Renin is an enzyme that cleaves renin substrate or angiotensinogen, which is synthesized by the liver, to produce the decapeptide, angiotensin I. Angiotensin I is rapidly cleaved by angiotensin-converting enzyme (ACE) in the lung and other tissues to form the octapeptide, angiotensin II. Moreover, angiotensinase cleaves the NH2-terminal Asp residue from angiotensin II and produces the heptapeptide, angiotensin III, whose circulating levels are 15 to 25% of those of angiotensin II. Angiotensin II and III stimulate aldosterone secretion and vasoconstriction, while angiotensin II is more potent for vasoconstriction. The angiotensins are inactivated within minutes by tissue and plasma peptidase. The levels of the circulating renin are the rate-limiting factor of this process.

Renin is synthesized by the juxtaglomerular cells in the renal cortex and its secretion is controlled by renal arterial blood pressure, sodium concentrations of tubular fluid sensed by the macula densa and renal sympathetic nervous activity (9). Factors that decrease renal blood flow, such as hemorrhage, dehydration, salt restriction, upright posture and renal artery narrowing, increase renin levels. In contrast, factors that increase blood pressure, such as high salt intake, peripheral vasoconstrictors and supine posture, decrease renin levels. Hypokalemia increases and hyperkalemia decreases renin release (10).

The effect of angiotensin II and III on the adrenal glomerulosa is initiated by binding to G-protein coupled receptors. The first mechanism of the intracellular signal transduction is activation of phospholipase C, which hydrolyzes PIP2 to IP3, which then releases intracellular calcium ions (11). Interestingly, angiotensin II does not stimulate adenylate cyclase activity (12). Angiotensin II stimulation leads to increased transfer of cholesterol to the inner mitochondrial membrane and increased conversion of cholesterol to pregnenolone and corticosterone to aldosterone. (12, 13).

Potassium directly increases aldosterone secretion by the adrenal cortex and aldosterone then lowers serum potassium by stimulating its excretion by the kidney. High dietary potassium intake increases plasma aldosterone and enhances the aldosterone response to a subsequent potassium or angiotensin II infusion (11). The primary action of potassium for stimulating aldosterone secretion is to depolarize the plasma membrane, which activates voltage-dependent calcium channels, that permit influx or exflux of extracellular calcium (11). The increased cytosolic calcium stimulates the same two steps in aldosterone biosynthesis that angiotensin II does (13).

ACTH and possibly other POMC-derived peptides, including α-MSH, α-MSH, β-LPH and β-END, influence aldosterone secretion, however, the role of ACTH in aldosterone secretion is minor (10). ACTH increases aldosterone secretion by binding to glomerulosa cell-surface melanocortin-2 receptor, by activating adenylate cyclase, and increasing intracellular cAMP (15). Like other agents, ACTH stimulates the same two early and late steps of aldosterone biosynthesis.

Vasopressin has a modest and transient stimulatory effect on aldosterone secretion from zona granulosa cells in vitro. This effect is probably mediated via V2 receptors and phospholipase C generating IP3 and diacylglycerol (16).

Sodium intake influences aldosterone secretion by an indirect effect through renin and to a minor extent by direct effects on zona glomerulosa responsiveness to angiotensin II. High sodium intake increases vascular volume, which suppresses renin secretion and angiotensin II generation and decreases the sensitivity of aldosterone response to angiotensin II (17).

Dopamine inhibits aldosterone secretion in humans by a mechanism that is independent of the effects of prolactin, ACTH, electrolytes and the renin-angiotensin system (18). This inhibitory effect may involve binding to D2 receptors on glomerulosa cells (19). Atrial natriuretic peptide (ANP) directly inhibits aldosterone secretion and blocks the stimulatory effects of angiotensin II, potassium and ACTH, at least in part, by interfering with extracellular calcium influx (20).

Aldosterone is crucial for sodium conservation in the kidney, salivary glands, sweat glands and colon. Aldosterone promotes active sodium transport and excretion of potassium in its major target tissues. It exerts its effects via the mineralocorticoid receptor (MR) and the resultant activation of specific amiloride-sensitive sodium channels (ENaC) and the Na-K ATP ase pump (21). Aldosterone and the MR may be involved in the regulation of genes coding for the subunits of the amiloride sensitive sodium channel and the Na-K ATP ase pump, as well as of other proteins (22,23). Aldosterone indeed increases the number of active sodium channels and augments the action and number of the Na-K ATP ase pump units in its target tissues (24).

The MR is found in the cytoplasm and nucleus and the sodium channels are expressed in the apical membrane of epithelial cells of the distal convoluted tubule as well as in cells of other tissues involved with conservation of salt, such as colon, sweat glands, lung and tongue. MR is a member of the nuclear receptor superfamily. Together with the glucocorticoid, progesterone and androgen receptors, MR forms the steroid receptor subfamily (25). Steroid receptors display a modular structure comprised of five regions (A-E). The N-terminal A/B region harbors an autonomous activation function. The central C region, corresponding to the DNA-binding domain, is highly conserved and is composed of two zinc fingers involved in DNA binding and receptor dimerization. D region is a hydrophilic region and it forms a hinge between DNA-binding domain and ligand-binding domain. E region corresponds to the C-terminal ligand-binding domain and mediates numerous functions, including ligand binding, interaction with heat-shock proteins, dimerization, nuclear targeting, and hormone-dependent activation (26) (Figure 2). The human MR (hMR) and human glucocorticoid receptor (hGR) have almost identical DNA-binding domains (94% homology in the amino acid) and very similar ligand-binding domains (57%), but divergent N-terminal A/B regions (<15%) (27). The hMR gene was mapped on chromosome 4q31.1-31.2 (28,29) and hMR cDNA encodes a 107 kilodalton polypeptide with 984 amino acids (27). The hMR gene consists of 10 exons, including two exons 1 that encode different 5'-untranslated sequences (30). Expression of the two different hMR variants is under the control of two different promoters that contain no obvious TATA element, but multiple GC boxes. Both hMRαand hMRβ mRNAs are expressed at approximately the same level in the mineralocorticoid target tissues (31).

MRs in its unliganded state is located in the cytoplasm, as part of hetero-oligomeric complexes containing heat shock proteins 90, 70 and 50 (32). Upon binding with their ligand, the receptor-ligand complex dissociates from the heat shock proteins, homo- or heterodimerizes and translocates into the nucleus. Homodimers or heterodimers of the MR interact with hormone-responsive elements (HRE) and/or other transcription factors in the promoter regions of target genes, including the subunits of the ENaC or other proteins related to this channel and sodium transport in general, and modulates the transcription rates of these genes (33) (Figure 3).

Since cortisol circulates at plasma concentrations several orders of magnitude higher than those of aldosterone does, and since it has a high affinity for the MR, it would be expected to overwhelm this receptor in mineralocorticoid target tissues and cause mineralocorticoid excess. A local enzyme, 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2), however, converts active cortisol to inactive cortisone, and protects the MRs from the effects of cortisol (34). 11β-HSD catalyzes the interconvension of hormonally active C11-hydroxylated corticosteroids (cortisol in humans or corticosterone in rodents) and their inactive C11-keto metabolites (cortisone in humans or 11-dehydrocorticosterone in rodents). Two isozymes of 11β-HSD have been identified, 11β-HSD type 1 (11β-HSD1) and 11β-HSD2, which differ in their biological properties and tissue distributions. 11β-HSD2, a potent NAD-dependent 11β-hydrogenase, rapidly inactivates glucocorticoids. The human 11β-HSD2 gene encodes 405 amino acids and its molecular weight is approximately 40-kilodalton (35). 11β-HSD2 has a hydrophilic N-terminal domain that is thought to anchor the protein into membranes (36). 11β-HSD2 is localized as a dimmer in the nucleus and cytoplasm of cells of the cortical collecting duct and colon (36,37). Prednisolone and prednisone are substrates for both 11β-HSD isozymes (38,39) and dexamethosone is metabolized slightly by 11β-HSD2 (40). Licorice derivatives, such as glycyrrhizic acid, and the hemisuccinate derivative carbenoxolone are inhibitors of 11β-HSD2. Inhibition of 11β-HSD2 with such agents derivatives confers mineralocorticoid potency to physiologic concentrations of endogenous glucocorticoids in the kidney and colon (41). Thus, in normal physiology, 11β-HSD2 protects the MR by converting cortisol to the inactive cortisone and allows aldosterone-selective access to the inherently nonselective MR in mineralocorticoid target tissues.

The cDNA of the α-subunit of the ENaC (αENaC) was cloned from the rat colon in 1993 (42) and soon after the cDNAs of the β- and γ-subunits of this channel were cloned for the same species (43). The human α-, β- and γ-subunits of ENaC were also cloned recently (44,45). In vitro studies demonstrated that the α subunit of the ENaC itself had the majority of Na channel function, while, the β- and γ- subunits alone were not shown to play as major a role in sodium transport (42). However, the β-and γ-subunits enhanced the function of the α-subunit. It appears then that this channel consists of the α-, β- and γ-subunits and an amiloride-binding protein (Figure 4). The proline-rich region of the C-terminal of the αENaC is important for binding to α-spectrin and for stabilization of the sodium channel in the membrane (46). Recently, several studies demonstrated abnormalities of the β- and γ-subunits of the ENaC in patients with Liddle's syndrome, characterized by mineralocorticoid excess (hypertension and hypokalemic alkalosis), and suppressed aldosterone secretion (47-50). The truncation caused by these mutations influenced the PY motif at the N-terminal of the molecule. This motif is responsible for the binding of the channel subunits with NEDD4, a carrier protein facilitating clearance of the channel (51). Moreover, a point mutation of the αENaC gene, located close to the N-terminal of the protein, was reported to cause a decrease of the probability of an open sodium channel, resulting in defective reabsorption (52,53).

The first category of conditions, which is characterized by defective stimulation of aldosterone secretion, includes the syndromes of congenital and acquired hyporeninemic hypoaldosteronism. One of these conditions is due to a defect of renin secretion such as hyporeninemia resulting from β-blockers, prostaglandin synthetase inhibitors, and calcium channel blockers. Another condition is due to decrease in the conversion of angiotensin I to angiotensin II mediated by converting enzyme inhibitor medications and is associated with hyperreninemia.

The second category of conditions, which are characterized by primary defects in adrenal synthesis or secretion of aldosterone, includes all causes of primary adrenal insufficiency and primary hypoaldosteronism caused by aldosterone synthase (CYP11B2) deficiency or as an acquired state. Primary adrenal insufficiency causes include congenital adrenal hypoplasia, congenital adrenal hyperplasia, adrenoleukodystrophy/adrenomyeloneuropathy, acquired adrenal insufficiency due to autoimmune, infectious and infiltrative disease, bilateral adrenalectomy and use of adrenolytic agents and enzyme inhibitors that block cortisol and aldosterone biosynthesis. These conditions are usually combined with defective cortisol synthesis. Aldosterone synthase (CYP11B2) deficiency leads to reduced aldosterone production associated with low or high levels of 18-hydroxycorticosterone, referred to as CMO I or CMO II deficiency, respectively. Several conditions may be associated with aldosterone biosynthetic activity. Heparin suppresses aldosterone synthesis. Critically ill patients with persistent hypovolemia and hypotension also have inappropriately low plasma aldosterone concentrations in relation to the activity of the renin-angiotensin system. Isolated primary hypoaldosteronism in occasionally associated with metastatic cancer of the adrenal gland.

The third category which is characterized by defective aldosterone action includes syndromes of aldosterone resistance such as pseudohypoaldosteronism type 1 and sodium-wasting states resulting from excessive amounts of circulating mineralocorticoid antagonists, such as spinololactone and its analogues, and synthetic progestin or natural agonists, such as progesterone or 17-hydroxyprogesterone. These mineralocorticoid antagonists may antagonize aldosterone at the levels of mineralocorticoid receptor (54) and frequently, these states are compensated for by elevated concentrations of plasma aldosterone.

The most common form of isolated hypoaldosteronism is caused by impaired renin release from the kidney. Hudson et al. first described this syndrome in 1957 (55), however, hyporeninemia was first recognized in 1972 (56,57). The typical patient is 50 to 70 years old and usually presents with chronic and asymptomatic hyperkalemia and mild to moderate renal insufficiency with a 40-70% decrease in the glomerular filtration rate when compared to that of age matched healthy subjects. Hyperchloremic metabolic acidosis is seen in approximately half of the patients. This acidosis is classified as a renal tubular acidosis type IV (58). The acidosis is a consequence of decreased renal ammonia neogenesis, reduced hydrogen ion-secretory capacity in the distal nephron and mild reduction in the proximal tubular threshold for bicarbonate reabsorption. Occasionally, muscle weakness or cardiac arrhythmias are present in some patients. More than a half of the patients have diabetes mellitus (59). Other frequently associated states include autonomic neuropathy, hypotension and various nephropathies such as glomerulonephritis, gouty nephropathy and pyelonephritis (60). Also, this syndrome is associated with nephropathies of multiple myeloma and systemic lupus erythematosus, mixed cryoglobulinemia, nephrolithiasis, analgesic nephropathy, renal amyloidosis, IgA nephropathy, cirrhosis, sickle cell anemia, acquired immune deficiency syndrome (AIDS), polyneuropathy, organomegaly, endocrinopathy, M protein and skin changes (POEMS) syndrome, lead poisoning, excess sodium bicarbonate and Sjögren’s symdrome (61-68, 68.1, 68.2). Moreover, this syndrome occurs transiently in association with use of non-steroidal anti-inflammatory drugs, cyclosporin A, mitomycin C, cosyntropin and other agents in susceptible individuals (69-72).

Urinary aldosterone excretion is low under basal conditions and fails to increase after sodium restriction. Plasma renin activity is also low and does not increase appropriately during sodium restriction, periods of prolonged upright posture, or diuretic administration (56). Interstitial renal disease and damage to the juxtaglomerular apparatus seems the most likely cause for the primary defect in renin generation or release and secondary deficiency of aldosterone. However, in some patients with this syndrome there is an absent or blunted aldosterone response to angiotensin II (73,74), suggesting a coexisting primary defect in aldosterone secretion or it reflects atrophy of the zona glomerulosa caused by chronic renin deficiency.

There are various mechanisms to be explain the hyporeninemia. First possible mechanism is the hypervolemia. The expanded extracellular fluid volume due to hypertension may suppress renin. In fact, long-term sodium restriction and diuretic administration increase plasma renin activity in these patients, however, the increments of plasma renin activity are less than those of normal subjects (75). A second possible mechanism is insufficiency of the autonomic nervous system, particularly in patients with diabetic neuropathy. Impaired adrenergic response to postural change may contribute to insufficient renin release. Besides, these patients exhibit decreased sensitivity to β-adrenergic agonists, suggesting defects in both production and action of catecholamines (76). A third proposed mechanism is secretion of abnormal forms of renin, such as a defect in the conversion of prorenin to renin. Insufficiency of autonomic nervous system may be associated with impaired conversion of prorenin to renin. Indeed, diabetic patients with autonomic neuropathy have elevated plasma levels of prorenin (77). A fourth possibility is prostaglandin deficiency. Production of prostaglandin I2 (prostacyclin), which mediates renin release (78), is apparently diminished in patients with hyporeninemic hypoaldosteronism as assessed by measurement of the stable urinary metabolite 6-keto-prostaglandin F1α (79). Furthermore, the prostaglandin I2 in these patients was unresponsive to the potent stimulators norepinephrine and calcium. Prostaglandin I2 deficiency may cause hyporeninemic hypoaldosteronism by causing defects in the conversion of prorenin to renin (80) and renin release.

The diagnosis of hyporeninemic hypoaldosteronism must be considered in any patient with unexplained hyperkalemia. Excess potassium intake from food or drugs does not cause sustained hyperkalemia, if renal function is normal. Renal function should be evaluated and drugs that impair renal potassium excretion should be excluded as a cause. The clinical diagnosis is confirmed by low plasma renin activity and low plasma concentrations or urinary aldosterone excretion under conditions that activate the renin-angiotensin-aldosterone axis by maintenance of upright posture and/or furosemide administration. A low random plasma renin concentration associated with a normal ratio of aldosterone to plasma renin activity is also useful for the diagnosis (81,82).

The therapeutic approach should be chosen after taking into consideration the age of the patients and other concurrent disorders. Only monitoring potassium concentrations is enough for patients with moderate hyperkalemia and without electro-cardiographic changes. Drugs that promote hyperkalemia, such as β-adrenergic antagonists, cyclooxygenase inhibitors, angiotensin-converting enzyme inhibitors, heparin and potassium-sparing diuretics, should be avoided. Dietary potassium intake should be reduced, if possible. Diuretics is the initial treatment for patients who have disorders associated with sodium retention, such as hypertension and congestive heart failure. Mineralocorticoid replacement with fludrocortisone is reserved for patients with severe hyperkalemia without hypertension and congestive heart failure.

Several conditions may be associated with aldosterone biosynthetic defects. The administration of heparin causes natriuresis and hyperkalemia (92). Heparin preparations suppress aldosterone synthesis, leading to a compensatory rise in plasma renin activity. However, it has been demonstrated that this suppression of enzyme activity is attributable to chlorbutol (1,1,1-trichloro-2-methyl-2-propanol), the preservative used in commercial heparin, rather than to pure heparin (93).

Persistently hypotensive, critically ill patients with sepsis, pneumonia, peritonitis, cholangiitis and liver failure, also have inappropriately low plasma aldosterone concentrations in relation to elevated plasma renin activity (94). The defect is at the level of the adrenal but has not been associated with any particular disease or therapy. Plasma cortisol levels are high, reflecting the stressed state. The response to angiotensin infusion is impaired, and the ratio of plasma 18-hydroxycorticosterone to aldosterone is increased, suggesting selective insufficiency of CMO II. It is possible that the hypoxia causes a relative zona glomerulosa insufficiency (95).

Mineralocorticoid resistance (pseudohypoaldosteronism type 1, PHA1) results from inability of aldosterone to exert its effect on its target tissues and was first reported by Cheek and Perry as a sporadic occurrence in 1958 (96). This disease, usually presents in infancy with severe salt-wasting and failure to thrive, accompanied by profound urinary sodium loss, severe hyponatremia, hyperkalemia, acidosis, hyperreninemia and paradoxically markedly elevated plasma and urinary aldosterone concentrations. Usually, renal and adrenal functions are normal. This disease has been reported in over 70 patients (97). Approximately one fifth of these cases were familial, and both an autosomal dominant and a recessive form of genetic transmission were observed. All patients had renal tubular unresponsiveness to aldosterone, while some had involvement of other mineralocorticoid target-tissues, including the sweat and salivary glands, and the colonic epithelium, as well.

The mechanism(s) by which aldosterone controls sodium transport in its target tissues involves the mineralocorticoid receptor (MR) and proteins that are associated with the amiloride-sensitive sodium channel (ENaC). The latter proteins are expressed in the apical membrane of epithelial cells of the distal convoluted tubule and in the membranes of cells of other tissues involved in the conservation of salt, such as colon, sweat gland, lung and tongue. Thus, the MR and the ENaC were considered as potential candidate molecules for the pathogenesis of PHA1. In fact, mutations of α- and β-subunits of the ENaC were reported in PHA patients from autosomal recessive kindreds (52,98). Mutation of the MR were also reported in the patients with autosomal dominant PHA1 (99, 100). However, no molecular defects were found in either MR or ENaC in some patients with PHA1, especially in those with the sporadic form PHA1, which suggests molecular heterogeneity in PHA1 (101-107).

Electrolyte profiles suggest mineralocorticoid deficiency or end-organ resistance, along with hyperkalemia, hyponatremia and metabolic acidosis associated with profound urinary salt loss. Renal and adrenal function are normal. The diagnosis is confirmed as markedly elevated plasma aldosterone concentrations and plasma renin activity.

The differential diagnosis of PHA1 includes salt-wasting states due to hypoaldosteronism, including several forms of congenital adrenal hyperplasia, isolated hypoaldosteronism due to corticosterone methyloxidase (CMO) I and II deficiencies and congenital adrenal hypoplasia. Normal cortisol and excessive aldosterone responses to adrenocorticotropin (ACTH) are expected in patients with congenital PHA.

The standard treatment of PHA has been replacement with high doses of salt, with a variable response among patients (97). Recently, carbenoxolone, an 11β-hydroxysteroid dehydrogenase inhibitor, was employed as therapy in PHA1 and an ameliorating effect was observed which was attributed to mediation by the MR (101). We studied a 17-yr-old male patient with congenital multifocal target-organ resistance to aldosterone. We examined his clinical response to carbenoxolone, expected to increase the intracellular level of cortisol in the kidney by preventing local conversion of cortisol to cortisone, and to high doses of fludrocortisone, a synthetic mineralocorticoid. Subsequently, and for a brief period of time, we administered dexamethasone, which has no intrinsic salt-retaining activity, in addition to carbenoxolone, to suppress endogenous cortisol, along with its intrinsic mineralocorticoid activity.

Carbenoxolone normalized the patient's serum electrolyte concentrations and decreased his urinary excretion of sodium within a week (Figure 6, above). Subsequent long-term therapy of this patient with carbenoxolone (450 mg/day p.o.) maintained his electrolyte concentrations within the normal range. His urinary 24 h free cortisol was increased during carbenoxolone therapy. Addition of dexamethasone suppressed his urinary free cortisol excretion and reversed the beneficial effect of carbenoxolone on serum and urinary electrolytes (Figure 6). These data suggest that an increase in urinary free cortisol observed during carbenoxolone therapy was due to a localized effect of this drug on the kidney rather than on tissues involved in the negative feedback effect of glucocorticoids. The effect of carbenoxolone does not seem to be mediated by GR, but seems to be exerted purely via the MR (Figure 7). There were no adverse effects of long-term carbenoxolone therapy in this patient. He also reported increased stamina, a better ability to concentrate and less anxiety. On treatment, the patient grew 6 cm/y and progressed from -4SD to -3SD scores for mean height for age. He also progressed in his pubertal development from Tanner stage III to IV for pubic hair, while his bone age advanced from 12 to 14 y.

Both carbenoxolone and fludrocortisone normalized the serum electrolytes of our patient, suggesting the presence of a functional, albeit possibly defective, renal MR. Interestingly, the same patient was unresponsive to intravenous infusion of aldosterone and fludrocortisone (up to 3 mg/day) when studied in infancy (108), suggesting that the clinical improvement that has been noted in the majority of PHA patients with age may be related to changes in their responsiveness to mineralocorticoid.

In 1996, Chang et al. reported homozygous mutations introducing a stop codon or frame shift in the αENaC gene of affected members of families with autosomal recessive PHA (52). More recently, in Swedish families with autosomal recessive PHA, homozygous or compound heterozygous mutations introducing a stop codon or a frame shift in the αENaC gene were associated with pulmonary disease as well (109). The truncation caused by these mutations influenced the PY motif at the N-terminal region of the molecule. This motif is responsible for the binding of the channel subunits with NEDD4, a carrier protein facilitating clearance of the channel (51). Moreover, a point mutation of the αENaC gene, located close to the N-terminal of the protein, was reported to cause a decrease of the probability of an open sodium channel, resulting in defective reabsorption (52,110). In other four families with autosomal recessive PHA, insertion of a T in exon 8 and nonsense mutation (R508X) in exon 11 of the αENaC gene, these resulting in a truncated αENaC subunit, were found (110.1). A splice site mutation in intron 12 of the βENaC gene, which preventing correct splicing of the mRNA was found in a Scottish patient (110.1). Also, other autosomal recessive families with PHA had a homozygous splice-site mutation in the γENaC, while a Japanese sporadic patient with the systemic form of PHA was a compound heterozygote for mutations in the αENaC, which resulted in the generation of a truncated channel subunit (98,111).

Recently, Geller et al. reported families with autosomal dominant PHA, who had molecular defects of the MR resulting in non-expression of one of the 2 alleles (99) (Figure 8). In addition, Viemann et al. reported a sporadic patient with PHA who had a heterozygous mutation in exon 9 of the MR that introduced a premature stop codon (107) (Figure 8). From these studies, one can conclude that expression of only one allele of the MR is insufficient to prevent salt loss. The Viermann et al. study did not identify any abnormalities of the MR in PHA patients from two families with the autosomal dominant form of the disease (107), while Tajima et al. reported a heterozygous missense mutation in exon 8 of the MR gene identified in PHA patients from a Japanese autosomal dominant family (100) (Figure 8). A heterozygous nonsense mutations in exon 2 (S163X, C436X) and in exon 9 (R947X) of the MR, leading to a premature stop codon of the MR gene were found in other patients with autosomal dominant PHA (107.1, 107.2, 107.3). Sartorato et al. reported that Q776R mutation in exon 5 or L979P mutation in exon 9, which located in the ligand-binding domain of the MR, presented reduced or absent aldosterone binding, respectively (107.4). These studies suggest major molecular heterogeneity in PHA.

We studied 5 unrelated cases of sporadic PHA (101,105,106). We found a nonconservative homozygous mutation (A241V) in the MR of 4 of the patients and a conservative heterozygous mutation (I180V) in one of these patients and his asymptomatic father, while no abnormalities were found in the DNA- or ligand-binding domains of the MR. The Val241 and Val180 substitutions were found also in the normal population. The heterozygosity and homozygosity frequencies of the Val241 and Val180 mutations were 48%, 38%, 22% and 1.5%, respectively. We also found a nonconservative amino acid substitution (T663A) in the αENaC, which was located close to the C-terminal (106). Of the 5 patients, 2 were homozygous and 3 heterozygous for this variation, respectively. This amino acid substitution was also present at high frequency in apparently normal controls. The homozygosity and heterozygosity frequencies of the αENaC Ala663 were 31% and 64%, respectively. Three of the 4 (75%) patients with multiple tissue resistance to aldosterone had both αENaC (heterozygous or homozygous) and MR (homozygous) mutations as described above, while only 7% of our controls with apparently normal salt conservation had the same concurrent abnormalities (Table 2, p < 0.025).

We also identified, in a Japanese patient with sporadic PHA, three homozygous substitutions in the MR gene: G215C, I180V or A241V, which had previously reported to occur in healthy populations. Luciferase activities induced by MR with either G215C, I180V or A241V substitution were significantly lower than those for wild-type MR with aldosterone at concentrations ranging from 10-11 to 10-9 M, 10-8M, or 10-11 to 10-6M, respectively. A homozygous A to G substitution of the donor splice site of αENaC intron 4 was found in the patient. These results suggest that each of three MR polymorphisms identified in our patient is functionally and structurally heterogeneous (111.1).

We cannot rule out the possibility that the above polymorphisms may confer vulnerability in salt conservation, which might be expressed fully only when concurrently present with other genetic defects of the MR or other proteins that participate in sodium homeostasis, such as NEDD4 (112). Our hypothesis, if true, would be compatible with a sporadic presentation or a digenic or multigenic expression and heredity as previously described in retinitis pigmentosa (113). In this case, hereditary transmission might be complex and appear either as a dominant and/or recessive trait with variable penetrance.