A Switch in the Mechanism of Hypertension in the Syndrome of Apparent Mineralocorticoid Excess

The syndrome of apparent mineralocorticoid excess arises from nonfunctional mutations in 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2), an enzyme that inactivates cortisol and confers aldosterone specificity on the mineralocorticoid receptor. Loss of 11βHSD2 permits glucocorticoids to activate the mineralocorticoid receptor, and the hypertension in the syndrome is presumed to arise from volume expansion secondary to renal sodium retention. An 11βHSD2 null mouse was generated on an inbred C57BL/6J genetic background, allowing survival to adulthood. 11βHSD2−/− mice had BP approximately 20 mmHg higher on average compared with wild-type mice but were volume contracted, not volume expanded as expected. Initially, impaired sodium excretion associated with increased activity of the epithelial sodium channel was observed. By 80 days of age, however, channel activity was abolished and 11βHSD2−/− mice lost salt. Despite the natriuresis, hypertension remained but was not attributable to intrinsic vascular dysfunction. Instead, urinary catecholamine levels in 11βHSD2−/− mice were double those in wild-type mice, and α1-adrenergic receptor blockade rescued the hypertensive phenotype, suggesting that vasoconstriction contributes to the sustained hypertension in this model. In summary, it is proposed that renal sodium retention remains a key event in apparent mineralocorticoid excess but that the accompanying hypertension changes from a renal to a vascular etiology over time. In 90% of hypertensive patients, the causal pathophysiology cannot be defined and, in 25%, BP fails to fall to target values with existing therapies. Improved understanding of pathogenesis is key to addressing this problem. Fundamental principles of BP homeostasis have been established through investigation of the rare Mendelian hypertensive disorders. These disorders result almost exclusively from mutations in genes encoding proteins influencing directly or indirectly sodium balance,1 suggesting that altered renal salt homeostasis is a key factor in the misregulation of BP2 The fine-tuning of sodium balance is achieved in the distal nephron, determined principally by the actions of aldosterone at the mineralocorticoid receptor (MR). In vitro, MR can be activated with equal potency by both mineralocorticoids and glucocorticoids.3In vivo, ligand access is determined by the enzyme 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2), which catalyzes the rapid conversion of the active glucocorticoid cortisol (corticosterone in mice) to inert cortisone (11-dehydrocorticosterone), thereby conferring on MR the specificity to aldosterone that it inherently lacks.4,5 In the kidney, 11βHSD2 is expressed in the connecting tubule and in the principal cells of the cortical (CCD) and outer medullary collecting duct.6 Acute pharmacologic inhibition of the enzyme promotes collecting duct sodium reabsorption7via increased activity of the epithelial sodium channel (ENaC).8 Inactivating mutations in the gene HSD11B2, which encodes 11βHSD2, cause the syndrome of apparent mineralocorticoid excess (SAME; OMIM #218030). In this setting, cortisol activates MR,9–12 resulting in severe hypertension thought to arise from volume expansion secondary to renal sodium retention.9,11,13,14 Suppression of cortisol by administration of synthetic glucocorticoids that do not activate MR is therapeutically effective,12 and in one case, full correction of the disorder was achieved by renal transplantation,15 highlighting the central role of the kidney in SAME. These human genetic studies suggest that 11βHSD2 is a physiologic regulator of BP, but investigation of key mechanisms is not possible in patients because the disease is rare and most patients are children. We originally modeled SAME by targeted disruption of the 11βHSD2 locus in an outbred (MF1) mouse strain,16 but the high neonatal mortality permitted only limited physiologic examination. We have now transferred the targeted mutation onto the C57BL/6J genetic background, which attenuates the neonatal mortality while retaining the cardinal features of the disease. Using a time course stretching from weaning to early adulthood, we have identified key mechanistic events in the development and maintenance of hypertension in SAME. RESULTS Sustained Hypertension but Transient Sodium Retention in 11βHSD2−/− Mice 11βHSD2−/− mice had elevated mean arterial BP (MABP) at each of the four time points studied (Figure 1A). Fractional sodium excretion was reduced by >50% in 11βHSD2−/− mice at 35 and 60 d of age (Figure 1B), during which time amiloride-sensitive sodium reabsorption was doubled (Figure 1C). Quantitative PCR showed increased renal expression of ENaC-α mRNA in 35- and 60-d-old 11βHSD2−/− mice (Figure 1D). Expression of ENaC-β and ENaC-γ were higher in 11βHSD2−/− mice than in controls, although this reached statistical significance only for ENaC-γ (P < 0.01; data not shown). The expression of MR was similar in both groups. ENaC-α immunoreactivity in the CCD and outer medullary collecting duct, not detectable in the C57BL/6J mice at any age (Figure 1E),17 was evident in 60-d-old 11βHSD2−/− mice, demonstrating increased protein abundance. Strong apical membrane localization was observed (Figure 1F). At 80 d of age, 11βHSD2−/− mice exhibited natriuresis, with fractional sodium excretion being normalized by 120 d (Figure 2A). The mechanism of escape was identified: the natriuretic impact of amiloride was lost in 80- and 120-d-old 11βHSD2−/− mice (Figure 2B). The downregulation of functional channel activity does not rely principally on attenuation of transcriptional or translational processes because both ENaC-α mRNA levels (Figure 2C) and renal immunoreactivity remained higher in 11βHSD2−/− mice than in controls (Figure 2D). Altered ENaC Trafficking in 11βHSD2−/− Mice In 120-d-old 11βHSD2−/− mice, ENaC-α immunoreactivity was displaced from the apical membrane of the collecting duct principal cells (Figure 2D), suggesting that altered channel trafficking underpins the transition from a sodium-retaining to sodium-shedding state. Western blotting analysis was therefore used to measure the renal abundance of two key ENaC trafficking proteins18: Serum glucocorticoid regulated kinase 1 (SGK1), which promotes insertion of the ENaC into the apical membrane of the principal cell; and neural precursor cell–expressed, developmentally downregulated protein type 4 (Nedd4), which causes channel removal by ubiquitination.18 SGK1 expression was approximately seven-fold higher in 11βHSD2−/− mouse kidney (P < 0.01) at 35 d of age (Figure 2E). Levels of Nedd4 were significantly reduced in 11βHSD2−/− mice at both 35 and 60 d of age (P < 0.01; Figure 2F). At 80 d, expression of both proteins was comparable between groups; however, by 120 d of age, renal expression of SGK1 was reduced whereas that of Nedd4 was enhanced in 11βHSD2−/− mice. 11βHSD2−/− Mice Exhibit Progressive Cytopathology of the Distal Convoluted Tubule In 35-d-old 11βHSD2−/− mice, the normally cuboidal (Figure 3A) epithelial cells of the distal nephron were columnar and the number of cross-sectional nuclei was increased, indicative of hypertrophy and hyperplasia, respectively (Figure 3B). From this time point, through 60 d (Figure 3, C and D), 80 d and 120 d (Figure 3E), there was progressive epithelial hypertrophy and hyperplasia with increasing prevalence of binucleation and hyperchromatic nuclei (indicative of polyploidy; Figure 3E). Many severely hypertrophied cells had loss of basal striations and had extensive accumulations of basophilic granules throughout the cytoplasm, changes suggestive of subcellular disorganization (Figure 3F). These marked changes of the renal epithelium were limited to the distal convoluted tubule: The segments of the collecting duct appeared anatomically normal during the course of study. Volume-Contracted Hypertension in 11βHSD2−/− Mice Renal sodium retention was not associated with volume expansion. Indeed, hematocrit was significantly increased in all cohorts of 11βHSD2−/− mice, indicating plasma volume contraction (Figure 4A). Blood volume was measured directly in a separate group of mice, and the approximately 30% reduction in 11βHSD2−/− mice confirmed (Figure 4B). The renin-angiotensin-aldosterone system, normally activated with plasma volume contraction, was suppressed: Plasma aldosterone was significantly lower in 11βHSD2−/− mice (Table 1) as was kidney renin mRNA, levels being <30% of controls in all age groups (P < 0.05). Plasma corticosterone concentrations were comparable between genotypes at all time points (Table 1). There was a significant hypernatremia (Figure 4C) and elevated plasma osmolality (Table 1) at all time points studied. Despite this, urine flow rate was approximately doubled and urine osmolality markedly reduced in 11βHSD2−/− mice (Table 1). The increase in free water clearance in 11βHSD2−/− mice (Table 1), inappropriate given the hypertonicity, is suggestive of a urine-concentrating defect. This may result from the progressive hypokalemia observed in 11βHSD2−/− mice (Figure 4D): Fractional potassium excretion was elevated in 11βHSD2−/− mice at 35 d (39.8 ± 4.9 versus 22.1 ± 3%; P < 0.05) and comparable to controls from 60 d onward (approximately 20% in both groups). No Evidence for Intrinsic Vascular Dysfunction in 11βHSD2−/− Mice We assessed endothelium-dependent vasodilation and sensitivity to catecholamines of sections of thoracic aorta from 11βHSD2−/− mice or age-matched controls. The effects of acetylcholine and noradrenaline in each of the four age groups are shown if Figure 5. The maximum response to acetylcholine was reduced in 11βHSD2−/− mice only in the 60-d-old group (Figure 5B, Table 2). Because vasodilator responses to sodium nitroprusside in 11βHSD2−/− mice were comparable to those in controls during this study (Table 2), the reduced response to acetylcholine at 60 d of age relates to endothelial cell dysfunction, rather than altered sensitivity to nitric oxide. The maximum response and sensitivity to noradrenaline was not increased in 11βHSD2−/− null mice at any age (Figure 5, Table 2). Indeed, there is trend toward a reduction in noradrenaline-mediated contraction in the 35- and 60-d-old groups (Figure 5, A and B). 11βHSD2−/− Mice Have Increased Catecholamine Levels In the absence of increased sensitivity to catecholamines of the vasculature ex vivo, urinary excretion of adrenaline and noradrenaline was measured and found to be substantially elevated in 11βHSD2−/− mice (Figure 6A). To assess directly the contribution of catecholamines to the maintenance of hypertension, we measured the impact on MABP of acute α1-adrenoreceptor blockade in vivo. A bolus of prazosin significantly reduced MABP in both groups of mice (>120 d of age; Figure 6, B and C, for sample traces), but the effect of the drug was substantially greater in the 11βHSD2−/− mice (−22.3 ± 1.4 versus −8.7 ± 1.1 mmHg; n = 5/6; P < 0.001). Importantly, α1-adrenoreceptor blockade rescued the hypertensive phenotype in 11βHSD2−/− mice (Figure 6D). DISCUSSION SAME is characterized by unregulated glucocorticoid activation of MR after ablation of 11βHSD2.9–12 Hypertension in this disorder is thought to be renal in origin, relating to enhanced ENaC-mediated sodium reabsorption and volume expansion.7,8,14 In this study, we used 11βHSD2−/− mice to model SAME and demonstrated that transient abnormal renal function can promote a chronic hypertensive cascade that is not sustained through volume expansion. Initially, 11βHSD2−/− mice had an impaired ability to excrete sodium, relating to increased expression of ENaC subunits and enhanced activity of the functional channel shown by the increased natriuretic potency of amiloride. We also found increased expression of SGK1 and reduced expression of Nedd4 during this period, which combined would increase the membrane retention time of ENaC.18 SGK1 is established as an early aldosterone response gene in the kidney.19 In contrast, Nedd4 protein levels are not effected by acute administration of aldosterone20,21; however, our findings and those in a recent study22 indicate that chronic activation of MR leads to decreased collecting duct expression of Nedd4-2. A key finding of our study is that the kidney is able to circumvent a genetic obligation for avid sodium reabsorption. By the 80-d time point, 11βHSD2−/− mice displayed enhanced sodium excretion. The escape from perceived mineralocorticoid excess does not rely on attenuation of transcriptional or translational processes because ENaC-α mRNA and CCD protein levels remained higher in 11βHSD2−/− mice than in controls during this period. Our data suggest that ENaC is rendered physiologically inactive because, in the CCD at least, it is not trafficked to the apical membrane. We have not confirmed altered ENaC trafficking in either the late distal convoluted tubule or the connecting tubule. That the natriuretic impact of amiloride was lost, however, indicates that functional channel activity is abolished throughout the distal nephron. The mechanisms underpinning trapping in the cytosol may, in the longer term, involve an alteration in the balance between insertion and retrieval, but ENaC is functionally inactivated before any changes in expression of SGK1 or Nedd4 are apparent. The loss of functional ENaC explains why amiloride is only an effective means of long-term BP control12,23 in SAME when used at the high dosages24 required to inhibit proximal tubule sodium transport.25 A generalized transport defect could be indicated, so extensive histopathologic examination of 11βHSD2−/− mouse kidneys was performed. In some patients with SAME, the kidney is grossly normal,10 but cysts, nephrocalcinosis, and hypertensive end-organ damage are seen in others, particularly as the disease progresses.26 We showed progressive changes of the renal epithelium in 11βHSD2−/− mice, but, surprisingly, these were limited to the distal convoluted tubule: the segments of the collecting duct appeared anatomically normal. Because the mouse distal convoluted tubule does not express 11βHSD2,6 these changes must be secondary to the deletion of the gene in other nephron segments. The structural changes are strongly reminiscent of those occurring in the rat after long-term exposure to loop diuretics such as furosemide.27 We propose that impaired sodium reabsorption in the loop of Henle, secondary to potassium depletion, promotes a compensatory hypertrophy of the distal convoluted tubule. The use of thiazide diuretics to inhibit sodium chloride co-transport in this segment would therefore be specifically indicated for long-term BP regulation in SAME.10,24 Our analysis suggests that downregulation of ENaC is not caused by gross changes in collecting duct structure. A striking outcome of this study is the marked volume contraction observed in 11βHSD2−/− mice, even during the period of enhanced sodium reabsorption. Human28 and animal studies29 have consistently shown volume expansion in the early, prehypertensive stage of experimental mineralocorticoidism. In this situation, volume expansion increases cardiac output, initiating hypertension, which is then maintained in the adaptive phase by peripheral vasoconstriction.30 In most forms of hypertension, peripheral resistance is high and blood volume is either normal or slightly low (reviewed by Hamlyn and Blaustein29 and Cowley31) during the chronic phase. The volume contraction observed in 11βHSD2−/− mice may therefore represent the postadaptive period of hypertension, and we cannot exclude a period of volume expansion before 35 d of age. To explain volume contraction in the face of sodium retention, we have two connected hypotheses. The first is that sodium retention is “water-free,” being held in osmotically inactive stores. This occurs in the DOCA-salt model of mineralocorticoid hypertension and minimizes the impact on extracellular fluid volume to buffer BP.32 The second hypothesis is that water homeostasis is in 11βHSD2−/− mice, being to or even blood The hypernatremia observed in 11βHSD2−/− mice throughout the experimental period the hypothesis but does not explain the 11βHSD2−/− mice are most as a result of secondary to potassium are also is why a volume balance is not in the of the are by chronic and pharmacologic inhibition of the enzyme promotes may be that a system, in this is required to to progressive potassium the human it of the hypertensive mechanism in 11βHSD2−/− mice. can BP through promotes contraction of vascular and vascular can be indirectly by impaired endothelium-dependent increased and of effects are also found with a in 11βHSD2−/− mice and one sustained the downregulation of ENaC sodium is the in hypertension, with potassium being a however, from both human such as the to or combined with in suggest that an in one or the other is of than In this study, the sodium reabsorption in the distal nephron would potassium and the resulting potassium would cause volume contraction and Because volume expansion does not to hypertension in 11βHSD2−/− mice, we measured the intrinsic of the Vascular both glucocorticoid and activation of which can vascular to the effects of 11βHSD2 is to the vascular and inhibition of the enzyme increases vascular sensitivity to and Vascular to noradrenaline is enhanced in a with and we have found significant endothelial in the outbred mouse model of SAME at 120 d of although not increased sensitivity to In this study, however, we found only limited for enhanced vascular in 11βHSD2−/− mice, and this was We that changes in the intrinsic of the vasculature do not a contribution to the development or maintenance of hypertension in this model of SAME. That the vascular phenotype is not observed on the C57BL/6J that the physiologic function of the MR Increased urinary excretion of catecholamines was observed in 11βHSD2−/− mice, which increased blockade was at to the hypertensive this to the situation, the vasodilator have been used to SAME in patients do not to either MR blockade or cortisol The mechanism increased activity is but could result from either potassium or sodium Our findings that renal sodium retention is transient and amiloride-sensitive sodium reabsorption is downregulated in 11βHSD2−/− mice. In the chronic hypertension is not sustained through either volume expansion or vascular dysfunction. We propose that the key hypertensive event is an that activates the system, with marked volume A mouse was generated by of the 11βHSD2−/− mouse with mice used in this study were from null after of onto C57BL/6J C57BL/6J were used as controls. this extensive the be to the inbred we not use as controls, it is that the the targeted disruption of in the In a separate group of were used to that the genotypes as were at in the normal Mendelian In to the outbred the 11βHSD2−/− mice exhibited abnormal of neonatal age groups of mice were used in this and 120 d of age. The time points were to the of renal function from weaning to adulthood, with the time being in the period of kidney function in the C57BL/6J were in with the In Renal function were as Mice were with a at a rate of A period was by two of renal function by of a blood the first amiloride was and, after a period, a second urine was MABP was measured throughout At the of the a blood sample was for of aldosterone and and the kidneys were In separate groups of mice, plasma volume was determined by of of a and blood volume was from plasma volume and the effect of acute blockade was measured by of the effect of a bolus of prazosin on and urinary excretion of and was measured by were in for by sections were with and and by a in protein was from one kidney by by of protein and to membrane. either Nedd4 or SGK1 was to the a protein of and of was to equal protein The localization of ENaC-α was determined by on sections of kidney from a separate group of age-matched mice, as a by of Quantitative PCR was from and mRNA of were by to the an were normalized to on a 11βHSD2−/− mice and age-matched controls were by and thoracic were into a physiologic mouse were for of as data are as means ± for were either or with as were MABP in 11βHSD2−/− mice and age-matched C57BL/6J mice Mice were studied at 35 d = controls, 60 d 80 d and 120 d of age. Fractional sodium excretion. The effect of amiloride on sodium excretion The renal expression of ENaC-α mRNA normalized to the expression of in 35- and 60-d-old 11βHSD2−/− mice and C57BL/6J mice. are means ± were with < < The expression of ENaC-α protein was the in kidneys from mice with a but was to the apical membrane in the CCD of 60-d-old 11βHSD2−/− mice Fractional sodium excretion in and 120-d-old 11βHSD2−/− mice and age-matched C57BL/6J mice The effect of amiloride on sodium excretion The renal expression of ENaC-α mRNA normalized to the expression of Expression of ENaC-α protein in the CCD of 120-d-old 11βHSD2−/− mice, that immunoreactivity is Western analysis the expression of SGK1 and Nedd4 in the kidney = for each of 11βHSD2−/− mice, as a of that in kidneys of age-matched C57BL/6J mice. The the of equal was by data are means ± were with for and a a sample mean of < < of epithelial in distal convoluted of 11βHSD2−/− mice. C57BL/6J mouse d The cuboidal epithelium apical nuclei and basal A 35-d-old 11βHSD2−/− The epithelium is and the number of cross-sectional nuclei is increased, indicative of hypertrophy and hyperplasia, A 60-d-old 11βHSD2−/− In to epithelial hypertrophy and hyperplasia, there are cells A 60-d-old 11βHSD2−/− into the A 120-d-old 11βHSD2−/− nuclei are as a result of and A hyperchromatic suggests A of epithelial cells into the with with the membrane A of epithelial cells in the tubule A 120-d-old 11βHSD2−/− In to striking hypertrophy and the there is loss of basal striations and indicative of subcellular and = in 11βHSD2−/− mice and age-matched C57BL/6J mice Mice were studied at 35 d = controls, 60 d 80 d and 120 d of age. Blood volume in 11βHSD2−/− mice = and C57BL/6J mice = normalized of Plasma Plasma are means ± and were either or with a < < Vascular in from 11βHSD2−/− mice and age-matched C57BL/6J mice Mice were studied at 35 60 80 and 120 d of age with mice used at each time to acetylcholine are shown on the and to on the are means ± of the to the response by were either or with a < urinary excretion of adrenaline and in 120-d-old 11βHSD2−/− mice and age-matched C57BL/6J mice expressed as of = in each of the α1-adrenoreceptor prazosin on MABP in 120-d-old 11βHSD2−/− mice n = and age-matched C57BL/6J mice n = are means ± and were either or with a < BP are shown for C57BL/6J mice and 11βHSD2−/− mice Plasma and urine osmolality free water clearance and plasma concentrations of potassium aldosterone and corticosterone in four cohorts of 11βHSD2−/− and C57BL/6J data from 11βHSD2−/− and C57BL/6J was by the and and the a and from the analysis was by the of for for which the of and is