Pathogenesis of pseudohypoaldosteronism type 2 by WNK1 mutations.

PURPOSE OF REVIEW: Pseudohypoaldosteronism type 2 (PHA2) is a rare autosomal dominant form of human arterial hypertension, associated with hyperkalemia and hyperchloremic metabolic acidosis. WNK1 and WNK4 are two of the genes mutated in PHA2 patients. This review focuses on the mechanisms by which deletions of the first intron of WNK1 found in PHA2 patients trigger the disease. RECENT FINDINGS: The WNK1 gene gives rise to a ubiquitous kinase (L-WNK1) and to a shorter kinase-defective isoform, KS-WNK1 (for kidney-specific WNK1), expressed only in the distal convoluted tubule (DCT) and connecting tubule. WNK1 first intron deletion leads to overexpression of L-WNK1 in the DCT and ubiquitous ectopic expression of KS-WNK1. The increased expression of L-WNK1 in the DCT results in increased activity of the Na-Cl cotransporter (NCC) and thus hypervolemia and hypertension. Contrarily, the mechanisms underlying the hyperkalemia and metabolic acidosis remain unclear. SUMMARY: As particularly small doses of thiazide diuretics, inhibitors of NCC activity, correct both the blood pressure and metabolic disorders in PHA2 patients, it was believed that increased NCC was directly responsible for all PHA2 features. Studies performed in mouse models of KS-WNK1 inactivation or WNK4-related PHA2, however, have revealed that the situation is much more complex.

Deletion of the NESP55 differentially methylated region causes loss of maternal GNAS imprints and pseudohypoparathyroidism type Ib.

Epigenetic defects in the imprinted GNAS cluster are associated with pseudohypoparathyroidism type Ib. In two kindreds with this disorder, we now report deletions that remove the differentially methylated region encompassing exon NESP55 and exons 3 and 4 of the antisense transcript. When inherited from a female, either deletion abolishes all maternal GNAS imprints and derepresses maternally silenced transcripts, suggesting that the deleted region contains a cis-acting element that controls imprinting of the maternal GNAS allele.

Immunochemical analysis of the alpha-subunit of the stimulatory G-protein of adenylyl cyclase in patients with Albright's hereditary osteodystrophy.

Albright's hereditary osteodystrophy (AHO) is an autosomal dominant disorder characterized by an unusual phenotypic appearance and reduced biological activity of the alpha-subunit of the stimulatory G-protein of adenylyl cyclase (Gs alpha). In most AHO patients deficient Gs alpha activity is associated with generalized target organ resistance to hormones that act via stimulation of adenylyl cyclase. This form of the disorder is termed pseudohypoparathyroidism type Ia (PHP Ia). By contrast, other patients with Gs alpha deficiency fail to demonstrate clinical evidence of hormone resistance and are considered to have the related disorder pseudopseudohypoparathyroidism (pseudoPHP). Previous studies demonstrating deficient Gs alpha bioactivity in cell membranes from patients with AHO used functional assays that were unable to distinguish between reduced amounts of normal Gs alpha protein and normal amounts of defective Gs alpha protein. In the present study we used specific Gs alpha antisera to analyze immunoactive Gs alpha protein in erythrocyte and fibroblast membranes from 20 patients with AHO who had either normal or reduced levels of Gs alpha mRNA. Cell membranes were subjected to immunoblot analysis using Gs alpha antisera developed against synthetic peptides corresponding to amino acid sequences in the amino- or carboxyl-terminus of the Gs alpha molecule. Fibroblast membranes from patients with AHO who had reduced or normal levels of Gs alpha mRNA contained both the 45- and 52-kDa forms of the Gs alpha protein in quantities that were significantly less [mean +/- SE, 52 +/- 6%; (n = 8) for reduced mRNA and 35 +/- 19% (n = 2) for normal mRNA, percentage of control values] than those present in membranes from normal subjects. Similar reductions were found in the level of the 45-kDa form of Gs alpha in erythrocyte membranes from all AHO patients studied [40 +/- 4% (mean +/- SE) of control values]. No abnormal forms of Gs alpha protein were detected. Cell membranes from patients with PHP type Ia and from patients with pseudoPHP contained levels of immunoactive Gs alpha that were equivalently reduced (43 +/- 4% vs. 42 +/- 5%, respectively). By contrast, erythrocyte membranes from patients with PHP type Ib, who have normal Gs alpha activity, had normal levels of Gs alpha immunoactivity (101 +/- 7%). These results indicate that most patients with AHO have reduced levels of Gs alpha protein as the basis for deficient Gs alpha bioactivity.

Effects of parathyroid hormone on urinary excretion of N-acetyl-beta-D-glucosaminidase in idiopathic hypoparathyroidism and pseudohypoparathyroidism.

N-acetyl-beta-D-glucosaminidase (NAG) is a lysosomal enzyme predominantly located in renal proximal tubules. In idiopathic hypoparathyroidism (IHP), 100 Units of human PTH (1-34) increased urinary excretion of NAG from 0.029 +/- 0.027 to 0.173 +/- 0.035 U/lGF (p less than 0.05) in two patients before treatment and from 0.025 +/- 0.004 to 0.189 +/- 0.092U/lGF (p less than 0.02) in four patients during treatment with active vitamin D3 (1,25(OH)2D3 or 1 alpha OHD3). In pseudohypoparathyroidism (PHP), PTH did not significantly increase the urinary excretion of NAG in one patient with before treatment (0.048 to 0.025 U/lGF) and four patients during treatment with active vitamin D3 (0.018 +/- 0.008 to 0.036 +/- 0.015 U/lGF). Increase in urinary excretion of NAG after injection of PTH may be a new indicator of renal effect of PTH.

New form of pseudohypoparathyroidism with abnormal catalytic adenylate cyclase.

Patients with pseudohypoparathyroidism type Ia have resistance to multiple hormones because of deficient activity of the stimulatory guanine nucleotide-binding protein (Gs) that couples membrane receptors to activation of adenylate cyclase. However, in a subset of patients with pseudohypoparathyroidism who have resistance to multiple hormones yet possess normal erythrocyte membrane Gs activity, the biochemical abnormality responsible for hormone resistance has remained undefined. Cultured skin fibroblasts were derived from a patient with this atypical form of pseudohypoparathyroidism. In the patient's fibroblast membranes, adenylate cyclase stimulation mediated by Gs after fluoride ion treatment produced only 52% of normal activity, yet fibroblast membrane Gs activity measured by cyc- complementation was normal. Activation of the catalytic unit of adenylate cyclase with manganese produced 49% of normal activity; manganese plus forskolin produced 54% of normal adenylate cyclase activity. beta-Adrenergic receptor coupling to Gs and phosphodiesterase activity were normal. A defect in the catalytic unit of adenylate cyclase can account for these results and may be a mechanism for clinical resistance to multiple hormones that act through adenylate cyclase.

Clinical investigation of olfactory and auditory function in type I pseudohypoparathyroidism: participation of adenylate cyclase system.

Olfactory and auditory function was examined in five patients with type I pseudohypoparathyroidism; in two patients the activity of stimulatory guanine nucleotide-binding protein was examined and found to have low values. The olfactory tests were performed by T & T olfactometer and intravenous injection of thiamine propyl disulphide. The four patients receiving olfactory test showed a disturbance in recognization of the odorants. All five patients had normal hearing at frequencies with the normal range. An adenylate cyclase system is thought to play an important role in olfactory transduction, whereas cochlear function may be unaffected by this system in the normal state.

The stimulatory and inhibitory guanine nucleotide-binding proteins of adenylate cyclase in erythrocytes from patients with pseudohypoparathyroidism type I.

Both the inhibitory and stimulatory guanine nucleotide-binding proteins of the adenylate cyclase complex were measured in erythrocyte membranes from patients with pseudohypoparathyroidism (PHP). The inhibitory guanine nucleotide-binding protein (Ni) of adenylate cyclase was measured by incorporation of [32P]ADP-ribose from [32P]NAD into the 39K subunit of Ni catalyzed by pertussis toxin. The ADP-ribosyltransferase activity of the toxin was expressed through incubation with dithiothreitol and erythrocyte membranes. Erythrocytes from 12 patients with PHP type I (PHP-I) had Ni values similar to those of 9 normal subjects and 2 patients with pseudopseudohypoparathyroidism. In 6 PHP-I patients, decreased activity of the stimulatory guanine nucleotide-binding protein (Ns) of adenylate cyclase, as determined by reconstitution of adenylate cyclase in the Ns-deficient membranes of cyc-S49 cells, corresponded with the reduced degree of ADP-ribosylation of the 42K subunit of Ns catalyzed by cholera toxin. These data suggest that the defect of Ns results in reduced stimulation of adenylate cyclase in some PHP-I patients, and that enhanced inhibition of the enzyme due to an increase in the 39K subunit of Ni does not account for the biochemical lesion in PHP-I patients.

Deficient adenylate cyclase regulatory protein in renal membranes from a patient with pseudohypoparathyroidism.

Recent studies have established that some patients with pseudohypoparathyroidism have a deficiency of the adenylate cyclase regulatory protein (the G unit) in plasma membranes from erythrocytes, platelets, and fibroblasts. We have directly measured the activity of the G unit in renal membranes from a patient with pseudohypoparathyroidism who, in addition to parathyroid hormone resistance, has resistance to thyrotropin and gonadotropins. Erythrocyte membrane G unit activity was 57% that of control erythrocyte membranes. Lubrol PX extracts of renal membranes had only 30% of the G unit activity of control renal membrane extracts, whether assayed with sodium fluoride or guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma-S). In cholate extracts, the G unit activity was 37 and 48% of control with fluoride or GTP-gamma-S, respectively. Cholera toxin-dependent incorporation of [32P]ADP-ribose into the 42,000-Mr subunit of the G unit was decreased in renal membranes from the patient compared with control renal membranes. The data demonstrate that the membrane G unit deficiency in pseudohypoparathyroidism extends to the cells of a clinically relevant parathyroid hormone target tissue.

[Pseudohypoparathyroidism: a generalized and multiple disorder in the adenyl cyclase system]. / Pseudohypoparathyreoidismus: eine generalisierte und multiple Störung im hormonsensitiven Adenylzyklasesystem.

Pseudohypoparathyroidism (PHP) is caused by non response of target organs (kidney and bone) to parathyroid hormone because of defects in adenylate cyclase-system. The often seen combination of other endocrine abnormalities with PHP speak for a generalized defect of hormonsensitive adenylate cyclases, but not for isolated resistance to parathyroid hormone. At least two defects are known: in Typ I-PHP the defect is before the formation of cyclic AMP (cAMP) because of a reduced activity of receptor-cyclase coupling protein. In Typ II-PHP the defect lies behind cAMP-formation (defect in the reception of cAMP-signal?).

Pseudohypoparathyroidism and hypocalcemic "myopathy". A case report.

A patient with the clinical features of pseudohypoparathyroidism and elevated concentrations of serum CK and LDH, which normalized after successful therapy, is described. Clinical signs of myopathy did not exist. The bioptical material from the m. tibialis anterior was microscopically normal. The biochemical analysis revealed a reduced phosphorylase-a-activity with the total phosphorylase-activity (a and b) being within the normal range. The significance of these findings as well as possible pathogenetic mechanisms are discussed.

Human mutation affecting hormone-sensitive adenylate cyclase.

Hormone-sensitive adenylate cyclase contains a recently discovered protein component that is required for stimulation of cyclic AMP synthesis by hormones and guanine nucleotides. We measured this protein in erythrocyte membranes of ten patients with pseudohypoparathyroidism (PHP), using assays of its biochemical activity and of its susceptibility to radiolabeling in the presence of 32P-NAD and cholera toxin. By both assays, the protein was reduced by 50% in erythrocytes of 4 PHP patients, as compared with normal and hypoparathyroid subjects. These 4 subjects, in contrast to the 6 PHP patients (5 in one family) whose erythrocytes contained apparently normal amounts of the cyclase component, exhibited the full spectrum of skeletal abnormalities found in PHP. We conclude that partial deficiency of the guanine nucleotide regulatory protein is a biochemical marker for a subset of PHP patients. If present in other tissues, this deficiency could explain the resistance of target organs in PHP to parathormone and other hormones that work via cyclic AMP.

Friedreich's ataxia forme fruste and elevated creatine phosphokinase in a child with pseudohypoparathyroidism.

A 14-year-old boy with focal seizures, syncopal attacks, and progressive intellectual deterioration had intracranial calcifications on a CT scan and hypocalcemia unresponsive to parathyroid hormone. The commonly recognized neurological manifestations of pseudohypoparathyroidism were complicated by signs of Friedreich's ataxia. A marked elevation of serum CPK was unexplained and was unrelated to seizures.

Altered activity of the nucleotide regulatory site in the parathyroid hormone-sensitive adenylate cyclase from the renal cortex of a patient with pseudohypoparathyroidism.

A series of clinical studies suggest that the primary defect underlying pseudohypoparathyroidism is an abnormality of the parathyroid hormone-receptor-adenylate cyclase complex of the renal cortical cell plasma membrane. In the present study we compared parathyroid hormone-stimulated adenylate cyclase activity in membrane preparations from the renal cortex of three controls and a patient with pseudohypoparathyroidism. In the pseudohypoparathyroid preparation the Km for ATP was significantly greater and parathyroid hormone elicited markedly diminished adenylate cyclase activity at a subsaturating concentration of ATP. In contrast, the dose-response effect of enzyme activity to parathyroid hormone was the same in the control preparations, and that of the pseudohypoparathyroidism kidney, at a saturating concentration of ATP. The apparent alteration in enzyme kinetics, however, was normalized upon addition of guanosine 5'-triphosphate to the reaction mixtures. These results indicate that the defect in the parathyroid hormone-receptor-adenylate cyclase complex of the renal cell membranes, in our patient with pseudohypoparathyroidism, is an abnormal nucleotide receptor site of decreased activity. Such a defect may result in partial uncoupling of the parathyroid hormone receptor and adenylate cyclase, rendering the organ refractory to hormonal stimulation.

[Pseudohypoparathyroidism. Disease of the second messenger]. / Pseudo-Hypoparathyreoidismus. Eine Erkrankung des "zweiten Boten"

Pseudo-hypoparathyroidism (PHP) is a hereditary disorder with typical dysmorphic signs and symptoms of hypoparathyroidism, which is resistant to parathyroid hormone (PTH). The knowledge of the role of cyclic adenosine monophosphate (cAMP) as a mediator of the action of PTH and the finding, that parathyroid extract in PHP could not stimulate the urinary excretion of cAMP, were necessary to explain the hitherto unknown pathogenesis of Pseudo-hypoparathyroidism. The PTH-sensitive adenylcyclase-system as second messenger is here unable to mediate the action of PTH on its target cells causing disturbances in calcium and phosphorus metabolism.