Martin Wolley

Role for Germline Mutations and a Rare Coding Single Nucleotide Polymorphism Within the KCNJ5 Potassium Channel in a Large Cohort of Sporadic Cases of Primary Aldosteronism

Primary aldosteronism (autonomous aldosterone production with suppressed renin) plays an important pathophysiological role in what has been previously labeled as essential hypertension. Besides the recently described germline mutations in the KCNJ5 potassium channel associated with familial primary aldosteronism, somatic mutations in the same channel have been identified within aldosterone-producing adenomas. In this study, we have resequenced the flanking and coding region of KCNJ5 in peripheral blood DNA from 251 white subjects with primary aldosteronism to look for rare variants that might be important for the pathophysiology of sporadic primary aldosteronism. We have identified 3 heterozygous missense mutations (R52H, E246K, and G247R) in the cohort and found that 12 (5% of the cohort) were carriers for the rare nonsynonymous single nucleotide polymorphism rs7102584 causing E282Q substitution of KCNJ5. By expressing the channels in Xenopus oocytes and human adrenal H295R cells, we have shown that the R52H, E246K, and E282Q substitutions are functional, but the G247R mutation is indistinguishable from wild type. Although the functional substitutions are remote from the selectivity filter, they affect the inward-rectification, the ability of the KCNJ5 channels to conduct Na+ currents and ATII-induced aldosterone release from the H295R cell line. Together these data suggest that germline variation in the KCNJ5 gene has a role to play in the common sporadic form as well as the much rarer syndromic forms of primary aldosteronism.

Does contralateral suppression at adrenal venous sampling predict outcome following unilateral adrenalectomy for primary aldosteronism? A retrospective study.

CONTEXT In primary aldosteronism (PA), adrenal vein sampling (AVS) distinguishes unilateral and bilateral disease by comparison of aldosterone/cortisol (A/F) ratios. There is controversy about the criteria for interpretation, however, and in particular it is not clear whether contralateral suppression (CS) (defined as A/F(adrenal) ≤ A/F(peripheral) on the unaffected side) is important. We therefore performed a retrospective study to determine whether CS in surgically treated unilateral PA was associated with blood pressure (BP) and biochemical outcomes. SETTING AND DESIGN Patients who underwent unilateral adrenalectomy for PA after successful AVS were included if the lateralization index (A/F(dominant):A/F(nondominant)) was ≥ 2. Cases were reviewed at 6 to 24 months follow-up for outcomes with respect to the presence and degree of CS. RESULTS Sixty-six of 80 patients had CS. Baseline characteristics were similar. At postoperative follow-up, those with CS had lower systolic BP (SBP) (128 mm Hg vs 144 mm Hg, P = .001), a greater proportion with cure or improvement of hypertension (96% vs 64%, P = .0034), a greater proportion with biochemical cure of PA on fludrocortisone suppression testing (43 of 49 [88%] vs 4 of 9 [44%], P = .002) and were taking a lower median number of antihypertensive medications (0 vs 1.5, P = .0032). In a multivariate model, the degree of CS and preoperative SBP were both significantly correlated with postoperative SBP, but the lateralization index, sex, and age were not. CONCLUSION In this study, the presence of CS correlated with good BP and biochemical outcomes from surgery. This finding suggests that CS should be a factor in deciding whether to offer surgery for treatment of PA.

Adverse Cardiovascular Outcomes of Corticosteroid Excess

Corticosteroid excess is associated with adverse cardiovascular outcomes. Patients with Cushingss syndrome, either caused by endogenous or exogenous glucocorticoid excess, and patients with primary aldosteronism have increased cardiovascular risk. The increase in risk is mediated partly by traditional cardiovascular risk factors including hypertension and metabolic syndrome but also by other, less well-characterized mechanisms. Experimental and human studies have shown that target organ deterioration induced by aldosterone depends on concomitant high dietary salt intake. Key ongoing research questions that warrant further study by both clinical and experimental approaches include the following: 1) beyond inducing the metabolic syndrome, what are the mechanisms by which glucocorticoids are associated with excess cardiovascular risk, 2) what are the cellular pathways by which excessive mineralocorticoid receptor activation brings about cardiovascular and renal damage, and 3) why is salt critical in this process?

Detection of mutations in KLHL3 and CUL3 in families with FHHt (familial hyperkalaemic hypertension or Gordon's syndrome)

The study of families with rare inherited forms of hypo- and hyper-tension has been one of the most successful strategies to probe the molecular pathophysiology of blood pressure control and has revealed dysregulation of distal nephron Na+ reabsorption to be a common mechanism. FHHt (familial hyperkalaemic hypertension; also known as Gordons syndrome) is a salt-dependent form of hypertension caused by mutations in the regulators of the thiazide-sensitive Na+–Cl− co-transporter NCC [also known as SLC12A3 (solute carrier family 12 member 3)] and is effectively treated by thiazide diuretics and/or dietary salt restriction. Variation in at least four genes can cause FHHt, including WNK1 [With No lysine (=K) 1] and WNK4, KLHL3 (kelch-like family member 3), and CUL3 (cullin 3). In the present study we have identified novel disease-causing variants in CUL3 and KLHL3 segregating in 63% of the pedigrees with previously unexplained FHHt, confirming the importance of these recently described FHHt genes. We have demonstrated conclusively, in two unrelated affected individuals, that rare intronic variants in CUL3 cause the skipping of exon 9 as has been proposed previously. KLHL3 variants all occur in kelch-repeat domains and so probably disrupt WNK complex binding. We have found no evidence of any plausible disease-causing variants within SLC4A8 (an alternative thiazide-sensitive sodium transporter) in this population. The results of the present study support the existing evidence that the CUL3 and KLHL3 gene products are physiologically important regulators of thiazide-sensitive distal nephron NaCl reabsorption, and hence potentially interesting novel anti-hypertensive drug targets. As a third of our non-WNK FHHt families do not have plausible CUL3 or KLHL3 variants, there are probably additional, as yet undiscovered, regulators of the thiazide-sensitive pathways.

Variation in and prognostic importance of troponin T measured using a high-sensitivity assay in clinically stable haemodialysis patients

Background A recently introduced high-sensitivity assay can measure troponin T (hsT) at low levels with greater precision than the fourth generation troponin T assay. As most patients with end-stage renal failure (ESRF) may have elevated hsT levels, data on biological variability and the impact of haemodialysis are needed for clinical interpretation of results. Methods This is a prospective observational cohort study aiming to identify baseline levels of hsT in stable haemodialysis patients in addition to examining variation in levels over time. Cardiovascular (CV) mortality was analysed at 6 months after the baseline hsT measurement. hsT was measured prior to the haemodialysis using the high-sensitivity Roche troponin T assay in 239 prevalent haemodialysis patients. In a subset of 78 patients, repeat measurements were made 1 month later, both before and after haemodialysis. Results hsT was above the 99th centile for the normal healthy population (14 ng/mL) in 98% of patients with a median level of 63 ng/L [Interquartile range (IQR) 37–108]. Higher hsT levels were associated with diabetes and left ventricular ejection fraction <50%. hsT was higher in patients who died from CV causes (median 418, IQR 109–776) compared with alive patients (median 59.5, IQR 36–96 P = 0.0027), and this association remained significant after adjustment for other predictors of mortality. In 95% of stable patients, variation in hsT over 1 month was <54%. In three patients with unstable coronary artery disease, hsT varied by >100% and >100 ng/L. Haemodialysis reduced hsT by a median of 24% (IQR 6–22, P = 0.0001). Conclusions hsT levels are elevated in almost all patients with ESRF. Variation in hsT over 1 month was <50% in most patients. Greater variation may indicate an acute coronary syndrome or worsening cardiac disease.

Repeating adrenal vein sampling when neither aldosterone/cortisol ratio exceeds peripheral yields a high incidence of aldosterone-producing adenoma

Objectives: In primary aldosteronism, adrenal vein sampling (AVS) suggests unilateral aldosterone-producing adenoma (APA) when the aldosterone/cortisol (A/F) ratio is less than or equal to peripheral on one side and at least two times peripheral on the other. When A/F ratios are lower bilaterally than peripheral despite adequate samples (adrenal venous cortisol ≥3 times peripheral), we recommend repeat AVS. This study aimed to determine the frequency of this occurrence and outcomes in such cases. Methods: We performed a retrospective observational study of all cases of primary aldosteronism undergoing initial AVS over a 34-year period. Results: Initial AVS in 1397 patients returned satisfactory and discriminatory results in 1066 (76.3%) but 37 patients (2.6%) had adequate samples but bilateral A/F ratios no higher than peripheral. Of the 22 of these 37 who agreed to repeat AVS, 10 demonstrated unilateral aldosterone production, and eight of these had unilateral adrenalectomy disclosing APAs and resulting in cure (3) or improvement (5) in hypertension. Eight had bilateral aldosterone production. Four studies were inconclusive. Patients with initial unsatisfactory AVS because of bilaterally low A/F ratios had significantly (P = 0.023) more unilateral disease [10 of 18 satisfactory repeat studies (55.6%) vs. 326 of 1066 satisfactory initial studies (30.6%)] and a significantly higher (67.6 vs. 49.9%, P = 0.034) percentage of males. Conclusion: As the incidence of APAs was high in a subgroup with low A/F bilaterally on initial AVS, these patients should be offered repeat AVS. This might reflect both a greater dependence of aldosterone production on adrenocorticotrophic hormone (ACTH) in APAs and the pulsatile nature of ACTH secretion.

Does ACTH improve the diagnostic performance of adrenal vein sampling for subtyping primary aldosteronism

Adrenal vein sampling (AVS) is used for determining treatment options for primary aldosteronism (PA), but is a difficult procedure. Adrenocorticotropic hormone (ACTH) infusion or bolus has been reported to improve AVS success rates by increasing cortisol secretion, but effects on lateralization are controversial. We therefore assessed the effects of ACTH in regard to AVS success and lateralization in our unit, after a change in protocol to ACTH‐stimulated AVS.

Should aldosterone suppression tests be conducted during a particular phase of the menstrual cycle, and, if so, which phase? Results of a preliminary study

As renin and aldosterone levels vary during the menstrual cycle, and are critical criteria for interpretation of aldosterone suppression tests to confirm or exclude primary aldosteronism, outcome of testing may vary depending on the menstrual cycle phase. We assessed the effect of timing within the menstrual cycle on levels of renin, aldosterone and female sex steroids during fludrocortisone suppression testing (FST).

In primary aldosteronism, mineralocorticoids influence exosomal sodium-chloride cotransporter abundance

Distal tubular sodium retention is a potent driver of hypertension, and the thiazide-sensitive sodium-chloride cotransporter (NCC) has a key role in this process. In humans, factors regulating NCC are unclear, but in animal models, aldosterone is a potent regulator, possibly via effects on plasma potassium. We studied the effects of the mineralocorticoid fludrocortisone on the abundance of NCC and its phosphorylated form (pNCC) as well as WNK lysine deficient protein kinase 4 (WNK4) and STE20/SPS1-related, proline alanine-rich kinase (SPAK) in human urinary exosomes. We isolated exosomes from daily urine samples in 25 patients undergoing fludrocortisone suppression testing (100 μg every 6 hours for 4 days) to diagnose or exclude primary aldosteronism. Over the course of the test, NCC levels increased 3.68-fold (P<0.01) and pNCC levels increased 2.73-fold (P<0.01) relative to baseline. The ratio of pNCC/NCC dropped by 48% (P<0.01). The abundance of WNK4 increased 3.23-fold (P<0.01), but SPAK abundance did not change significantly (P=0.14). Plasma potassium concentration strongly and negatively correlated with pNCC, NCC, and WNK4 abundance (P<0.001 for all). This study shows that, in humans, mineralocorticoid administration is associated with a rapid increase in abundance of NCC and pNCC, possibly via the WNK pathway. These effects may be driven by changes in plasma potassium.

Effect of moxonidine on the aldosterone/renin ratio in healthy male volunteers

Background The most popular screening test for primary aldosteronism is the plasma aldosterone/renin ratio (ARR). Medications, dietary sodium, posture, and time of day all affect renin and aldosterone levels and can result in false-negative or false-positive ARRs if not controlled. Most antihypertensive medications affect the ARR and can interfere with interpretation of results. To our knowledge, no study has been undertaken to evaluate the effects of moxonidine on the ARR. Methods Normotensive, nonmedicated male volunteers (n = 20) underwent measurement (seated, midmorning) of plasma aldosterone (by high-performance liquid chromatography-tandem mass spectrometry), direct renin concentration (DRC), plasma renin activity (PRA), cortisol, electrolytes and creatinine; and urinary aldosterone, cortisol, electrolytes and creatinine at baseline and after 1 week of moxonidine at 0.2 mg/d and a further 5 weeks at 0.4 mg/d. Results Compared with baseline, despite the expected significant falls in both systolic and diastolic blood pressure, levels of plasma aldosterone [median, 134 (range, 90 to 535) pmol/L], DRC [20 (10 to 37) mU/L], PRA [2.2 (1.0-3.8) ng/mL/h], and ARR using either DRC [8.0 (4.4 to 14.4)] or PRA [73 (36 to 218)] were not significantly changed after either 1 [135 (98-550) pmol/L, 20 (11-35) mU/L, 2.0 (1.2-4.1) ng/mL/h, 8.8 (4.2 to 15.9), and 73 (32-194), respectively] or 6 weeks [130 (90-500) pmol/L, 22 (8 to 40) mU/L, 2.1 (1.0 to 3.2) ng/mL/h, 7.7 (4.3 to 22.4), and 84 (32 to 192), respectively] of moxonidine. There were no changes in any urinary measurements. Conclusion Moxonidine was associated with no significant change in the ARR and may therefore be a good option for maintaining control of hypertension when screening for primary aldosteronism.