Pathogenesis of hypertension in a mouse model for human CLCN2 related hyperaldosteronism

Abstract

Human primary aldosteronism (PA) can be caused by mutations in several ion channel genes but mouse models replicating this condition are lacking. We now show that almost all known PA-associated CLCN2 mutations markedly increase ClC-2 chloride currents and generate knock-in mice expressing a constitutively open ClC-2 Cl− channel as mouse model for PA. The Clcn2op allele strongly increases the chloride conductance of zona glomerulosa cells, provoking a strong depolarization and increasing cytoplasmic Ca2+ concentration. Clcn2op mice display typical features of human PA, including high serum aldosterone in the presence of low renin activity, marked hypertension and hypokalemia. These symptoms are more pronounced in homozygous Clcn2op/op than in heterozygous Clcn2+/op mice. This difference is attributed to the unexpected finding that only ~50 % of Clcn2+/op zona glomerulosa cells are depolarized. By reproducing essential features of human PA, Clcn2op mice are a valuable model to study the pathological mechanisms underlying this disease.

CUL4-DDB1-CRBN E3 Ubiquitin Ligase Regulates Proteostasis of ClC-2 Chloride Channels: Implication for Aldosteronism and Leukodystrophy

“…Importantly, we also provide the first direct evidence indicating that an equivalent aldosteronism-causing mutant in mouse ClC-2 (G32D) is associated with a more than 70% increase in the total protein level, suggesting that this disease-causing mutation may affect the biochemical property of ClC-2 channels by promoting protein expression. This apparent gain in ClC-2 proteostasis is reminiscent of the gain-of-function phenotype of aldosteronism-causing human ClC-2 mutant channels that manifest as enhanced membrane Cl − currents in aldosterone-producing adrenal glomerulosa cells [ 12 , 13 , 14 ]. We therefore propose that, in addition to promoting voltage-dependent channel activation, aldosteronism-causing CLCN2 mutations may also facilitate ClC-2 protein folding and consequently reduce ER-associated degradation of the Cl − channel ( Figure 12 ).…”

“…Various mutations in the human CLCN2 gene, which encodes the ClC-2 channel, have been associated with distinct types of genetic diseases. In primary aldosteronism, gain-of-function mutations in the CLCN2 gene lead to enhanced Cl − efflux and therefore membrane depolarization in aldosterone-producing adrenal glomerulosa cells, manifesting as constitutive aldosterone secretion, hypertension, and hypokalemia [ 11 , 12 , 13 , 14 , 15 ]. On the other hand, loss-of-function mutations in the CLCN2 gene have been linked to a type of leukodystrophy (white matter disorder), CLCN2 -related leukoencephalopathy, characterized by intramyelinic edema in the brain [ 16 , 17 , 18 ], which is reminiscent of the myelin vacuolation found in ClC-2 knockout mice [ 10 ].…”

“…Biophysical analyses indicate that functional expression of Cl − currents is notably enhanced and diminished in aldosteronism- and leukodystrophy-associated ClC-2 mutant channels, respectively. The mechanism underlying the enhanced cell surface Cl − conductance in aldosteronism can be attributed to altered voltage-dependent gating properties that increase the current amplitude of mutant ClC-2 channels [ 12 , 13 , 14 ]. In contrast, leukodystrophy-associated mutations result in altered voltage-dependent gating properties that reduce the current amplitude of mutant ClC-2 channels [ 18 ].…”

NKCC1, an Elusive Molecular Target in Brain Development: Making Sense of the Existing Data

“…(d) NKCC1 regions used for producing the antibodies (green). (1) Affinity-purified rabbit antibody produced against the entire N-terminus of human NKCC1 [27], (2) polyclonal rabbit antibody raised against amino acids 3-202 of rat NKCC1 [28], (3) α-NT, rabbit polyclonal antibody against aa 238-261 or rat NKCC1 [29], (4) TEFS-2, affinity-purified rabbit polyclonal antibody produced against the entire C-terminus of human NKCC1 (characterized in [30]), (5) α-wCT, rabbit polyclonal antibody, aa 750-1203 of rat NKCC1 [29], (6) T4, mouse monoclonal antibody, aa 902-1212 of human NKCC1 [31], (7) rabbit polyclonal antibody, aa 938-1011 of mouse NKCC1 [32], (8) Chemicon International, affinity-purified rabbit polyclonal antibody, aa 967-988 of mouse NKCC1 [29,33], (9) affinity-purified rabbit polyclonal antibody raised against aa 977-991 of mouse NKCC1 [34]. Linear protein sequences of mouse, human, and rat NKCC1 are aligned and depicted in gray, with mismatches shown by vertical lines and gaps as dashes.…”

Identification of Somatic Mutations in CLCN2 in Aldosterone-Producing Adenomas

“…The consequential rise in cytoplasmic Ca 2+ leads to upregulation of CYP11B2 and increased aldosterone biosynthesis [ 27 , 28 ]. Recent mouse models expressing an open ClC-2 Cl − channel ( Clcn2 op ) [ 32 ] and a heterozygous Clcn2 mutation ( Clcn2 R180Q/+ ) at the location homologous to the p.R172Q mutation in humans [ 33 ] also demonstrated a PA phenotype.…”