase in intercalated cells or indirectly via activation of ENaC-dependent Na ⁺ transport in principal cells, which increases the electrochemical driving force for H ⁺ secretion by intercalated cells. Functional studies on isolated collecting ducts indicated that aldosterone rapidly stimulates intercalated cells via non-genomic pathways that may involve PKC and PKA signaling, and perhaps require the vasopressin V1 receptor. Nevertheless, it is uncertain whether the observed effects depend on a yet-unknown putative membrane-bound corticosteroid-binding receptor or the classical MR. Although expression of the MR in intercalated cells was long disputed, a recent immunohistochemical study established rather strong expression of the MR, not only in the ASDN segment-specific cells, but also in all subtypes of intercalated cells. However, this study did also provide evidence that the rather low expression levels of the 11β-HSD2 in TAL, early DCT, and intercalated cells, makes it unlikely that aldosterone has significant direct genomic effects on these cell types in the physiological context of several-fold higher plasma levels of corticosterone than aldosterone. Nevertheless, mineralocorticoid-selectivity might be conferred to the MR also in an 11β-HSD2-independent way by a functional preference of the receptor to aldosterone, as indicated in cell culture assays and in vivo experiments. Moreover, MR expression in TAL, early DCT, and intercalated cells may become relevant in patients with pharmacological (e.g., by spironolactone) or genetic (i.e., PHA-1) MR inactivation. Under these conditions, the loss of MR function in these cells may contribute to the salt-losing phenotype with hyperkalemia and metabolic acidosis.

Aldosterone and the Epithelial Sodium Channel ENaC

The apical entry of Na ⁺ involves the low conductance, highly selective (Na ⁺ over K ⁺ ), and amiloride-sensitive epithelial Na ⁺ channel (ENaC). ENaC was cloned from rat distal colon by functional expression in Xenopus oocytes. The channel is composed of three homologous subunits denoted α-, β-, and γ-EnaC, which share 30 to 40% identity at the level of their amino acid sequences. In addition to these three well-characterized ENaC subunits, a fourth EnaC subunit, δ-ENaC, has been cloned from a human kidney cDNA library. However, this subunit has been described so far only in humans, and its relevance for transepithelial Na ⁺ reabsorption in the ASDN is unclear. When the three classical α-, β-, and γ-ENaC subunits are co-expressed in Xenopus laevis oocytes, the subunits form a channel with functional characteristics similar to the channel identified by Palmer in the rat CCD, with a low 5 pS conductance (for Na ⁺ ), a high selectivity ratio of Na ⁺ over K ⁺ (>20), and a high sensitivity for amiloride with a K _i in the submicromolar range. In the Xenopus oocyte expression system, the co-expression of all three ENaC subunits is required for maximal cell surface abundance and activity of the channel. The subunit stoichiometry of the active channel is not finally resolved. Various technical approaches suggested models with four, eight or nine subunits. Recently, the crystal structure of the closely related acid-sensing ion channel isoform ASIC1 _A has been reported, and points to a trimeric oligomerization of the channel. However, this trimeric model still awaits further confirmation by establishing the crystal structure of ENaC itself.

ENaC is expressed in several organs including the kidney, the colon, the salivary and sweat glands. ENaC-mediated Na ⁺ transport helps to adjust Na ⁺ excretion in the urine, feces, and sweat. In lung and airways, ENaC activity is important for alveolar liquid clearance and regulation of mucous fluidity. In the taste buds of the tongue, ENaC is likely involved in salt tasting, whereas ENaC expression in the eye and inner ear may help to control the ionic composition of the aqueous humor and the endolymph, respectively. In the kidney the distribution of ENaC is fully compatible with its involvement in aldosterone-dependent Na ⁺ transport. Morphological and functional studies on rodent and human kidneys clearly showed that the ENaC subunits are highly abundant along the entire ASDN, starting in the late DCT (DCT2), and extending through the CNT down to the medullary CD. In the ASDN cells, increased plasma aldosterone levels induced either by dietary Na ⁺ restriction or by exogenous application appear to stimulate ENaC mainly by three mechanisms. First, aldosterone increases the abundance of the α-ENaC mRNA and protein, without much affecting the mRNAs encoding for the β- and γ-subunits. In contrast, the β- and γ-subunit mRNAs are upregulated in the colon by aldosterone, while the α-subunit mRNA is expressed constitutively. Second, aldosterone induces the appearance of low molecular weight forms of α- and γ-ENaC, which are thought to represent proteolytically activated channel subunits. Recently, Frindt and co-workers performed cell surface biotinylation experiments on rat kidneys perfused with biotin in situ . These experiments showed that feeding a low-Na ⁺ diet or the infusion of aldosterone for one week increases the cell surface pool of cleaved α- and γ-ENaC, but not β-ENaC subunits at the cell surface. Third, aldosterone causes a redistribution of ENaC channel subunits from intracellular storage compartments to the apical plasma membrane. In fact, the subcellular localization of ENaC along the axis of the ASDN changes drastically with altered plasma aldosterone levels in response to altered dietary Na ⁺ intake. In rodents kept under a high dietary Na ⁺ intake with low plasma aldosterone levels, ENaC subunits are barely detectable at the luminal membrane, and are found almost exclusively at intracellular sites. On a standard dietary Na ⁺ intake with moderate plasma aldosterone levels, ENaC subunits are traceable at the luminal membrane in the late DCT and the early CNT, but in downstream segments (i.e., late CNT and CD), particularly β- and γ-ENaC are found almost exclusively at intracellular sites. Only the α-EnaC-subunit was reported to have a more pronounced apical localization and can be found at the apical plasma membrane in CNT and CD. In response to dietary Na ⁺ restriction, the apical abundance of ENaC subunits drastically increases due to a translocation of channel subunits from intracellular compartments to the apical plasma membrane. Nevertheless, the axial gradient for apical ENaC prevails, and the apical localization of ENaC subunits remains more prominent in early ASDN than in late ASDN. The activation and apical translocation of ENaC occurs rapidly (within hours), suggesting that they are relevant for the renal adaptation to day-to-day variations of Na ⁺ intake. Likewise, injection of aldosterone in adrenalectomized rats induces a rapid (within 2 hours) induction of α-ENaC-subunit expression along the ASDN which is followed by a translocation of all three ENaC subunits to the apical plasma membrane ( Figure 35.3 ). Again, the apical shift occurs predominantly in the early and not in the late ASDN, although the induction of α-ENaC was seen to be similar along the entire segment. Based on previous studies in Xenopus laevis A6 cells, it has been proposed that the induction of α-ENaC might be a prerequisite for full assembly of ENaC in the ER and its subsequent delivery to the cell surface. However, in the kidney in vivo the induction of α-ENaC protein by aldosterone is rather small and, at least in the CCD, not necessarily followed by an apical redistribution of all three ENaC subunits. Moreover, apical targeting of ENaC apparently occurs even without previous α-ENaC induction. Likewise, when ENaC activity was assessed as whole-cell amiloride-sensitive current, ENaC activity in rat CCD was drastically increased within 15 hours of salt restriction, and thus in a timeframe in which no changes in the abundance of ENaC mRNA or protein could be detected. Thus, the induction of α-ENaC alone cannot account for the apical targeting of ENaC. Other co-stimulatory factors are needed. One of these factors was proposed to be the serum- and glucocorticoid-regulated kinase (Sgk1). Sgk1 is a member of the PKB/Akt family of serine/threonine kinases, and is rapidly induced by aldosterone in ASDN model epithelia in vitro , as well as in the kidney in vivo . Likewise, prolonged dietary Na ⁺ restriction increases Sgk1 mRNA expression in the kidney. Co-expression of ENaC with Sgk1 in heterologous expression systems profoundly increases ENaC-mediated Na ⁺ -currents, most likely by an accelerated insertion-rate of ENaC into the plasma membrane. Consistently, the induction of Sgk1 by aldosterone precedes the apical translocation of ENaC in ASDN cells in vitro and Sgk1-deficient mice ( Table 35.1 ) show mild urinary Na ⁺ -wasting that becomes aggravated on dietary Na ⁺ restriction. At least part of the effects of Sgk1 on ENaC cell surface abundance is mediated by an Sgk1-dependent phosphorylation of the ubiquitin-protein ligase Nedd4-2. Nedd4-2 phosphorylation blocks the interaction of this ligase with the C-terminus of ENaC subunits, and thereby presumably prevents Nedd4-2-induced ubiquitylation and subsequent endocytosis and degradation of ENaC subunits. Interestingly, immunohistochemical studies showed that the Nedd4-2 abundance increases along the ASDN, which is inverse to the axial gradient of apical ENaC abundance, and further supports the idea that Nedd4-2 is a negative regulator of ENaC cell surface abundance.

Patch-clamp studies corroborated the immunohistochemically traceable axial gradient of the apical localization of ENaC. The ASDN segments isolated from rats kept on a standard lab chow exhibited no amiloride-sensitive currents at the single channel level, whereas CNTs and CDs isolated from rats with elevated plasma aldosterone levels revealed significant single channel ENaC currents ( Figure 35.4 ). Mice appear to have sizeable ENaC whole-cell currents already on a standard diet, which further increase when animals are kept on low dietary Na ⁺ intake. In general, amiloride-sensitive currents decrease in the following order: CNT > initial CD > CD. These findings on apical ENaC localization and activity are consistent with previous studies on microperfused rat tubules, and on isolated rabbit tubules that established several times higher Na ⁺ transport rates in early ASDN (i.e., DCT and CNT) than in further downstream ASDN segments (i.e., CD). The axial gradient of ENaC also parallels the progressive decrease of the basolateral Na ⁺ ,K ⁺ -ATPase activity along the ASDN. Taken together, these data clearly indicate that the aldosterone-dependent adaptation of renal Na ⁺ excretion to dietary Na ⁺ intake occurs through ENaC, predominantly in the early ASDN, while the late ASDN gets recruited only under high plasma aldosterone levels. Using whole-cell current data, and considering the anatomical length of the two segments, Frindt and Palmer estimated that the Na ⁺ transport capacity of the CNT is at least 10 times higher than that of the CD.

The importance of the early ASDN versus late ASDN for the maintenance of Na ⁺ balance was highlighted by the development of a mouse model with targeted inactivation of α-ENaC in the collecting duct. These mice survive well and are able to maintain Na ⁺ and K ⁺ balance, even when challenged by salt restriction or K ⁺ -loading ( Figure 35.5 a). Moreover, these mice show a normal urinary acidification in response to furosemide and thiazide treatment, suggesting that functional expression of ENaC in the CNT is sufficient for maintaining ion homeostasis. In contrast, mice with targeted inactivation of α-ENaC in CD and CNT show symptoms of renal salt-wasting ( Figure 35.5 b). Already under control conditions, the mice have significantly higher urinary Na ⁺ excretion and increased plasma aldosterone levels. With dietary Na ⁺ restriction, the mice start to decompensate and exhibited at day 15 a more than 12% reduction in body weight and exceedingly high plasma aldosterone levels (~20,000 pg/ml in knockout mice versus ~1200 pg/ml in control mice). Moreover, the mice show hyperkalemia, that worsens with dietary K ⁺ -loading. Furthermore, the mice are polyuric, consistent with the idea that ENaC is important not only for renal Na ⁺ and K ⁺ handling, but also for the control of water reabsorption.

Similar to this CNT and CD α-ENaC knockout model, mice with targeted deletion of the MR in the CD and CNT, but intact MR expression and ENaC regulation in early CNT and DCT2, show impaired Na ⁺ balance ( Figure 35.6 ). Although these mice develop normally under a standard diet, they continuously lose body weight and show signs of severe extracellular volume contraction under a low-Na ⁺ diet. These studies did also show that the MR is crucial for the increased expression of αENaC and apical targeting of all three ENaC subunits in response to dietary Na ⁺ restriction. Interestingly, these observations in mice with genetic deletion of the MR contrast with findings in rodents with pharmacological inhibition of the MR. In rats, blockade of the MR by spironolactone does not prevent the apical translocation of ENaC in response to dietary Na ⁺ restriction. Likewise, the combined inhibition of the MR and GR by spironolactone and RU486 does not block the induction of αENaC and ENaC trafficking induced by aldosterone-infusion. Together with the observation that the severe symptoms of pseudohypoaldosteronism in MR-deficient mice can be improved by glucocorticoid treatment, and that mice with CNT/CD-specific MR-deficiency have a milder phenotype than mice with CNT/CD-specific deletion of αENaC, the data point to some redundancies in the MR and GR-dependent signaling pathways that control ENaC function.