Primary aldosteronism, a common cause of severe hypertension1, features constitutive production of the adrenal steroid aldosterone. We analyzed a multiplex family with familial hyperaldosteronism type II (FH-II)2 and 80 additional probands with unsolved early-onset primary aldosteronism. Eight probands had novel heterozygous variants in CLCN2, including two de novo mutations and four independent occurrences of the identical p.Arg172Gln mutation; all relatives with early-onset primary aldosteronism carried the CLCN2 variant found in probands. CLCN2 encodes a voltage-gated chloride channel expressed in adrenal glomerulosa that opens at hyperpolarized membrane potentials. Channel opening depolarizes glomerulosa cells and induces expression of aldosterone synthase, the rate-limiting enzyme for aldosterone biosynthesis. Mutant channels cause gain of function, with higher open probabilities at the glomerulosa resting potential. These findings for the first time demonstrate a role of anion channels in glomerulosa membrane potential determination, aldosterone production and hypertension. They establish the cause of a substantial fraction of early-onset primary aldosteronism.

Gain-of-function mutations in the chloride channel ClC-2 were recently described as a cause of familial hyperaldosteronism type II (FH-II). Here, we report the generation of a mouse model carrying a missense mutation homologous to the most common FH-II-associated CLCN2 mutation. In these Clcn2R180Q/+ mice, adrenal morphology is normal, but Cyp11b2 expression and plasma aldosterone levels are elevated. Male Clcn2R180Q/+ mice have increased aldosterone:renin ratios as well as elevated blood pressure levels. The counterpart knockout model (Clcn2−/−), in contrast, requires elevated renin levels to maintain normal aldosterone levels. Adrenal slices of Clcn2R180Q/+ mice show increased calcium oscillatory activity. Together, our work provides a knockin mouse model with a mild form of primary aldosteronism, likely due to increased chloride efflux and depolarization. We demonstrate a role of ClC-2 in normal aldosterone production beyond the observed pathophysiology.

Primary aldosteronism is characterized by at least partially autonomous production of the adrenal steroid hormone aldosterone and is the most common cause of secondary hypertension. The most frequent subforms are idiopathic hyperaldosteronism and aldosterone-producing adenoma. Rare causes include unilateral hyperplasia, adrenocortical carcinoma and Mendelian forms (familial hyperaldosteronism). Studies conducted in the last eight years have identified somatic driver mutations in a substantial portion of aldosterone-producing adenomas, including the genes KCNJ5 (encoding inwardly rectifying potassium channel GIRK4), CACNA1D (encoding a subunit of L-type voltage-gated calcium channel Ca V 1.3), ATP1A1 (encoding a subunit of Na + /K +-ATPase), ATP2B3 (encoding a Ca 2+-ATPase), and CTNNB1 (encoding ß-catenin). In addition, aldosterone-producing cells were recently reported to form small clusters (aldosterone-producing cell clusters) beneath the adrenal capsule. Such clusters accumulate with age and appear to be more frequent in individuals with idiopathic hyperaldosteronism. The fact that they are associated with somatic mutations implicated in aldosterone-producing adenomas also suggests a precursor function for adenomas. Rare germline variants of CYP11B2 (encoding aldosterone synthase), CLCN2 (encoding voltage-gated chloride channel ClC-2), KCNJ5, CACNA1H (encoding a subunit of T-type voltage-gated calcium channel Ca V 3.2), and CACNA1D have been reported in different subtypes of familial hyperaldosteronism. Collectively, these studies suggest that primary aldosteronism is largely due to genetic mutations in single genes, with potential implications for diagnosis and therapy.

Background: Microbial infections are a relevant problem for patients with liver cirrhosis. Different types of bacteria are responsible for different kinds of infections: Escherichia coli and Klebsiella pneumoniae are frequently observed in spontaneous bacterial peritonitis or urinary tract infections, and Streptococcus pneumoniae and Mycoplasma pneumoniae in pulmonary infections. Mortality is up to 4-fold higher in infected patients with liver cirrhosis than in patients without infections. Key Messages: Infections in patients with liver cirrhosis are due to three major reasons: bacterial translocation, immune deficiency and an increased incidence of systemic infections. Nonparenchymal liver cells like Kupffer cells, sinusoidal endothelial cells and hepatic stellate cells are the first liver cells to come into contact with microbial products when systemic infection or bacterial translocation occurs. Kupffer cell (KC) activation by Toll-like receptor (TLR) agonists and endothelial sinusoidal dysfunction have been shown to be important mechanisms increasing portal pressure following intraperitoneal lipopolysaccharide pretreatment in cirrhotic rat livers. Reduced intrahepatic vasodilation and increased intrahepatic vasoconstriction are the relevant pathophysiological pathways. Thromboxane A₂ and leukotriene (LT) C₄/D₄ have been identified as important vasoconstrictors. Accordingly, treatment with montelukast to inhibit the cysteinyl-LT₁ receptor reduced portal pressure in cirrhotic rat livers. Clinical studies have demonstrated that activation of KCs, estimated by the amount of soluble CD163 in the blood, correlates with the risk for variceal bleeding. Additionally, intestinal decontamination with rifaximin in patients with alcohol-associated liver cirrhosis reduced the portal pressure and the risk for variceal bleeding. Conclusions: TLR activation of nonparenchymal liver cells by pathogens results in portal hypertension. This might explain the pathophysiologic correlation between microbial infections and portal hypertension in patients with liver cirrhosis. These findings are the basis for both better risk stratifying and new treatment options, such as specific inhibition of TLR for patients with liver cirrhosis and portal hypertension.

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Gain-of-function mutations in the CACNA1H gene (encoding the T-type calcium channel CaV3.2) cause autosomal-dominant familial hyperaldosteronism type IV (FH-IV) and early-onset hypertension in humans. We used CRISPR/Cas9 to generate Cacna1hM1560V/+ knockin mice as a model of the most common FH-IV mutation, along with corresponding knockout mice (Cacna1h−/−). Adrenal morphology of both Cacna1hM1560V/+ and Cacna1h−/− mice was normal. Cacna1hM1560V/+ mice had elevated aldosterone:renin ratios (a screening parameter for primary aldosteronism). Their adrenal Cyp11b2 (aldosterone synthase) expression was increased and remained elevated on a high-salt diet (relative autonomy, characteristic of primary aldosteronism), but plasma aldosterone was only elevated in male animals. The systolic blood pressure of Cacna1hM1560V/+ mice was 8 mmHg higher than in wild-type littermates and remained elevated on a high-salt diet. Cacna1h−/− mice had elevated renal Ren1 (renin-1) expression but normal adrenal Cyp11b2 levels, suggesting that in the absence of CaV3.2, stimulation of the renin-angiotensin system activates alternative calcium entry pathways to maintain normal aldosterone production. On a cellular level, Cacna1hM1560V/+ adrenal slices showed increased baseline and peak intracellular calcium concentrations in the zona glomerulosa compared to controls, but the frequency of calcium spikes did not rise. We conclude that FH-IV, on a molecular level, is caused by elevated intracellular Ca2+ concentrations as a signal for aldosterone production in adrenal glomerulosa cells. We demonstrate that a germline Cacna1h gain-of-function mutation is sufficient to cause mild primary aldosteronism, whereas loss of CaV3.2 channel function can be compensated for in a chronic setting.

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Kupffer cells (KCs) have a major role in liver injury, and cysteinyl-leukotrienes (Cys-LTs) are known to be involved as well. The KC-mediated pathways for the production and secretion of Cys-LT in cholestatic liver injury have not yet been elucidated. Here, we hypothesized that KC activation by Toll-like receptor ligands results in Cys-LT-mediated microcirculatory alterations and liver injury in acute cholestasis. We hypothesized further that this situation is associated with changes in the secretion and production of Cys-LT. One week after bile duct ligation (BDL), livers showed typical histological signs of cholestatic liver injury. Associated microcirculatory disturbances caused increased basal and maximal portal pressure following KC activation. These differences were determined in BDL livers compared with shamoperated livers in vivo (KC activation by LPS 4 mg/kg b.w.) and in isolated perfused organs (KC activation by Zymosan A, 150 mg/ml). Treatment with the 5-lipoxygenase inhibitor MK-886 alone did not alter portal perfusion pressure, lactate dehydrogenase (LDH) efflux, or bile duct proliferation in BDL animals. Following KC activation, portal perfusion pressure increased. The degree of cell injury was attenuated by MK-886 (3 mM) treatment as estimated by LDH efflux. In normal rats, a large amount of Cys-LT efflux was found in the bile. Only a minor amount was found in the effluent perfusate. In BDL livers, the KC-mediated Cys-LT efflux into the sinusoidal system increased, although the absolute Cys-LT level was still grossly lower than the biliary excretion in sham-operated livers. In conclusion, our results indicate that treatment with Cys-LT inhibitors might be a relevant target for attenuating cholestatic liver damage.