European Journal of Endocrinology 10.1530/EJE-16-0369 Peter H Nissen Medicine, AarhusUniversity, Aarhus,Denmark, Leif Mosekilde parathyroid glands, bone, kidney and the cardiovascular parathyroid glands,bone,kidney andthecardiovascular to be expressed in many different tissues including the ( by Brownet al. wasfirstdescribed The calcium-sensingreceptor (CASR) Introduction Conclusions: FHHdoesnotseemtobeassociatedwithanincreased riskofCVD. osmolality ifanalyseswererestrictedtothosenotontreatments withantihypertensivedrugs. aldosterone andrenindidnotdiffer betweengroups. FHHpatientshadalowerurinaryvolumewithanincreased higher renalexcretionofepithelialsodiumchannels(ENaCs)(P associated withasignificantlyhigherplasmaosmolality(P 66 ±9mmHg, vs (60 ±5 females FHH in ambulatory BPorpulsewavevelocity. However, compared with controls,diastolicBPduringnighttimewaslower (33%) wereontreatmentwithantihypertensivedrugs.Overall,nodifferences werefoundbetweengroupsin24-h Results: Studiedsubjects(69%women)hadameanageof56 Design: Cross-sectionalstudycomparing50patientswithFHHtoage-andgender-matched controls. system bymeasuring24-hbloodpressure(BP),arterialstiffness andvasoactivehormones. health. We, thereforeinvestigatedwhetherFHHisassociatedwithchanges intheregulationofcardiovascular The CASRmayalsoinfluenceelectrolyteandwaterhomeostasis.ItisunknownwhetherFHHaffects cardiovascular hypocalciuric hypercalcemia(FHH),causingwithhighnormalorelevatedparathyroidhormonelevels. Objective: Loss-of-functionvariantsinthegeneencodingcalcium-sensingreceptor(CASR)resultfamilial Abstract 2 1 Niels Frederik Breum Jakobsen receptor gene inactivating variantsinthecalcium-sensing sectional studyonphysiologicaleffects of hypocalciuric hypercalcemia: across- The cardiovascular systeminfamilial Holstebro Hospital,HospitalJutlandWest, Holstebro,Denmark Aarhus UniversityHospital,Aarhus,Denmark,and Danish DiabetesAcademy, OdenseUniversityHospital,Odense,Denmark, Department ofEndocrinologyandInternalMedicine,AarhusUniversityHospital,Aarhus,Denmark, DOI: 10.1530/EJE-16-0369 www.eje-online.orgwww.eje-online.org Clinical Study 5 1 , PerLøgstrup Poulsen 1 andLars Rejnmark ) in1993.Today, thereceptorisknown © 2016EuropeanSociety ofEndocrinology © 2016EuropeanSociety ofEndocrinology N FBJakobsenandothers 1 , Esben Laugesen 1,3 4 Departments ofSurgeryand P 6

University ClinicinNephrologyandHypertension, < 1 , ErlingBjerregaard Pedersen 0.01) andhigherinFHHmales(69 Printed inGreatBritain 1,23 , Lars Rolighed

3 < Department ofClinical 0.01), higherplasmalevelsofvasopressin(P 5 Clinical Biochemistry, changes inplasma-ionizedcalcium (Ca system (2 pathways that modify parathyroid hormone (PTH) pathways thatmodifyparathyroid hormone(PTH) and tocouplethisinformation tointracellularsignaling years. Asimilarnumberofpatientsandcontrols FHH andcardiovascularhealth Published byBioscientifica Ltd.

= 0.03), whereasurineaquaporin-2andplasmasodium, ). Its major function is to sense even small ). Itsmajorfunctionisto senseevensmall 4 , 3,6 , ± 6 vs64 ± Downloaded fromBioscientifica.com at09/29/202110:58:26AM 5 mmHg, P [email protected] Email to LRejnmark should beaddressed Correspondence (2016) Endocrinology European Journal of 175 .2. H was FHH =0.02). 175:4 : 4

175, 299–309 2 + ) concentrations ) concentrations .1 ad a and <0.01)

299–309 via freeaccess Clinical Study N F B Jakobsen and others FHH and cardiovascular health 175:4 300

secretion and renal cation handling (3). Loss-of-function and BP has not yet been fully elucidated (7). Moreover, in variants in the gene encoding the CASR on chromosome primary hyperparathyroidism (PHPT), the state of 3q21.1 are known to cause familial hypocalciuric hyperparathyroid hypercalcemia is associated with hypercalcemia (FHH) type 1 (OMIN #145980). The an increased risk of cardiovascular diseases, including condition is inherited in an autosomal dominant manner hypertension and increased arterial stiffness (20, 21). As and causes an increase in the set point of the parathyroid the biochemical characteristics of FHH and PHPT are to glands, i.e. the plasma Ca2+ concentration at which PTH some extent similar, it may be speculated whether the secretion is half-maximal. Accordingly, higher than hyperparathyroid hypercalcemia in FHH affects risk of normal plasma Ca2+ levels are necessary to inhibit the cardiovascular diseases. release of PTH, resulting in hypercalcemia associated with The aim of this study is to investigate surrogate inappropriately normal or elevated plasma levels of PTH. markers of cardiovascular health in terms of vasoactive The degree of hypercalcemia depends on how severely hormones 24-h BP, and arterial stiffness in FHH compared the genetic variant affects the function of the CASR (4). with a sex- and age-matched control group. Similarly, calcium reabsorption is increased in the renal tubules causing a relatively low renal calcium excretion (5, 6). Subjects and methods There is considerable physiologic and genetic Design and participants evidence indicating that the CASR takes part in the renal salt and water regulation by influencing the function of In a cross-sectional study, we compared 50 FHH patients the renal outer medullary potassium channel (ROMK), with 51 sex- and age-matched population-based controls the expression of aquaporin-2 (AQP2) and the secretion of aged 18–85 years. The design of the study has previously renin (7). It is presumably due to the action of the CASR been detailed (22). In brief, patients with FHH were that renal calcium excretion is increased in response to recruited from our out-patient clinic and all except a high calcium load. Furthermore, the receptor causes a one had a genetically verified diagnosis. The patient decreased blood pressure (BP) following a high dietary without a genetically verified diagnosis was included intake of calcium in spontaneously hypertensive rats and based on a family history of hypercalcemia and a low humans with hypertension (8, 9). Finally, it has been renal calcium excretion as determined by calculating the shown that calcimimetics may exert a hypotensive effect calcium–creatinine clearance ratio. Age- (±2 years) and which is believed to be mediated through their effects on sex-matched controls were randomly selected from the European Journal European of Endocrinology the CASR in uremic rats (10), spontaneously hypertensive general background population. rats (11), and in kidney transplant humans (12). We excluded patients and controls with major The CASR is also expressed by cells in the cardiovascular medical or social problems including impaired renal system including the intimal and medial layers of large function (plasma creatinine > 125 μmol/L), malignancies, elastic arteries (13, 14). Loss-of-function of the CASR untreated intestinal malabsorption, chronic disabling has been suggested to be involved in the development disease or prior hospital admission due to chronic drug of vessel wall calcification which may be inhibited by or alcohol abuse. In addition, we excluded pregnant treatment with calcimimetics (15). Conflicting results women and patients treated with drugs known to affect have, however, been reported on the potential role of the the CASR (lithium, strontium or cinacalcet), as well as CASR on risk of CVD. By comparing 2561 patients with subjects diagnosed with calcium metabolic disorders coronary artery disease (CAD) with 698 controls, März except osteoporosis. et al. (16) reported an association between the common All participants gave verbal and written informed A986S CASR polymorphism and risk of angiographic consent and the study was conducted in accordance with CAD, previous myocardial infarction and cardiovascular the Declaration of Helsinki II. The study was approved mortality. In contrast, Babinsky et al. (17) found no by The Central Denmark Region Committees on Health effects of six common CASR polymorphisms (including Research Ethics (#M-2010-0296) and notified to The A986S) on risk of CAD in a cohort of 284 renal transplant Danish Data Protection Agency (#2011-41-5733). patients. In FHH, it has been speculated whether BP We asked participants to fill in a questionnaire regulation is affected (18, 19). It is well known that FHH concerning their state of health (Table 1). We assessed causes a resistance to the diuretic effect of calcium, but daily total calcium intake according to reported dietary the effect of the inactivating variant on sodium balance intakes of milk, cheese, milk products and use of calcium

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Calcium andvitaminDintake Diuretics Beta-blockers Medication, n(%) Switzerland). Thelowerlimit ofdetectiontheassayis e601 immunoassay analyzers (Roche Diagnostics, Basel, intact PTHbyasecond-generation assayusingCobas 41.2 and 25.3 25OHD2, theCVvalueswere8.8and9.4%atlevels of 6.4 and 9.1% at levels of 66.5 and 21.1 Coefficient ofvariance(CV)values(%) for25OHD3were SRM 972(Chromsystems,Münich,Germany)wereused. both 25OHD2and25OHD3.CalibratorstraceabletoNIST (LC-MS/MC)(25).Thismethodquantifies spectrometry by isotopedilutionliquidchromatography-tandemmass albumin (24). (P-Ca) wascorrectedforindividualvariationsinplasma Cobas c-systems(Cobasc501).Plasmatotalcalcium methodsusingRoche/Hitachi by standardlaboratory analysis. Generalbiochemicalparameterswereanalyzed were analyzedimmediatelyorstoredat−80°Cuntil participants collecteda24-hurinesample.Bloodsamples overnight fastandfollowing1 Blood sampleswerecollectedinthemorningafteran Biochemistry Sa-med, Kvistgaard,Denmark). in lightindoorclothingusingthesamescale(Seca, supplements (23).We measuredheightandbodyweight Intake fromdietandsupplements. Clinical Study Body massindex(kg/m Weight (kg) Height (cm) Total calciumintake(mg/day Dietary calciumintake(mg/day) User ofcalciumsupplements,n(%) Vitamin-D intakefromsupplement(μg/day) Use ofvitamin-Dsupplements,n(%) Calcium channelblockers Angiotensin receptorblockers Angiotensin-converting-enzyme inhibitors Any antihypertensivedrug Cholesterol-lowering drugs Plasma 25-hydroxyvitaminD(25OHD)wasanalyzed Characteristics ofpatientswithfamilialhypocalciurichypercalcaemia(FHH)andtheirmatchedcontrols.Datais nmol/L respectively. We measured plasma- n (%) 2 s ) ±( . d . s ) . d . † ) ormedianwithInterquartile(25;75%)range. ) h ofbedrest.Inaddition, N FBJakobsenandothers nmol/L, and for 170.1 (8.4) 1000 (800;1300) 1250 (970;1750) 77.0 (14.0) 26.6 (4.4) Controls (n 7.5 (0.0;20.0) 23 (65.7) 35 (68.6) 56 (23–82) 23 (45.1) 35 (68.6) 10 (20) 17 (33.3) 28 (54.9) 8 (16) 1 (2) 8 (16) 7 (14) 8 (15.7) with C assay). Vasopressin (AVP) wereextractedfromplasma The CVvalueswere9.0%(interassay)and8.5%(intra- Kiel, Germany. Minimaldetectionlevelwas25 by RIAusingakitfromDemeditecDiagnosticsGmbH, range of4–263 0.9–3.6% (intra-assay) and 3.7–5.0% (inter-assay) in the Minimal detectionlevelwas1 CIS BioInternational,Gif-Sur-Yvette Cedex,France. determined usinganimmunoradiometricassayfrom frozen until assayed. Plasma renin concentration was 4°C. Plasmawasseparated from bloodcellsandkept hormones werecentrifugedfor10 3.7 and26.6 0.127 pmol/L,withaCVof3.3and2.7%atPTHlevels in humanAQP2towhich was addedanNH COOH-terminalaminoacids corresponding tothe15 Antibodies wereraisedinrabbits toasyntheticpeptide radioimmunoassay as described previously (28, 29). assayed. Urineaquaporin-2(AQP2)wasmeasuredby (intra-assay). 0.2 Jacques Dürr, Miami,Fl,USA.Minimaldetectionlevelwas (26, 27).TheantibodyagainstAVP wasagiftfromProf and subsequentlydeterminedbyradioimmunoassay FHH andcardiovascularhealth pmol/L. TheCVvalueswere13%(interassay)and9% Blood samplesformeasurementsofvasoactive Urine sampleswerekeptfrozenat−20°Cuntil = 51) 18 Sep-Pak(Water Associates,Milford,MA,USA), pmol/L respectively. pg/mL. Plasmaaldosteronewasdetermined 168.7 (8.6) 1000 (750;1250) 1050 (763;1575) 75.7 (16.7) 26.4 (4.7) 5.0 (0.0;24) 23 (68.0) 34 (68.0) 56 (23–82) 11 (22.0) 13 (26.0) 28 (56.0) 17 (34.0) 28 (56.0) FHH (n =50) 7 (14) 6 (12) 7 (14) 6 (12) 6 (12) Downloaded fromBioscientifica.com at09/29/202110:58:26AM pg/mL. TheCVvalueswere 175:4 min at2200 www.eje-online.org P-value 2 0.61 0.66 0.43 0.46 0.84 0.09 0.31 0.81 0.30 0.03 0.59 0.80 0.94 0.69 0.54 0.74 0.28 -terminal – pmol/L. g and 301 via freeaccess

Clinical Study N F B Jakobsen and others FHH and cardiovascular health 175:4 302

cysteine for conjugation and affinity purification. Minimal (Millar, SPT-301B, Houston, Texas, USA) and SphygmoCor detection level was 34 pg/tube. The CV values were 11.7% equipment and software, version 8.0 (AtCor Medical, (interassay) and 5.9% (intra-assay). The urinary excretion Sydney, Australia). After a minimum of 5 min of rest in of epithelial sodium channels (ENaCs) was measured the supine position, three office blood pressures were by radioimmunoassay as described previously (30). measured at the right arm with an appropriately sized Antibodies were raised against the synthetic ENaC peptide cuff using a Riester Champion N automatic blood pressure in rabbits and affinity purified as described previously monitor (Riester GmbH, Jungingen, Germany). The mean (31). Minimal detection level was 48 pg/tube. The CV of three systolic and diastolic BP was used for calibration values were 14% (interassay) and 6.7% (intra-assay). of the SphygmoCor for the PWV and PWA measurements. The carotid-femoral PWV was determined by sequential ECG referenced recordings of the pulse wave at the carotid Genetics and the femoral artery by the tonometer. The transit time FHH patients were characterized by molecular genetic was determined by the intersecting tangent algorithm analysis of the CASR gene as described previously (4, 32). method and the path length calculated by subtracting Sequences were aligned to GenBank reference sequence the distance between the carotid artery measurement site NM_000388.2. Findings were confirmed in a second and sternal notch from the distance between the femoral analysis on separately drawn blood. Identified variants artery site and the sternal notch (33), all measured directly of the CASR within the group of FHH patients have been by a tape measure. The mean of two PWV measurements reported previously (22). was calculated for each patient. The peripheral pulse wave was assessed by applanation tonometry at the right radial artery, and the central pulse waves were derived by the Blood pressure transfer function of the SphygmoCor device. Only values with an operator index >80 were accepted for analysis. Twenty-four-hour ambulatory BP (ABP) monitoring was performed using Spacelabs 90217 (Spacelabs Healthcare, Issaquah, Washington, USA). The measurements were Statistical analysis scheduled at 20-min intervals during day and night. Day We assessed differences between groups using χ2 test for and night periods were defined based on subjects’ diaries categorical variables and a two-sample t-test or Mann– of awake and sleeping hours. During the recordings, the Whitney U-test for continuous variables, as appropriate

European Journal European of Endocrinology subjects were asked to attend their usual daily schedule, after testing for normal distributions. Correlations refraining from unusual activities and from excessive between variables were tested by bivariate correlation physical exercise. They were also instructed to keep their analysis (Pearson’s or Spearman’s correlation coefficients, arm still at the time of each automated measurement, as appropriate). In our primary analyses, we included according to usual procedures. all studied subjects. As some of the participants were on treatment with antihypertensive and cholesterol-lowering drugs, we also performed analyses including only untreated Arterial stiffness participants. Furthermore, males and females were Arterial stiffness and central hemodynamics were compared separately. Results are reported as mean ± s.d. assessed noninvasively with the SphygmoCor device or as median with interquartile range (25 and 75% (AtCor Medical, Sydney, Australia). Arterial stiffness was percentiles), as appropriate according to the distribution assessed by carotid-femoral pulse wave velocity (PWV), of data. Before initiating the study, we calculated that 45 the gold-standard method for noninvasive assessment participants were needed in each group in order to be able of arterial stiffness (33). Hemodynamics at the level of to detect a 9 mmHg difference in systolic blood pressure the aorta was assessed by pulse wave analysis (PWA). between groups, assuming a mean (s.d.) blood pressure of Before measurements, study subjects had abstained 130 (15) mmHg (80% power, α = 0.05). Post hoc, we found from smoking and intake of tea, coffee or other that 50 participants in each groups provided the study caffeine-containing beverages for at least 3 h before the with a 92% statistical power to detect a between-group examinations. At least 2 h passed between food intake and difference of 1.0 m/s in PWV, which was considered as the examinations. Measurements of pulse wave velocity clinically relevant, as previous studies have shown that the (PWV) were performed using an applanation tonometer risk of total and cardiovascular risk increases with 15% for

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Urine

*After excludingparticipantsreceiving cholesterol-loweringdrugs. Plasma mean ± Table 2 the controls (14%vs2%,P treatment withanangiotensinreceptorblockerthan patients intheFHHgroupweremorefrequentlyon in generaldidnotdifferbetweengroups,exceptthat alsoshowstypesofAHDswhichdrugs (AHD).Table 1 with cholesterol-loweringdrugsandantihypertensive number ofpatientsandcontrolswereontreatment calcium fromdietandsupplements(Table 1). Asimilar did notdifferinheight,weight,BMIortotalintakeof mean ageof56years.69%werefemales.Thegroups . Ourparticipantshada controls areshowninTable 1 Characteristics oftheFHHpatientsandtheirmatched Results Social Sciences(SPSS21.0)forWindows (IBM). analyses wereperformedusingtheStatisticalPackagefor <0.05 wasconsideredstatisticallysignificant.Statistical increase of 1 every oasu (mmol/24 h) Potassium oim (mmol/24 h) Sodium Total CO Osmolality (mmol/kg) Sodium (mmol/L) raiie (mmol/24 h) Creatinine acu (mmol/24 h) Calcium Clinical Study Potassium (mmol/L) oue (mL/24 h) Volume Osmolality (mmol/kg) Triglycerides* (mmol/L) Creatinine clearance(mL/min) Triglycerides (mmol/L) Creatinine (μmol/L) LDL cholesterol*(mmol/L) 25OHD (nmol/L) PTH (pmol/L) LDL cholesterol(mmol/L) Phosphate (mmol/L) HDL cholesterol*(mmol/L) Calcium, albuminadj(mmol/L) HDL cholesterol(mmol/L) Calcium, total(mmol/L) Total cholesterol*(mmol/L) Calcium, ionized(mmol/L) Total cholesterol(mmol/L) Albumin (g/L) s . d . Biochemistry infamilialhypocalciurichypercalcaemia(FHH)patientsandmatchedcontrols.Dataispresentedas orasmedianwithinterquartile(25;75percentiles)range. 2 (mmol/L) m/s in PWV (34). A two-sided

< 0.03). A similarnumber of N FBJakobsenandothers Reference interval 0.71–1.53 2.18–2.52 2.20–2.55 1.18–1.32 300–900 137–145 280–300 30–130 50–150 2.0–9.0 3.5–4.6 45–105 50–150 1.6–6.9 34–48 23–37 8–22 >1.0 >1.0 >90 <2 <2 <3 <3 <5 <5 P-value compared withthecontrols.Otherbiochemicalindices 421.5 ±144.1mmol/kg, vs (518.6 ±208.8 U-osm 2115 ±55mL, vs (1802 ±529 volume AHD, FHHpatientshadalower24-hurinary 2).However, afterexcludingthosetaking groups (Table volume andosmolality(U-osm)didnotdifferbetween P not ontreatmentwithAHD(288 which wasalsofoundifanalysisrestrictedtothose groups. PlasmaosmolalitywashigherintheFHH-group, 2).Plasma25OHDlevelsdidnotdifferbetween (Table and PTH,whereasplasmaphosphatelevelswerelower ficantly higherplasmalevelsofcalcium,magnesium Compared with controls, patients with FHH had signi­ Biochemical variables or threeAHD(Table 1). patients andcontrolswereontreatmentwithone,two FHH andcardiovascularhealth = 0.03). Within theentiregroupofparticipants,urinary Control (n =51) 3.6 (2.4;5.6) 3.9 (3.2;4.8) 2012 ±562 1.03 ±0.16 2.50 ±0.10 2.33 ±0.09 1.24 ±0.03 145 ±63 438 ±153 140 ±2 289 ±4 123 ±39 3.9 ±0.3 1.3 ±0.7 1.2 ±0.6 3.0 ±0.9 2.8 ±0.9 1.7 ±0.4 1.7 ±0.4 5.1 ±0.9 5.0 ±1.0 71 ±25 12 ±4 69 ±15 52 ±25 37 ±3 26 ±2 Downloaded fromBioscientifica.com at09/29/202110:58:26AM 2.9 (1.9;5.1) 4.7 (3.7;6.4) 1955 ±623 51.5 ±21.4 0.87 ±0.15 2.84 ±0.20 2.69 ±0.20 1.45 ±0.12 FHH (n =50) 159 ±52 475 ±201 140 ±2 291 ±3 117 ±31 4.0 ±0.3 1.1 ±0.6 1.3 ±0.7 3.1 ±0.8 2.9 ±0.9 1.5 ±0.4 1.5 ±0.5 5.2 ±0.9 5.0 ±0.9 68 ±23 12 ±4 69 ±13 38 ±2 27 ±2 P = ± 0.03) with a higher 3 vs290 175:4 www.eje-online.org ± 3 <0.01 <0.001 <0.001 <0.001 <0.001 P-value mmol/kg, 0.65 0.25 0.32 0.46 0.66 0.25 0.12 0.64 0.20 0.44 0.25 0.93 0.38 0.92 0.79 0.01 0.04 0.11 0.80 0.90 0.11 0.05 P =0.04) 303 via freeaccess Clinical Study N F B Jakobsen and others FHH and cardiovascular health 175:4 304

shown in Table 2 did not change after excluding those who received AHD (data not shown). 0.01 0.34 0.62 0.79 0.03 P value Plasma lipids and cholesterol status did not differ between groups. However, after exclusion of participants on treatment with cholesterol-lowering drugs, HDL or median with . = 34)

d cholesterol was lower in FHH patients (Table 2). . s

± Stratification by sex did not affect indices shown in 407 ± 191 FHH ( n 81 (53; 140) 0.4 (0.2; 0.7) 5.0 (3.0; 7.5) Table 2 (data not shown). Female 0.71 (0.61; 1.00)

= 35) Markers of volume regulation

Table 3 shows measured vasoactive biochemical markers. 315 ± 143 93 (50; 170)

Control ( n In the entire group of participants as well as within the 0.2 (0.1; 0.4) 3.9 (2.5; 6.2) 0.75 (0.56; 0.97) group not on treatment with AHD, FHH patients had significantly higher plasma levels of AVP and a higher 0.18 0.90 0.65 0.36 renal excretion of ENaC than controls, whereas urinary P value >0.99 AQP2 and plasma levels of aldosterone and renin did not differ between groups. After stratification by sex, AVP levels and renal = 16) ENaC excretion were higher in women but not in men (Table 3), which was also found if analyses were restricted 469 ± 271 FHH ( n Male 0.9 (0.3; 1.2) 5.1 (3.9; 8.8)

107 (59; 148) to participants not on treatment with AHD (data 0.93 (0.64; 1.46) not shown). Within the group of participants not on treatment with AHD, urinary ENaC excretion correlated

= 16) positively with urinary AQP2 (r = 0.61, P < 0.01), urinary osmolality (r = 0.41, P < 0.01), plasma osmolality (r = 0.30,

395 ± 164 P = 0.02), ionized calcium levels (r = 0.36, P < 0.01) and Control ( n 94 (57; 150) 0.5 (0.3; 1) 5.7 (3.5; 9.2) 1.1 (0.89; 1.28) PTH (r = 0.31, P = 0.02), but not with plasma levels of

European Journal European of Endocrinology aldosterone, renin or AVP. 0.49 0.65 0.03 0.76 P value <0.01

Blood pressure

Office BP and 24-h ABP did not differ between FHH and = 50) controls (Table 4). However, after stratification by sex, 24-h 426 ± 217 5 (3.4; 7.7) FHH ( n averages as well as day- and night-time systolic-, diastolic- All 87 (54; 140) 0.5 (0.2; 0.9)

0.74 (0.62; 1.06) and mean arterial (MAP) ABP was significantly lower in FHH females compared with controls (Table 4). If patients on treatment with AHD were excluded, the direction of the differences remained the same even though only diastolic BP at nighttime was statistically significantly 340 ± 153 94 (53; 165) Control ( n = 51) 0.3 (0.1; 0.6) 4.2 (2.6; 8.0) lower in FHH females (61 ± 5 vs 66 ± 9 mmHg, P < 0.05) 0.85 (0.59; 1.13) (Supplementary Table 1, see section on supplementary data given at the end of this article). In FHH males, a significantly higher diastolic BP and MAP at nighttime were observed. This difference

Vasoactive biochemical agents in familial hypocalciuric hypercalcaemia (FHH) patients and matched controls. Data is presented as mean Vasoactive remained significant after exclusion of participants on treatment with AHD (nighttime diastolic BP 70 ± 4 vs 63 ± 5 mmHg, P = 0.01 and MAP 84 ± 5 vs 78 ± 7 mmHg, P-vasopressin (pg/mL) P-renin (pg/mL) S-aldosteron (pmol/L) Table 3 Table U-ENaC (pg/mL) interquartile (25; 75 percentiles) range. U-aquaporin (ng/mL) P < 0.05).

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Downloaded from Bioscientifica.com at 09/29/2021 10:58:26AM via free access European Journal of Endocrinology MAP, meanarterialbloodpressure. Systolic Office bloodpressure hypocalciuric hypercalcaemia(FHH)andmatchedcontrols.Dataispresentedasmean Table 4 Diastolic MAP Systolic 24-h measurement (P changed byexclusionofpatientsontreatmentwithAHD and night)BPinmalesvsfemales(Table 4). Thiswasnot found significantly higher systolic anddiastolic (24-h, day wave velocity(PWV) and pulsewaveanalyses (PWA) did Overall, indices of arterial stiffness in terms of pulse Pulse waveanalysesandpulsevelocity Diastolic MAP

Systolic Day Diastolic MAP

Systolic Night Diastolic MAP

*Adjusted forheartrate(Aix@HR75). Carotid-femoral pulsewave Estimated aorticmean Estimated aorticdiastolic Estimated aorticsystolic Adjusted augmentationindex* Augmentation index(%) matched controls.Dataispresentedasmean Table 5 velocity (m/s) pressure (mmHg) BP (mmHg) BP (mmHg) Clinical Study Heart rate Heart rate Heart rate < 0.02 forallcomparisons). In theFHHgroup,butnotincontrolwe Office blood pressure and 24-h ambulatory blood pressure (mmHg) and heart rate (per min) in patients with familial Office bloodpressureand24-hambulatory(mmHg)heartrate(permin)inpatientswithfamilial Pulse waveanalysisandpulsevelocityinpatientswithfamilialhypocalciurichypercalcaemia(FHH)their 129 ±14 123 ±13 128 ±14 110 ±13 Controls (n 79 ±10 97 ±11 75 ±7 91 ±8 69 ±9 79 ±8 95 ±9 72 ±10 65 ±8 81 ±9 62 ±8 = 36) 129 ±17 118 ±12 124 ±13 106 ±10 (n =38) 79 ±12 97 ±13 72 ±9 88 ±9 69 ±7 76 ±9 93 ±9 73 ±8 63 ±7 79 ±8 61 ±7 118 ±14 7.5 ±1.8 All FHH 19 ±11 23 ±11 97 ±11 80 ±10 Control N FBJakobsenandothers (n = 38) ± 119 ±15 8.0 ±1.9 P value s 22 ±9 27 ±11 97 ±13 80 ±12 (n =39) 0.90 0.73 0.94 0.10 0.15 0.20 0.97 0.19 0.23 0.23 0.87 0.11 0.18 0.28 0.63 . d FHH All . forallparticipantsandstratifiedbysex. 131 ±11 123 ±11 129 ±12 108 ±10 P value Controls 0.16 0.13 0.94 0.74 0.32 0.76 (n =13) 81 ±8 98 ±9 75 ±6 90 ±7 68 ±9 79 ±8 95 ±8 70 ±10 64 ±5 79 ±6 60 ±8 with antihypertensivedrugs,FHHpatientscompared present (P with antihypertensivedrugs,thetendencywasnolonger 6.9 ±1.0, 81.7 ±7.5, vs 80.5 ±7.5, for higherperipheraldiastolicpressure(88.5 Within themalegroup,FHHpatients had atendency 5). not differbetweenFHHpatientsandcontrols(Table FHH andcardiovascularhealth 117 ±11 6.9 ±1 Control (n =16) 98 ±9 82 ±8 11 ±6 16 ±7 136 ±16 105 ±11 126 ±13 131 ±15 112 ±9 Males In females, after exclusion of participants on treatment (n =13) 89 ±10 79 ±9 94 ±8 71 ±9 83 ±9 98 ±9 74 ±10 69 ±6 84 ±6 63 ±8 FHH P > 105 ±11 124 ±12 .5, eta datlc rsue (89.6 ±10.2 pressure diastolic central P =0.05), 7.6 ±0.6 = (n =16) 90 ±10 16 ±10 18 ±12 Male Supplementary Table 10.10 forallanalyses,Supplementary ). 0.09). Afterexclusionofthoseontreatment FHH P ± = s P value . d 0.06) andpulsewavevelocity(7.6 0.39 0.05 0.10 0.60 0.15 0.20 0.30 0.67 0.24 0.31 0.31 0.28 0.02 0.04 0.26 forallparticipantsandalsostratifiedbysex.. P value 0.10 0.06 0.21 0.69 0.09 0.21 Downloaded fromBioscientifica.com at09/29/202110:58:26AM 128 ±16 123 ±15 128 ±15 111 ±15 Controls (n 79 ±11 96 ±12 76 ±8 91 ±9 70 ±9 79 ±8 95 ±9 73 ±10 66 ±9 82 ±10 63 ±8 = 36) 118 ±15 7.8 ±2 Control (n =35) 96 ±12 80 ±11 22 ±12 26 ±11 175:4 Females 126 ±17 114 ±10 120 ±10 102 ±10 (n 75 ±11 94 ±12 69 ±7 85 ±7 68 ±6 73 ±7 90 ±7 72 ±6 60 ±5 76 ±7 60 ±6 www.eje-online.org FHH 117 ±16 Female = 25) 8.1 ±2.1 (n =34) 94 ±12 76 ±11 24 ±8 30 ±9 FHH ± 10.2 vs P value <0.01

<0.01 ± P value 0.61 0.19 0.40 0.02 0.02 0.39 0.05 0.02 0.02 0.50 0.01 0.02 0.11 0.40 0.21 0.35 0.12 0.82 0.58 0.6 vs 305 via freeaccess Clinical Study N F B Jakobsen and others FHH and cardiovascular health 175:4 306

controls had a tendency for lower peripheral diastolic in the tubular fluid are accordingly not increased (38). pressure (74 ± 10 vs 80 ± 10 mmHg, P = 0.08) and central However, our study does not suggest an increased activity diastolic pressure (75 ± 10 vs 80 ± 10 mmHg, P = 0.09). of AQP2 in FHH, as renal excretion of AQP2 did not differ In the FHH group, females had a significantly lower between groups. estimated aortic diastolic (but not systolic) BP compared In accordance with our findings, previous studies have with males (76 ± 11 vs 90 ± 10 mmHg, P < 0.01), and this was shown that individuals with inactivating variants of the not changed by exclusion of FHH patients on treatment CASR are able to concentrate their urine quite normally with AHD (P < 0.01), whereas measurements did not differ despite hypercalcemia (38). In a direct comparison between sexes in the control group. between patients with FHH and PHPT, PHPT was associated with an impaired ability to concentrate urine compared with FHH (5). Similarly, nonparathyroid hypercalcemia is Discussion associated with a nephrogenic concentrating defect (39). Our findings support intact urinary concentration ability To the best of our knowledge, this study is one of the first in FHH. Actually, urinary osmolality was increased in the to investigate possible effects in humans of inactivating FHH group after excluding the group of participants on variants in the CASR on blood pressure and volume treatment with antihypertensive drugs. regulation. After exclusion of participants on therapy Our study showed an increased renal excretion of with AHD, our study showed higher plasma levels of the epithelial sodium channel (ENaC) in FHH compared AVP and a higher plasma and urinary osmolality, as well with controls. ENaC is responsible for the reabsorption as a higher renal excretion of ENaC and a lower 24-h of sodium through the apical membrane of the principal urinary volume in the FHH group compared with the cells in the distal part of the nephron and plays a key controls. In addition, compared with their respective role in controlling sodium balance, extracellular fluid controls, males with FHH had a higher diastolic BP and volume and blood pressure. Although aldosterone is MAP at nighttime, whereas FHH females had a lower considered as the principal regulator of the ENaC, AVP DBP at nighttime. However, PWV and PWA did not differ has also been shown to activate ENaC. Similar to AQP2, between groups. the sodium transport via the epithelial sodium channels The CASR is considered to be primarily of importance is supposed to be reflected by the level of urinary to the regulation of calcium balance, but a number of excretion of a protein fraction from the channels (35). studies have suggested that the receptor also may be invol­ In a recent study, activation of ENaC by AVP was shown European Journal European of Endocrinology ved in salt and water transport in the kidney. The CASR is to facilitate urine concentration and dilution of plasma localized to segments of the nephron that participate in (40). This may be of importance in FHH, as APQ2 levels urinary concentration. In principal cells of the collecting did not differ between groups despite increased AVP duct, CASR co-localizes with AQP2 in vesicles. Increased levels with no differences between groups in plasma u-AQP2 reflects increased water transport from the tubular levels of sodium and aldosterone. However, we found no lumen to the intracellular space via the aquaporin2 water significant correlations between urinary ENaC and plasma channels, located in the principal cells in the distal part levels of AVP. On the other hand, we found a significant of the nephron (28, 29, 35). Activation of the CASR causes positive correlation between ionized calcium levels and a reduced AQP2 expression and AVP-stimulated insertion renal excretion of ENaC. Further studies are needed to of AQP2 into the apical membrane (36). Consequently, determine whether the CASR is directly involved in the activation of the CASR results in dilution of the urine and regulation of ENaC. may through this mechanism reduce the risk of kidney Our findings suggest diurnal differences in BP stones in patients with hypercalcemia (37). Our findings regulation in patients with FHH, with opposite effects in agree to some extent with these observations as we found males and females. In females, FHH was associated with an increased urinary osmolality in FHH patients compared a lower 24-h, day and night BP compared with controls, with controls after excluding participants on treatment whereas we found an opposite association in males, in with AHD. Accordingly, the inactivating variant of the whom FHH was associated with higher diastolic BP at CASR causes less-diluted urine. Apparently, patients with night. In both the 24-h ABP measurements and the central FHH are not at an increased risk of renal stones, which diastolic BP measurements, we observed a significant may be explained by the fact that inactivating variants difference between sexes in the FHH group with higher increases tubular calcium reabsorption and calcium levels values in the FHH males. Accordingly, it seems that the

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Downloaded from Bioscientifica.com at 09/29/2021 10:58:26AM via free access European Journal of Endocrinology major differenceswerefound incentralbloodpressure adverse effectsoncardiovascular healthofFHH.No In conclusion, our resultsdo notsuggestmajoroverall Conclusion larger cohortsareneededtotestsuchhypothesis. importance to cardiovascular health. Further studies in whether such non-FHH polymorphismsare of levels (43,44). Our study was not designed to study have beenassociatedwithslightlyincreasedcalcium nonpathologic. However, someofthepolymorphism been identifiedwhich,ingeneral,areconsideredto be polymorphisms inthecarboxylterminushavealso In additiontothevariantscausingFHH,anumberof analyses werenotperformedinthecontrolgroup. the sameinsubgroupsnotreceivingAHD.Genetic differencesbetweenmenandwomenwere the observed antihypertensive treatment.However, thedirectionsof differs fromtheeffectsofvariantsinthosenoton of variantsintheCASRthoseontreatmentwithAHD ( in affectedindividualsvariesbetweendifferentvariants associated withFHH,andthedegreeofhypercalcemia revealed morethan100differentvariantswhichare results toanymajordegree.AnalysesoftheCASRhave of participantsontreatmentwithAHDdidnotchange analyses accordingtotreatment.Ingeneral,exclusion controls toinvestigatesucheffects.Instead,westratified ethically acceptabletostoptherapyinFHHpatientsand being ondrugtreatment.However, wedidnotfindit found ifallparticipantswereinvestigatedwithout that furtherdifferencesbetweengroupshadbeen on thephysiologicaleffectsstudied.We cannotexclude measured indicescontributeswithsomeuncertainties our participantswereontreatmentwithdrugsaffecting carried outbytrainedpersonnel.Thefactthatsomeof general backgroundpopulation.Allinvestigationswere the factthatcontrolswererandomlyrecruitedfrom a balanced matching of FHH patients with controls and conclusions inthisregard. patients, yetthecross-sectionalstudydesignpreclude a tendencytolowercardiovascularriskinfemaleFHH potential difference.The24-hABPdatacouldindicate FHH (41).Furtherstudiesareneededtoelucidatesuch reported similargenderdifferencesonboneeffectsof differ between males and females.We have previously consequences ofinactivatingvariantsintheCASRmay 4, 42).We cannotexcludethatthephysiologicaleffects Clinical Study The majorstrengthofourstudyisthedesignwith N FBJakobsenandothers Research Center, InstituteofAnatomy, AarhusUniversity, Denmark. The anti-AQP2antibody was a gift fromSørenNielsen, The Water and Salt Acknowledgements Diabetes AcademysupportedbytheNovoNordiskFoundation. Council forindependentResearchinMedicalSciences(FSS)andtheDanish This studywassupportedbyaGrant(#10-094047)fromTheDanish Funding perceived asprejudicingtheimpartialityofresearchreported. The authorsdeclarethatthereisnoconflictofinterestcouldbe Declaration ofinterest EJE-16-0369. This islinkedtotheonlineversionofpaperathttp://dx.doi.org/10.1530/ Supplementary data major increasedriskofcardiovasculardiseases. the geneencodingCASRdonotseemtobeatany Accordingly, individualswithaninactivatingvariantin female couldprotectagainstcardiovasculardiseases. in the between men and women with an inactivating variant suggest differentialeffectsonvolumeandBPregulation or measuresofarterialstiffness.However, ourdata References

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