Nephrol Dial Transplant (2022) 37: 2474–2486 https://doi.org/10.1093/ndt/gfac029 Advance Access publication date 7 February 2022 Parathyroid hormone and phosphate homeostasis in patients ORIGINAL ARTICLE with Bartter and Gitelman syndrome: an international cross-sectional study Maartje F.A. Verploegen1 , Rosa Vargas-Poussou 2 , Stephen B. Walsh3 , Harika Alpay4 , Atefeh Amouzegar 5 , Gema Ariceta6 , Bahriye Atmis7 , Justine Bacchetta 8 , Peter Bárány9 , Stéphanie Baron10 , Umut Selda Bayrakci11 , Hendrica Belge12 , Martine Besouw13 , Anne Blanchard 14,15 , Arend Bökenkamp16 , Olivia Boyer17 , Kathrin Burgmaier 18 , Lorenzo A. Calò19 , Stéphane Decramer20 , Olivier Devuyst 21 , Maria van Dyck22 , Pietro Manuel Ferraro 23,24 , Marc Fila25 , Telma Francisco26 , Gian Marco Ghiggeri 27 , Leire Gondra28 , Stefano Guarino29 , Nakysa Hooman30 , Ewout J. Hoorn 31 , Pascal Houillier15,32 , Konstantinos Kamperis33 , Jameela A. Kari34 , Martin Konrad35 , Elena Levtchenko36 , Laura Lucchetti37 , Francesca Lugani27 , Pierluigi Marzuillo 29 , Barian Mohidin3 , Thomas J. Neuhaus38 , Abdaldafae Osman39 , Svetlana Papizh40 , Manel Perelló41 , Maarten B Rookmaaker42 , Valerie Said Conti43 , Fernando Santos44 , Ghalia Sawaf45 , Erkin Serdaroglu46 , Maria Szczepanska47 , Francesca Taroni48 , Rezan Topaloglu 49 , Francesco Trepiccione50 , Enrico Vidal51 , Elizabeth R. Wan3 , Lutz Weber18 , Zeynep Yuruk Yildirim52 , Selçuk Yüksel 53 , Galia Zlatanova54 , Detlef Bockenhauer3,39 , Francesco Emma 37 and Tom Nijenhuis 1 1 Department of Nephrology, Radboud University Medical Center, Nijmegen, The Netherlands, 2 Department of Genetics, Centre de Références MARHEA, Hôpital Européen Georges Pompidou Assistance Publique Hôpitaux de Paris, Paris, France, 3 Department of Renal Medicine, University College London, London, UK, 4 Division of Paediatric Nephrology, Faculty of Medicine, Marmara University, Istanbul, Turkey, 5 Division of Nephrology, Department of Medicine, Firoozgar Clinical Research Development Center, Iran University of Medical Sciences, Tehran, Iran, 6 Paediatric Nephrology Department, University Hospital Vall d’Hebron, Barcelona, Spain, 7 Department of Paediatric Nephrology, Cukurova University Faculty of Medicine, Adana, Turkey, 8 Department of Paediatric Nephrology, Rheumatology and Dermatology, Reference Centre for Rare Renal Diseases, Reference Centre for Rare Diseases of Calcium and Phosphate Metabolism. University Children’s Hospital, Lyon, France, 9 Division of Renal Medicine, Department of Clinical Science, Intervention and Technology, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden, 10 Department of Physiology, Hôpital Européen Georges Pompidou Assistance Publique Hôpitaux de Paris, Paris, France, 11 Department of Paediatric Nephrology, Ankara City Hospital, Üniversiteler Mahallesi Bilkent Caddesi, Çankaya/Ankara, Turkey, 12 Center for Human Genetics, Institute of Pathology and Genetics, Gosselies, Belgium, 13 Department of Paediatric Nephrology, University of Groningen, University Medical Centre Groningen, Groningen, The Netherlands, 14 Clinical Research Centre 1418, Centre de Références MARHEA, Hôpital Européen Georges Pompidou Assistance Publique Hôpitaux de Paris, Paris, France, 15 Centre de Recherche des Cordeliers, INSERM, Sorbonne Université, Université de Paris, Paris, France, 16 Department of Paediatric Nephrology, Emma Children’s Hospital, Amsterdam UMC, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands, 17 Department of Paediatric Nephrology, Necker Hospital, APHP, MARHEA, Imagine Institute, Paris University, Paris, France, 18 Faculty of Medicine, University Hospital of Cologne, Children’s and Adolescents’ Hospital, Paediatric Nephrology, University of Cologne, Cologne, Germany, 19 Department of Medicine (DIMED), Nephrology, Dialysis, Transplantation, University of Padova, Padova, Italy, 20 Department of Paediatric Nephrology. Centre de Références SORARE, Toulouse University Hospital, Toulouse, France, 21 Division of Nephrology, UCLouvain Medical School, Brussels, Belgium; Institute of Physiology, Mechanism of Inherited Kidney Disorders Group, University of Zurich, Zurich, Switzerland, 22 Department of Paediatric Nephrology, University Hospital Leuven, Leuven, Belgium, 23 U.O.S. Terapia Conservativa della Malattia Renale Cronica, U.O.C. Nefrologia, Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy, 24 Dipartimento Universitario di Medicina e Chirurgia Traslazionale, Università Cattolica del Sacro Cuore, Rome, Italy, 25 Pediatric Nephrology, CHU Arnaud de Villeneuve, Montpellier University Hospital, Montpellier, France, 26 Department of Paediatric Nephrology, Centro Hospitalar Universitário de Lisboa Central, Lisbon, Portugal, 27 Division of Nephrology, Dialysis and Transplantation, Istituto Giannina Gaslini, IRCCS, Genoa, Italy, 28 Pediatric Nephrology Department, Cruces University Hospital, Barakaldo, Spain; Biocruces Bizkaia Health Research Institute, Barakaldo, Spain; Paediatric Department, University of the Basque Country UPV/EHU, Leioa, Spain, 29 Department of Woman, Child and of General and Specialized Surgery, Università degli Studi della © The Author(s) 2022. Published by Oxford University Press on behalf of the ERA. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Campania ‘Luigi Vanvitelli’, Naples, Italy, 30 Ali-Asghar Clinical Research Development Centre, Iran University of Medical Sciences, Tehran, Iran, 31 Department of Internal Medicine, Division of Nephrology and Transplantation, Erasmus Medical Centre, University Medical Centre Rotterdam, Rotterdam, The Netherlands, 32 Department of Physiology, Centre de Références MARHEA, Hôpital Européen Georges Pompidou Assistance Publique Hôpitaux de Paris, Paris, France, 33 Department of Paediatrics and Adolescent Medicine, Aarhus University Hospital, Aarhus, Denmark, 34 Pediatric Nephrology Centre of Excellence and Paediatric Department, Faculty of Medicine, King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia, 35 Department of General Paediatrics, Paediatric Nephrology, University Hospital Münster, Munster, Germany, 36 Department of Paediatric Nephrology & Department of Development and Regeneration, University Hospital Leuven, KU Leuven, Leuven, Belgium, 37 Department of Paediatric Subspecialties, Division of Nephrology, Bambino Gesù Children’s Hospital – IRCCS, Rome, Italy, 38 Department of Paediatrics, Children’s Hospital of Lucerne, Cantonal Hospital of Lucerne, Lucerne, Switzerland, 39 Paediatric Nephrology Unit, Great Ormond Street Hospital, London, UK, 40 Department of Hereditary and Acquired Kidney Diseases, Research and Clinical Institute for Pediatrics, Pirogov Russian National Research Medical University, Moscow, Russia, 41 Nephrology Department, Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain, 42 Department of Nephrology and Hypertension, University Medical Centre Utrecht, Utrecht, The Netherlands, 43 Department of Paediatrics, Mater Dei Hospital Malta, Msida, Malta, 44 Department of Paediatrics, Asturias Central University Hospital, University of Oviedo, Oviedo, Spain, 45 Department of Paediatric Nephrology, Damascus Hospital, Damascus, Syria, 46 Department of Paediatric Nephrology, Dr Behçet Uz Children’s Hospital, Izmir, Turkey, 47 Department of Paediatrics, Faculty of Medical Sciences in Zabrze, SUM in Katowice, Katowice, Poland, 48 Paediatric Nephrology, Dialysis and Transplant Unit, Fondazione IRCCS Ca’ Granda-Ospedale Maggiore Policlinico, Milan, Italy, 49 Department of Paediatric Nephrology, Hacettepe University School of Medicine, Ankara, Turkey, 50 Department of Translational Medical Sciences, University of Campania “L. Vanvitelli”, Naples, Italy, 51 Division of Paediatrics, Department of Medicine, University of Udine, Udine, Italy, 52 Department of Paediatrics, Division of Paediatric Nephrology, Istanbul University Istanbul Faculty of Medicine, Istanbul, Turkey, 53 Department of Paediatric Nephrology, Pamukkale University School of Medicine, Denizli, Turkey and 54 University Children’s Hospital Medical University, Sofia, Bulgaria Correspondence to: Francesco Emma; E-mail: francesco.emma@opbg.net; Tom Nijenhuis; E-mail: Tom.Nijenhuis@radboudumc.nl GRAPHICAL ABSTRACT PTH and phosphate homeostasis in Bartter and Gitelman syndrome 2475 KEY LEARNING POINTS What is already known about this subject? - High parathyroid hormone (PTH) levels have been observed in patients with Bartter syndrome in small cohort studies. - In patients with Gitelman syndrome, low serum phosphate levels have anecdotally been reported. - Data from large cohort studies on PTH and phosphate homeostasis in Bartter and Gitelman syndrome are lacking. What this study adds? - Up till now, this is the largest cohort of patients with salt-losing tubulopathies reported, comprising 285 patients with Bartter syndrome and 304 patients with Gitelman syndrome. - Elevated PTH is frequently observed in patients with Bartter syndrome, especially in type I and II. - In a significant number of patients with Bartter and Gitelman syndrome low serum phosphate was reported and is most likely related to a primarily PTH-independent renal phosphate leak. What impact this may have on practice or policy? - This study provides unique insights into the prevalence of aberrant PTH and phosphate homeostasis in patients with Bartter and Gitelman syndrome. - Further studies are necessary to understand the pathophysiology of these abnormalities and to demonstrate whether PTH and/or phosphate should be a treatment target in these salt-losing tubulopathies. ABSTR ACT Background. Small cohort studies have reported high parathy- I N T RODU C T ION roid hormone (PTH) levels in patients with Bartter syndrome and lower serum phosphate levels have anecdotally been Bartter and Gitelman syndrome are genetically distinct reported in patients with Gitelman syndrome. In this cross- hereditary salt-losing tubulopathies that are characterized sectional study, we assessed PTH and phosphate homeostasis by hypokalaemic metabolic alkalosis secondary to impaired in a large cohort of patients with salt-losing tubulopathies. reabsorption of sodium chloride (NaCl) in different segments Methods. Clinical and laboratory data of 589 patients with of the renal tubule [1, 2]. Bartter syndrome is further classified Bartter and Gitelman syndrome were provided by members into different subtypes. Bartter syndrome type I and II are of the European Rare Kidney Diseases Reference Network caused by biallelic mutations in the SLC12A1 gene encoding (ERKNet) and the European Society for Paediatric Nephrology the NKCC2 sodium-potassium-chloride cotransporter and by (ESPN). biallelic mutations in the KCNJ1 gene encoding the ROMK Results. A total of 285 patients with Bartter syndrome potassium channel, respectively, both of which are expressed and 304 patients with Gitelman syndrome were included for in the thick ascending limb of the loop of Henle (TAL). Bartter analysis. Patients with Bartter syndrome type I and II had syndrome type III results from biallelic mutations in the the highest median PTH level (7.5 pmol/L) and 56% had CLCNKB gene, which encodes the ClC-Kb chloride channel. hyperparathyroidism (PTH >7.0 pmol/L). Serum calcium was Bartter syndrome type IV is caused by biallelic mutations slightly lower in Bartter syndrome type I and II patients with in the BSND gene that encodes Barttin, or by molecular hyperparathyroidism (2.42 versus 2.49 mmol/L; P = .038) abnormalities affecting the two adjacent CLCNKA (encoding compared to those with normal PTH levels and correlated the chloride channel ClC-Ka) and CLCNKB genes. Barttin inversely with PTH (rs −0.253; P = .009). Serum phosphate and ClC-Kb are expressed in the TAL and distal convoluted and urinary phosphate excretion did not correlate with tubule (DCT); ClC-Ka is only expressed in the TAL. Finally, a PTH. Overall, 22% of patients had low serum phosphate transient X-linked form of Bartter syndrome (type V Bartter levels (phosphate—standard deviation score < −2), with syndrome) has been described in male patients harbouring the highest prevalence in patients with Bartter syndrome mutations in the MAGED2 gene, which alters the expression type III (32%). Serum phosphate correlated with tubular of the NKCC2 and NCC, the sodium chloride cotransporter. maximum reabsorption of phosphate/glomerular filtration rate Gitelman syndrome is caused by biallelic mutations in the (TmP/GFR) (rs 0.699; P < .001), suggesting renal phosphate SLC12A3 gene, encoding the NCC, of which the expression is wasting. restricted to the DCT. Conclusions. Hyperparathyroidism is frequent in patients While Bartter and Gitelman syndromes share similarities, with Bartter syndrome type I and II. Low serum phosphate including hypokalaemia, metabolic alkalosis, hyperreninemic is observed in a significant number of patients with Bartter hyperaldosteronism and low/normal blood pressure, differ- and Gitelman syndrome and appears associated with renal ences in the tubular segments where NaCl reabsorption is phosphate wasting. compromised produce different clinical pictures [2]. Patients with Gitelman syndrome usually present in late childhood or Keywords: Bartter syndrome, Gitelman syndrome, parathy- early adulthood. Typically, patients with Gitelman syndrome roid hormone, phosphate, salt losing tubulopathies have hypomagnesemia and hypocalciuria. Conversely, type I 2476 M. F. A. Verploegen et al. and II Bartter syndrome patients usually present antenatally 770 patients from with polyhydramnios and postnatally with severe polyuria, 48 centers hypercalciuria and early onset nephrocalcinosis. Patients with type III Bartter syndrome have variable phenotypes that, in some cases, are difficult to distinguish from Gitelman 706 patients with syndrome. The large phenotypic spectrum of Bartter III complete data* patients is most likely explained by variable expression of the pathogenic gene product in the TAL and DCT and by the possibility of partial compensation by other chloride channels, 634 patients with creatinine below in particular, ClC-Ka in the TAL. threshold In recent years, several investigators have observed ab- normal levels of parathyroid hormone (PTH) and serum • 9 patients with Bartter phosphate in patients with Bartter and Gitelman syndrome syndrome type IV [3–13]. Patients with Bartter syndrome tend to have a high • 4 patients with Bartter PTH [3–9]. However, the prevalence and pathophysiology syndrome type V • 32 patients with Gitelman of hyperparathyroidism in these Bartter patients are unclear. syndrome without genetic Furthermore, data have been in part contradictory since a confirmation tendency towards both hyper- and hypophosphatemia has been described in Bartter syndrome [3, 5]. In patients with 589 patients included Gitelman syndrome, high PTH levels have only been described for analysis in patients that had parathyroid adenoma [14, 15], while hypophosphatemia has been reported in several small studies [10–13] and has been attributed to renal phosphate wasting Bartter Bartter Bartter Gitelman [12, 13]. syndrome syndrome syndrome syndrome To verify whether these observations from case reports and type I and II type III NOS 304 patients small series are prevalent in Bartter and Gitelman syndromes 107 patients# 88 patients 90 patients or just incidental findings, we analysed PTH and phosphate levels in a very large cross-sectional cohort of patients with FIGURE 1: Patient cohort. NOS, not otherwise specified. Patients Bartter and Gitelman syndrome collected across Europe, were grouped for analysis according to the genetic confirmation of including both paediatric and adult subjects. disease (subtype). *Creatinine and PTH had to be provided. # 51 patients with Bartter syndrome type I and 56 patients with Bartter syndrome type II. M AT E R IA L S A N D M E T HOD S On behalf of the European Rare Kidney Diseases Refer- of low GFR on phosphate and PTH levels, patients with ence Network (ERKNet) Tubulopathies Working Group and decreased kidney function were excluded. Since estimated European Society for Paediatric Nephrology (ESPN) Work- GFR (eGFR) data were not available, patients were excluded ing Group on Inherited Renal Disorders, members of the based on serum creatinine. To this end, we excluded all patients ERKNet and ESPN were invited to provide retrospective aged >15 years with serum creatinine >100 μmol/L and clinical and laboratory data on patients with Bartter and patients aged ≤15 years with serum creatinine levels above Gitelman syndrome. Patients had either signed an informed an arbitrary value calculated with the following formula: 2.7 consent form, allowing an anonymized transfer of clinical x age (years) + 60. Using these conservative cut-off values, data within the ERKNet network, or consent was obtained we estimated by preliminary testing that most patients with according to local regulations and under the responsibility eGFR < 65 mL/min/1.73m2 have been excluded. Overall, we of local investigators. The data collection form included 38 excluded 76 patients with elevated creatinine (Figure 1). A data items, of which some were mandatory, in particular those completeness list is provided in Supplementary data, Table S2. related to kidney function, PTH levels, and calcium-phosphate Serum phosphate, alkaline phosphatase and TmP/GFR are metabolism (Supplementary data, Table S1). To minimize strongly influenced by age in the paediatric population. To biases in estimating the prevalence of signs and symptoms, be able to compare these variables in all patients, standard data collection was cross-sectional and centres were asked to deviation scores (SDS) were calculated based on age-related provide only information corresponding to the last available reference values [16–18]. We used the following formula patient visit. For example, if a given patient had transiently to calculate SDS for phosphate, alkaline phosphatase and developed high PTH in the past, this information would TmP/GFR: not be captured to avoid overestimating the prevalence of   SDS = level − mean level for age /SD for age. hyperparathyroidism. Laboratory data were converted to SI units. Outlier data Each formula was based on published age-related reference were identified and verified with the referring clinician. values for phosphate, alkaline phosphatase and TmP/GFR. Patients with incomplete data on creatinine or PTH were (Supplementary data and methods, Tables S3 and S4, excluded from the analysis. To preclude measuring the effects Figure S1–S3). PTH and phosphate homeostasis in Bartter and Gitelman syndrome 2477 Patients were stratified into 4 groups, based on the un- All disease subgroups had a negative median phosphate- derlying genetic cause. These include patients with Bartter SDS, demonstrating that median serum phosphate was lower syndrome type I or II (n = 107), Bartter syndrome type III in all subgroups than median age-specific serum phosphate (n = 88), Bartter syndrome ‘not otherwise specified’ (NOS) levels (Table 1; Figure 2). Patients with Bartter syndrome type including patients with Bartter syndrome for which genetic III had the lowest median phosphate-SDS of −1.34 (−2.31 to data were not available (n = 90) and Gitelman syndrome −0.15), which was significantly lower compared to phosphate- (n = 304). Patients with Bartter syndrome type IV (n = 9), SDS in Bartter syndrome type I and II and Gitelman syndrome type V (n = 4), or Gitelman syndrome NOS (n = 32) were (P = .032 and P = .011 respectively; Figure 2). No differences excluded from the final analysis because these tests were were observed in tubular reabsorption of phosphate (TRP) severely underpowered (Figure 1). (P = .254) between different disease subgroups. TmP/GFR- Data were analysed using IBM SPSS Statistics Version 25. SDS was lowest in patients with Gitelman syndrome [−0.98 Categorical data are described as numbers and %. Contin- (−1.47 to −0.46); Figure 2]. This was significantly lower uous data are presented as median with interquartile range compared to Bartter syndrome type I and II (P = .006). (IQR). Data were analysed using Chi-squared test, Fisher’s Median and IQR of serum calcium were within the normal exact test, Mann–Whitney U test, and Kruskal–Wallis test range for all subgroups (Table 1; Figure 2). Serum calcium as appropriate. Spearman’s rank correlation was used for the was significantly higher in patients with Bartter syndrome analysis of the correlation between variables. Univariable and compared to Gitelman syndrome (P < .001). No significant multivariable logistic regression were used to identify variables differences in serum calcium were observed between Bartter associated with elevated PTH; variables that reached a P-value syndrome subtypes. < .10 at the univariable level were used in the multivariable Although data were available only in part of the entire model. cohort (Supplementary data, Table S2), 25-hydroxy vitamin D (25OH vitamin D) levels were lower in patients with Bartter syndrome compared to Gitelman syndrome (P = .001) and R E SU LT S patients with Bartter syndrome used vitamin D supplements Overall characteristics more often (P < .001; Table 1). No difference in serum After excluding subjects with incomplete data, with serum calcium levels was found between patients who used vitamin creatinine above the threshold, or underpowered subgroups of D supplements and non-supplemented patients (data not patients, 589 patients from the original 770 patients collected shown). were included for analysis (Figure 1). Patient characteristics are in agreement with the known phenotypic characteristics of the diseases and are detailed in Table 1. The median age was 16.6 years (IQR 8.2–33.5). Hyperparathyroidism and hypophosphatemia Patients with Bartter syndrome were significantly younger than Analysis of the entire cohort. The characteristics and patients with Gitelman syndrome [median age 9.5 years (3.5– laboratory data of patients according to the absence or 16.3) versus 28.8 years (16.4–43.9) respectively; P < .001). presence of hyperparathyroidism are shown in Supplementary Sex distribution was not significantly different in the four data, Table S6. Overall, 84% of patients with elevated iPTH subgroups (P = .113). Overall, 52% of the patients were had Bartter syndrome. Notably, 45% of the patients with female. Nephrocalcinosis was reported more frequently in hyperparathyroidism were patients with Bartter syndrome patients with Bartter syndrome type I and II compared to type type I and II, while this disease subtype comprises only 18% of III, with a prevalence of 89 and 18% respectively and very patients in the entire cohort. Several parameters, among which rarely in patients with Gitelman syndrome, as expected. On urinary calcium/creatinine ratio and serum magnesium, were average, patients with Bartter syndrome had higher urinary significantly different when comparing patients with and with- calcium/creatinine ratios than Gitelman patients (P < .001) out hyperparathyroidism (Supplementary data, Table S6) and and patients with Gitelman syndrome had lower serum mag- iPTH was correlated with several parameters (Supplementary nesium levels than patients with Bartter syndrome (P < .001). data, Table S7). However, these analyses appear markedly Indomethacin or other nonsteroidal anti-inflammatory drugs influenced by the unequal prevalence of hyperparathyroidism (NSAIDs) were prescribed more often to patients with Bartter among different disease subgroups, and therefore, may reflect syndrome than Gitelman syndrome (P < .001). differences related to the underlying disease, rather than differ- Elevated PTH levels were observed in a significant propor- ences related to high iPTH. The same limitation applies to the tion of patients. Hyperparathyroidism (iPTH >7.0 pmol/L) multivariable logistic regression reported in Supplementary was present in 23% of patients (Supplementary data, data, Table S8 and is underscored by the finding that the Table S5). Patients with Bartter syndrome type I and II strongest predictor for hyperparathyroidism is the Bartter had the highest median intact parathyroid hormone (iPTH) syndrome type I and II sub-class (OR: 3.20, 95% CI 1.68– levels [7.5 pmol/L (4.8–11.2)] (Table 1; Figure 2) and the 6.08; P < .001). Other significant factors associated with prevalence of hyperparathyroidism was 56% in this group. hyperparathyroidism included higher serum magnesium [OR: Conversely, Gitelman patients had the lowest prevalence of 18.47 (2.67–127.93) per mmol/L, P = .003], lower serum hyperparathyroidism (7%) and even showed a 20% prevalence calcium [OR: 0.03 (0.01–0.21) per mmol/L, P < .001), higher of hypoparathyroidism (iPTH < 2.0 pmol/L). alkaline phosphatase-SDS [OR: 1.260 (1.11–1.431) per SDS, 2478 M. F. A. Verploegen et al. Table 1. Patient characteristics Bartter syndrome Bartter syndrome Bartter syndrome Gitelman type I and II type III NOS syndrome Total Number of patients 107 88 90 304 589 Characteristics Male: Female ratio, n (%) 51: 56 (48: 52) 50: 38 (57: 43) 48: 42 (53: 47) 133: 171 (44: 56) 282: 307 (48: 52) Age (years) 9.0 [3.5–16.0] 10.4 [3.3–17.2] 9.6 [3.6–16.3] 28.8 [16.4–43.9] 16.6 [8.2–33.5] Nephrocalcinosis, n (%) 90/101 (89) 15/85 (18) 46/82 (56) 4/257 (2) 155/525 (30) Laboratory results iPTH (pmol/L) 7.5 [4.8–11.2] 4.4 [3.0–6.7] 5.1 [2.9–8.3] 3.1 [2.3–4.0] 3.8 [2.5–6.6] Creatinine (umol/L) 55 [37–76] 43 [–62] 48 [–66] 58 [47–71] 55 [41–70] Sodium (mmol/L) 140 [138–142] 139 [137–141] 139 [137–140] 139 [138–141] 139 [138–141] Potassium (mmol/L) 3.6 [3.3–3.8] 3.2 [2.8–3.6] 3.4 [3.0–3.9] 3.0 [2.7–3.4] 3.2 [2.8–3.7] Chloride (mmol/L) 99 [97–101] 97 [92–100] 96 [93–101] 98 [95–100] 98 [95–100] Bicarbonate (mmol/L) 26.2 [24.3–28.1] 29.0 [26.3–32.0] 28.4 [27.0–30.7] 29.0 [27.0–31.0] 28.3 [26.3–31.0] Phosphate (mmol/L) 1.42 [1.21–1.63] 1.28 [1.09–1.44] 1.30 [1.12–1.53] 1.07 [0.92–1.25] 1.21 [0.99–1.42] Phosphate-SDS -0.67 [-1.76–0.43] −1.34 [-2.31– −0.15] −1.32 [−2.32–0.05] -0.77 [−1.51–0.29] −0.91 [−1.82–0.18] Calcium (mmol/L) 2.44 [2.37–2.53] 2.48 [2.40–2.58] 2.50 [2.38–2.60] 2.41 [2.31–2.50] 2.43 [2.34–2.53] Magnesium (mmol/L) 0.89 [0.80–0.97] 0.77 [0.68–0.85] 0.81 [0.75–0.95] 0.63 [0.56–0.71] 0.71 [0.61–0.84] Uric acid (μmol/l) 315 [246–405] 220 [162–319] 286 [190–409] 250 [190–321] 262 [190–350] Alkaline phosphatase (IU/L) 227 [161–287] 212 [114–274] 230 [160–303] 66 [51–136] 148 [62–245] Alkaline phosphatase-SDS 0.79 [−0.05–1.52] 0.21 [−0.50–0.99] 0.65 [−0.36–1.89] −1.00 [−1.72–0.00] −0.13 [−1.18–0.93] Total protein (g/L) 73 [70–77] 73 [69–76] 75 [71–78] 73 [69–76] 73 [70–77] Albumin (g/L) 46 [43–49] 45 [43–48] 46 [43–49] 46 [44–49] 46 [43–49] 25OH vitamin D (nmol/L) 39 [–68] 40 [–82] 43 [–64] 61 [–85] 51 [–80] Urinary calcium/creatinine 1.55 [0.94–2.62] 0.33 [0.14–0.71] 0.55 [0.23–1.14] 0.08 [0.04–0.17] 0.21 [0.07–0.75] (mmol/mmol) TRP 0.90 [0.81–0.92] 0.90 [0.86–0.93] 0.88 [0.82–0.95] 0.91 [0.86–0.94] 0.90 [0.85–0.94] TmP/GFR (mmol/L) 1.41 [1.10–1.48] 1.17 [1.02–1.33] 1.11 [0.84–1.35] 0.95 [0.82–1.10] 1.04 [0.84–1.23] TmP/GFR-SDS 0.23 [−0.46–0.78] −0.62 [−1.48–0.44] −0.66 [−2.03–0.14] −0.98 [−1.47– −0.46] −0.86 [−1.54– −0.02] Treatment NSAIDs, n (%) 69 (65) 52 (59) 55 (61) 26 (9) 202 (34) Indomethacin, n (%) 53 (50) 48 (55) 54 (60) 26 (9) 181 (31) Ibuprophen, n (%) 16 (15) 4 (5) 2 (2) 0 (0) 22 (4) Other NSAIDs, n (%) 5 (5) 7 (8) 4 (4) 1 (0.3) 17 (3) Potassium-sparing diuretics, n (%) 2 (2) 12 (14) 4 (4) 63 (21) 81 (14) Amilorid, n (%) 2 (2) 12 (14) 4 (4) 58 (19) 76 (13) Triamterene, n (%) 0 (0) 0 (0) 0 (0) 5 (2) 5 (1) Aldosterone antagonists, n (%) 5 (5) 17 (19) 23 (26) 54 (18) 99 (17) Eplerenone, n (%) 1 (1) 2 (2) 6 (7) 15 (5) 24 (4) Spironolactone, n (%) 4 (4) 15 (17) 16 (18) 37 (12) 72 (12) Canrenone, n (%) 0 (0) 0 (0) 1 (1) 2 (1) 3 (1) ACE inhibitors/ARBs, n (%) 4 (4) 5 (6) 0 (0) 6 (2) 15 (3) Hydrochlorothiazide, n (%) 3 (3) 0 (0) 6 (7) 0 (0) 9 (2) Potassium supplements, n (%) 71 (66) 83 (94) 76 (84) 280 (92) 510 (87) Sodium supplements, n (%) 28 (26) 44 (50) 21 (23) 43 (14) 136 (23) Magnesium supplements, n (%) 11 (10) 21 (24) 18 (20) 226 (74) 276 (47) Phosphate supplements, n (%) 0 (0) 2 (2) 3 (3) 1 (0,3) 6 (1) Proton pump inhibitors, n (%) 20 (19) 24 (27) 14 (16) 39 (13) 97 (17) Other gastric protectors, n (%) 12 (11) 10 (11) 8 (9) 11 (4) 41 (7) Oral contraceptive, n (%) 1 (1) 1 (1) 1 (1) 20 (7) 23 (4) Vitamin D supplements, n (%) 41 (38) 20 (23) 17 (19) 26 (9) 104 (18) iPTH, intact parathyroid hormone; phosphate-SDS, age-related phosphate standard deviation score; alkaline phosphatase-SDS, age-related alkaline phosphatase standard deviation score; 25OH vitamin D, 25-hydroxy vitamin D; TRP, tubular reabsorption of phosphate; TmP/GFR, ratio of tubular maximum reabsorption of phosphate to GFR; TmP/GFR-SDS: age-related TmP/GFR standard deviation score; NSAIDs, nonsteroidal anti-inflammatory drugs; ACE inhibitors, angiotensin converting enzyme inhibitors; ARBs, angiotensin II receptor blockers. Categorical values are presented as number and % given in parentheses. Continuous values are presented as median with interquartile range (IQR) given in square brackets. Nota bene: some patients are using more than one NSAID according to the provided data. P < .001] and the use of NSAIDs [OR: 1.88 (1.08–3.28); TmP/GFR-SDS (Supplementary data, Table S10). Phosphate- P = .026]. SDS correlated with TRP (rs = 0.201; P = .004) and with Hypophosphatemia (phosphate-SDS < −2) was observed TmP/GFR-SDS (rs = 0.699; P < .001) (Supplementary data, in 22% of patients (Supplementary data, Table S9). The preva- Table S11). To exclude an effect of age and/or the necessary lence of hypophosphatemia was 19% in patients with Bartter modulation of serum phosphate to SDS score to be able to syndrome type I and II, 32% in patients with Bartter syndrome compare paediatric age groups and adults, we also evaluated type III, and 16% in patients with Gitelman syndrome. Patients serum phosphate only in adults. Hypophosphatemia was with hypophosphatemia had higher serum calcium and lower observed in 14.3% of adult patients (Supplementary data, Table PTH and phosphate homeostasis in Bartter and Gitelman syndrome 2479 A B 40 3.5 *** * * *** 30 3.0 Calcium (mmol/L) iPTH (pmol/L) 20 2.5 10 2.0 0 1.5 Bartter I and II Bartter III Bartter NOS Gitelman Bartter I and II Bartter III Bartter NOS Gitelman C D * ** * * 5.0 5.0 Phosphate-SDS TmP/GFR-SDS 0 0 –5.0 –5.0 Bartter I and II Bartter III Bartter NOS Gitelman Bartter I and II Bartter III Bartter NOS Gitelman FIGURE 2: Boxplots of iPTH, calcium, phosphate-SDS and TmP/GFR-SDS according to disease subtype iPTH, intact parathyroid hormone; phosphate-SDS, age-related phosphate standard deviation score; TmP/GFR-SDS, age-related ratio of tubular maximum reabsorption of phosphate to GFR standard deviation score; Boxplot graphs represent the median and IQR; the upper and lower whiskers represent data points within 25th percentile − 1.5x IQR and 75th percentile + 1,5x IQR. Outliers are plotted individually. *P < .05; **P < .01; ***P < .001. A. iPTH in pmol/L; statistically significant difference in iPTH level between patients with Bartter syndrome type I and II and patients with Bartter syndrome type III, Bartter syndrome NOS and Gitelman syndrome. B. Calcium in mmol/L, statistically significant difference in calcium level between patients with Bartter syndrome type I and II and patients with Gitelman syndrome. C. Phosphate-SDS; statistically significant difference in age-adjusted phosphate level between patients with Bartter syndrome type III and patients with Bartter syndrome type I and II and Gitelman syndrome. D. TmP/GFR-SDS; statistically significant difference between Bartter syndrome type I and II and Bartter syndrome NOS and Gitelman syndrome. S12). The prevalence of hypophosphatemia was 7% in patients differences were observed in serum creatinine, phosphate-SDS, with Bartter syndrome type I and II, 5% in patients with 25OH vitamin D, TmP/GFR-SDS, urinary calcium/creatinine Bartter syndrome type III, and 15% in patients with Gitelman ratio, or the prevalence of nephrocalcinosis. No differences syndrome. However, patient numbers in the Bartter subtype were observed in the prescription of NSAIDs or vitamin D groups were low. supplements between patients with and without hyperparathy- We finally performed a sensitivity analysis, demonstrating roidism. that the overall results of Bartter patients were not significantly In this cohort of patients with Bartter syndrome type I influenced by the inclusion of the Bartter syndrome NOS and II, a correlation was observed between iPTH and alkaline cohort (data not shown). Overall, patients included in the phosphatase-SDS (rs 0.268, P = .015) and an inverse correla- NOS subgroup tended to resemble more patients with type tion between iPTH and calcium (rs −0.253, P = .009) (Table 3; I and II Bartter syndrome, especially if they had elevated Figure 3). No correlation was observed between iPTH and PTH. phosphate-SDS, TmP/GFR-SDS, or urinary calcium/creatinine ratio. By multivariable logistic regression analysis, higher Analysis of disease subgroups. When restricting the anal- alkaline phosphatase-SDS [OR: 1.54 (1.09–2.19) per SDS, ysis to patients with Bartter syndrome type I and II, patients P = .015] was associated with high iPTH (Table 4). A trend with hyperparathyroidism had lower serum calcium levels was observed for lower serum calcium [OR: 0.025 (0.001– (2.42 versus 2.49 mmol/L; P = .038) and higher alkaline 1.16) per SDS, P = .059], which did not reach statistical phosphatase-SDS (1.08 versus 0.27; P = .010) (Table 2). No significance. 2480 M. F. A. Verploegen et al. Table 2. Characteristics of patients with Bartter syndrome type I and II with and without hyperparathyroidism Variable Hyperparathyroidisim No hyperparathyroidism P-value Number of patients, n (%) 60 (56) 47 (44) Characteristics Age (years) 9.6 [3.8–16.3] 8.9 [2.7–15.5] 0.476 Sex (male) 27 (45) 24 (51) 0.563 Nephrocalcinosis, n (%) 49/57 (86) 41/44 (93) 0.340 Laboratory results iPTH (pmol/L) 10.7 [8.6–14.0] 4.5 [3.2–5.6] NT Creatinine (μmol L) 54 [–71] 55 [37–77] 0.863 Sodium (mmol/L) 140 [138–142] 140 [138–143] 0.838 Potassium (mmol/L) 3.6 [3.2–3.8] 3.6 [3.3–3.9] 0.538 Chloride (mmol/L) 99 [97–102] 99 [97–101] 0.390 Bicarbonate (mmol/L) 26.0 [24.0–28.0] 26.4 [24.8–28.8] 0.414 Phosphate-SDS −0.66 [−1.56–0.47] −0.82 [−1.94–0.21] 0.418 Calcium (mmol/L) 2.42 [2.34–2.51] 2.49 [2.39–2.60] 0.038 Magnesium (mmol/L) 0.90 [0.81–0.98] 0.89 [0.79–0.98] 0.651 Alkaline phosphatase-SDS 1.08 [0.17–1.89] 0.27 [−0.28–1.09] 0.010 Uric acid (μmol/L) 315 [240–403] 327 [262–421] 0.697 Total protein (g/L) 74 [72–78] 72 [68–75] 0.147 Albumin (g/L) 46 [43–48] 47 [43–50] 0.321 25OH vitamin D (nmol/L) 40 [24.5–62.9] 35 [–94] 0.761 Urinary calcium/creatinine (mmol/mmol) 1.46 [0.91–2.42] 1.73 [1.02–2.76] 0.452 TRP 0.87 [0.79–0.93] 0.91 [0.81–0.93] 0.673 TmP/GFR-SDS −0.13 [−1.26–0.67] 0.56 [−0.36–0.90] 0.481 Treatment Indomethacin or other NSAID, n (%) 37 (62) 32 (68) 0.545 Potassium-sparing diuretics, n (%) 2 (3) 0 (0) 0.503 Aldosterone antagonists, n (%) 2 (3) 3 (6) 0.652 ACE inhibitors/ARBs, n (%) 2 (3) 2 (4) 1.000 Hydrochlorothiazide, n (%) 2 (3) 1 (2) 1.000 Potassium supplements, n (%) 42 (70) 29 (62) 0.413 Sodium supplements, n (%) 17 (28) 11 (23) 0.660 Magnesium supplements, n (%) 9 (15) 2 (4) 0.108 Phosphate supplements, n (%) 0 (0) 0 (0) N/A Proton pump inhibitors, n (%) 10 (17) 10 (21) 0.621 Other gastric protectors, n (%) 5 (8) 7 (15) 0.360 Oral contraceptives, n (%) 1 (2) 0 (0) 1.000 Vitamin D supplements, n (%) 26 (43) 15 (32) 0.238 iPTH, intact parathyroid hormone; phosphate-SDS, age-related phosphate standard deviation score; alkaline phosphatase-SDS, age-related alkaline phosphatase standard deviation score; 25OH vitamin D, 25-hydroxy vitamin D; TRP, tubular reabsorption of phosphate; TmP/GFR-SDS, age-related ratio of tubular maximum reabsorption of phosphate to GFR standard deviation score; NSAID, nonsteroidal anti-inflammatory drug; ACE inhibitors, angiotensin converting enzyme inhibitors; ARBs, angiotensin II receptor blockers; NT, not tested; N/A, not applicable Categorical data is presented as number and % and was analysed by Fisher’s exact test. Continuous data is presented as median and interquartile range and was analysed by Mann–Whitney U test. In the Gitelman cohort, we observed a significant cor- of patients had overt hypophosphatemia (phosphate-SDS relation between iPTH and serum magnesium (rs = 0.191, < −2). P = .001) and an inverse correlation between iPTH and 25OH To date, hyperparathyroidism in these disorders has only vitamin D (rs = −0.207, P = .004) (Supplementary data, been described in case reports and small series [3–9]. For Table S13). example, Landau et al. observed high PTH levels in 10/14 In addition, as in the entire cohort, a significant correlation patients with Bartter syndrome type II [5]. Additional small was observed between serum phosphate-SDS and TmP/GFR- studies did not include genetic characterization [3, 6], while SDS in patients with Bartter syndrome type III (rs = 0.744; others included only type I and II Bartter syndrome [4, 7– P < .001) and Gitelman syndrome (rs = 0.845; P < .001) 9]. Owing to the large number of patients that were recruited, (Figure 4). we were able to analyse subgroups of patients, demonstrating that hyperparathyroidism developed primarily in patients with Bartter syndrome type I and II and to a lesser degree, in DI S C U S SION patients with Bartter syndrome type III. A major confounding This cross-sectional study analysed PTH and phosphate home- factor when analysing PTH levels is kidney function. To ostasis in patients with Bartter and Gitelman syndrome. We prevent overestimating hyperparathyroidism, we excluded observed high PTH (>7.0 pmol/L) in 56% of patients with type from the analysis all patients with elevated creatinine, using I and II disease. All disease subgroups, particularly patients a very conservative threshold. In addition, we observed no with Bartter syndrome type III, had a tendency towards correlation between PTH and creatinine levels. We did not lower serum phosphate levels and a significant proportion observe age-dependent changes in PTH. PTH and phosphate homeostasis in Bartter and Gitelman syndrome 2481 Table 3. Correlation coefficients of iPTH in patients with Bartter syndrome and Gitelman syndrome. As expected, hypercalciuria was a type I and II consistent feature in patients with Bartter syndrome type I and Variable N rs P-value II, but no correlation between urinary calcium excretion and Sex (male) 107 −0.056 0.569 PTH levels were observed in this subgroup. However, the cross- Age 107 0.116 0.236 sectional design of the study and significant heterogeneity Nephrocalcinosis 101 −0.064 0.526 of patient treatments, do not allow excluding a possible role Creatinine 107 0.171 0.078 of hypercalciuria in promoting PTH secretion. Patients with Sodium 104 0.003 0.974 hyperparathyroidism in this subgroup had on average lower Potassium 107 −0.112 0.249 Chloride 98 −0.027 0.792 serum calcium levels and, as expected, serum calcium was Bicarbonate 99 0.044 0.663 negatively correlated with PTH. These findings are concordant Phosphate-SDS 104 0.104 0.293 with the reports by Landau et al. and Bettinelli et al. [3, 5]. The Calcium 105 −0.253 0.009 cause of this lower serum calcium and whether it is sufficient to Magnesium 93 0.014 0.897 trigger PTH secretion in these patients remains undetermined. Uric acid 56 0.002 0.987 Alkaline phosphatase-SDS 82 0.268 0.015 In our study, high PTH levels in type I and II Bartter Total protein 48 0.115 0.435 syndrome patients were not associated with lower serum Albumin 75 −0.126 0.283 phosphate or with higher urinary excretion of phosphate, 25OH vitamin D 60 −0.044 0.739 as also observed by Landau et al. [5]. Taken together, these Urinary calcium/creatinine ratio 82 −0.083 0.459 TRP 17 −0.409 0.103 findings raise the possibility of renal hyporesponsiveness to TmP/GFR-SDS 17 −0.085 0.747 PTH, which could contribute to PTH secretion. Yet, with Indomethacin or other NSAID 107 −0.095 0.331 renal hyporesponsiveness increased serum phosphate could be Potassium-sparing diuretics 107 0.137 0.158 expected, which we did not observe. Aldosterone antagonists 107 −0.115 0.239 Hyperparathyroidism in Bartter syndrome could also be ACE inhibitors/ARBs 107 −0.049 0.619 Hydrochlorothiazide 107 −0.003 0.978 secondary to prostaglandin secretion [4, 7]. Patients with type Potassium supplements 107 0.102 0.295 I and II disease produce large amounts of prostaglandins due Sodium supplements 107 0.042 0.668 to decreased intracellular NaCl concentration in the macula Magnesium supplements 107 0.175 0.072 densa caused by impaired salt reabsorption via the NKCC2 Proton pump inhibitors 107 0.010 0.918 pathway [6, 20, 21]. Treatment of Bartter syndrome patients Other gastric protectors 107 −0.083 0.393 Oral contraceptives 107 0.108 0.266 with NSAIDs reduces renal calcium excretion [19, 20, 22, 23] Vitamin D supplements 107 0.102 0.294 and in one report, reduces PTH levels [19]. It is unclear in this latter study, if NSAIDs directly inhibit the parathyroid glands, iPTH, intact parathyroid hormone; phosphate-SDS, age-related phosphate standard deviation score; alkaline phosphatase-SDS, age-related alkaline phosphatase standard or reduce PTH secretion indirectly, by inhibiting renal calcium deviation score; 25OH vitamin D, 25-hydroxy vitamin D; TRP, tubular reabsorption excretion. In our study, we did not observe an association of phosphate; TmP/GFR-SDS, age-related ratio of tubular maximum reabsorption of between NSAID prescription and PTH levels, nor between phosphate to GFR standard deviation score; NSAID, nonsteroidal anti-inflammatory drug; ACE inhibitors, angiotensin converting enzyme inhibitors; ARBs, angiotensin II NSAID prescription and urinary calcium excretion. receptor blockers. It has also been hypothesized that higher PTH secretion rs, Spearman’s rank correlation coefficient. in Bartter syndrome may be secondary to hyperaldosteronism [5]. Parathyroid glands express mineralocorticoid receptors The aim of this study was to evaluate the prevalence of allowing stimulation of PTH secretion by aldosterone [24]. hyperparathyroidism and phosphate homeostasis abnormali- Accordingly, mild hyperparathyroidism has been reported ties in patients with Bartter and Gitelman syndrome. To this in patients with primary hyperaldosteronism [25] and PTH end, we designed a cross-sectional study, favouring collection levels in these patients decrease after adrenalectomy [26]. of data on a very large number of patients, over collecting This hypothesis seems unlikely in patients with salt-losing longitudinal data on fewer patients which would have been tubulopathies since patients with Gitelman syndrome, who more informative on pathophysiology, but would have limited typically have very high aldosterone levels, do not develop our ability to estimate disease prevalence. However, some hyperparathyroidism. In addition, PTH levels in our co- hypotheses on the pathophysiology and possible clinical hort were not associated with treatment with aldosterone implications can be proposed and discussed. antagonists. It has previously been hypothesized that hypercalciuria, a Finally, it has been suggested that hypokalaemia could cause characteristic feature of patients with Bartter syndrome type hyperparathyroidism and that potassium supplementation I and type II, may contribute to increased PTH secretion, as could normalize PTH [27]. Here again, we did not observe an a physiological response to maintain serum calcium within inverse correlation between serum potassium and PTH. More- the normal range despite excessive renal calcium loss [4, 5, over, Bartter and Gitelman syndromes are both characterized 7, 19]. Indeed, in our whole cohort, a correlation between by hypokalaemia, whereas we observed hyperparathyroidism urinary calcium excretion and PTH was found. Calciuria is primarily in patients with Bartter syndrome. a crucial distinction between Bartter and Gitelman syndrome Another noteworthy finding in our cohort was that 20% and our observational study cannot distinguish between of patients with Gitelman syndrome had hypoparathyroidism. a causative role of calciuria for hyperparathyroidism or a The calcium-sensing receptor signalling is known to be simple association because of other differences between Bartter dependent on extracellular magnesium levels. Unlike calcium, 2482 M. F. A. Verploegen et al. A B 30 30 20 20 iPTH (pmol/L) iPTH (pmol/L) 10 10 0 0 1.5 2.0 2.5 3.0 3.5 0 2.0 4.0 6.0 Calcium (mmol/L) Urinary Ca/Cr ratio C D 30 30 20 20 iPTH (pmol/L) iPTH (pmol/L) 10 10 0 0 –4.0 –2.0 0 2.0 4.0 –4.0 –2.0 0 2.0 4.0 Phosphate-SDS TmP/GFR-SDS E 30 20 iPTH (pmol/L) 10 0 –4 –2 0 2 4 6 8 Alkaline phosphatase-SDS FIGURE 3: Scatterplots of calcium, urinary calcium/creatinine ratio, phosphate-SDS, TmP/GFR-SDS and alkaline phosphatase-SDS with iPTH of patients with Bartter syndrome type I and II. iPTH, intact parathyroid hormone; phosphate-SDS, age-related phosphate standard deviation score; alkaline phosphatase-SDS, age-related alkaline phosphatase standard deviation score; TmP/GFR-SDS: age-related ratio of tubular maximum reabsorption of phosphate to GFR standard deviation score; A. Scatterplot of calcium with iPTH. B. Scatterplot of urinary calcium/creatinine ratio with iPTH. C. Scatterplot of phosphate-SDS with iPTH. D. Scatterplot of TmP/GFR-SDS with iPTH. E. Scatterplot of alkaline phosphatase-SDS with iPTH. PTH secretion is inhibited by supraphysiologically high mag- these data, we observed a significant correlation between PTH nesium levels via stimulation of the calcium-sensing receptor, and serum magnesium in patients with Gitelman syndrome, but also by low magnesium concentrations [28]. This latter suggesting that low serum magnesium might be, at least in part, response is suggested to result from intracellular disinhibition responsible for hypoparathyroidism. of G-protein signalling downstream from the calcium-sensing Hypophosphatemia was common in all disease subgroups, receptor, precluding PTH release [29]. Accordingly, decreased in particular in Bartter syndrome type III with a prevalence PTH secretion and decreased responsiveness of PTH to ionized of 32%. The prevalence of hypophosphatemia in Bartter calcium have been suggested in small cohorts of patients syndrome type III patients decreased in adulthood to 5%. with Gitelman syndrome [30, 31, 32]. In agreement with In Gitelman syndrome, the prevalence of hypophosphatemia PTH and phosphate homeostasis in Bartter and Gitelman syndrome 2483 Table 4. Multivariable logistic regression of patients with Bartter syndrome hypokalaemia can cause proximal tubular cell injury, resulting type I and II for hyperparathyroidism (PTH >7.0 pmol/L) in a phosphate leak [33]. However, serum potassium and serum Variable Units N OR 95% CI P-value phosphate were not correlated in our cohort. Furthermore, Calcium mmol/L 80 0.03 0.001–1.16 0.059 FGF23 could play a role. Interestingly, a recent study demon- Phosphate-SDS 80 1.29 0.93–1.81 0.128 strated that in the NCC knockout mouse model for Gitelman Alkaline phosphatase-SDS 80 1.54 1.09–2.19 0.015 syndrome, FGF23 levels were increased [34]. In contrast to our Indomethacin or other NSAID Yes 80 1.12 0.38–3.34 0.833 Gitelman cohort, these mice also showed increased PTH levels Vitamin D supplements Yes 80 1.34 0.49–3.66 0.566 which, however, did not alter fractional phosphate excretion or Phosphate-SDS, age-related phosphate standard deviation score; alkaline phosphatase- serum phosphate levels. SDS, age-related alkaline phosphatase standard deviation score; NSAID, nonsteroidal anti- inflammatory drug; OR, Odds ratio; 95% CI, 95% confidence interval. We also observed significantly higher serum calcium levels in patients with Bartter syndrome, compared to patients was not age-dependent. Low serum phosphate levels were with Gitelman syndrome. Theoretically, since patients with associated with lower TmP/GFR in all subgroups, indicating Gitelman syndrome have hypocalciuria, they are expected to that hypophosphatemia was most likely related to renal have higher serum calcium levels than patients with Bartter phosphate wasting. However, renal hypophosphatemia did syndrome that have hypercalciuria. Furthermore, compared not appear to be driven by hyperparathyroidism, since PTH to healthy controls, patients with Gitelman syndrome have did not correlate with phosphate-SDS or TmP/GFR-SDS. higher serum calcium levels [13] and patients with Bartter Furthermore, patients with Gitelman syndrome did not have syndrome tend to have lower serum calcium levels [3]. Vitamin high PTH levels, while they often had hypophosphatemia D supplementation may have influenced our results. Although and renal phosphate wasting. The exact mechanism(s) of supplements were prescribed more frequently to patients with hypophosphatemia in these patients remains unclear. Notably, Bartter syndrome, patients treated with vitamin D did not have low serum phosphate levels have already been observed in significantly higher serum calcium levels, compared to non- anecdotal reports on Gitelman syndrome [10–13], including a supplemented patients. possible link with lower renal tubular phosphate reabsorption Whether in clinical practice hyperparathyroidism and [12, 13]. Interestingly, it has been hypothesized that chronic hypophosphatemia require further diagnostic evaluation and A 4 B 4 2 2 Phosphate-SDS Phosphate-SDS 0 0 –2 –2 –4 –4 –6 –6 –8 –8 –8 –6 –4 –2 0 2 4 –8 –6 –4 –2 0 2 4 TmP/GFR-SDS TmP/GFR-SDS C 4 2 Phosphate-SDS 0 –2 –4 –6 –8 –8 –6 –4 –2 0 2 4 TmP/GFR-SDS FIGURE 4: Scatterplots of TmP/GFR with age-adjusted phosphate Phosphate-SDS, age-related phosphate standard deviation score; TmP/GFR-SDS, age-related ratio of tubular maximum reabsorption of phosphate to GFR standard deviation score; A. Scatterplot of TmP/GFR with age-adjusted phosphate of all patients. B. Scatterplot of TmP/GFR with age-adjusted phosphate of patients with Bartter syndrome type III. C. Scatterplots of TmP/GFR with age-adjusted phosphate of patients with Gitelman syndrome. 2484 M. F. A. Verploegen et al. treatments is unclear. In our cohort, high PTH was associated aspects were probably well compensated for by the large size with higher alkaline phosphatase levels. This is likely due to of our cohort. Since the age range of our patients spanned increased bone turnover. Reduced bone mineral density has from small children to late adulthood, some values had to be previously been observed in patients with Bartter syndrome normalized for age using reference data from the literature. [35, 36]. It may therefore be appropriate that Bartter syndrome This may have introduced some distortions in our data. patients undergo a regular assessment of their bone density In conclusion, this cross-sectional study demonstrates that to detect incipient osteopenia or osteoporosis, particularly if PTH is frequently elevated in patients with Bartter syndrome, they have high PTH. Since vitamin D deficiency stimulates especially in those with type I and II disease. The cause PTH secretion, supplements could be prescribed in patients of hyperparathyroidism in this population remains unclear. with low vitamin D levels, keeping in mind that they may Hypophosphatemia was observed in a significant proportion worsen hypercalciuria in patients with Bartter syndrome. Of of patients with Bartter and Gitelman syndrome and ap- note, 25-OH vitamin D concentrations are usually normal peared to be primarily related to a PTH-independent renal in patients with Bartter syndrome [23], while high levels of phosphate leak. Further studies are needed to understand 1,25-OH vitamin D have been reported [23, 36]. The use of better the pathophysiology and the clinical relevance of these calcimimetics in patients with hyperparathyroidism associated observations. with Bartter syndrome has been described in five patients [7, 8]. In four of them, PTH levels normalized. However, all had high-normal or even high serum calcium levels. In SU P P L E M E N TA RY DATA theory, normocalcemic patients with Bartter syndrome could Supplementary data are available at ndt online. be at increased risk of developing symptomatic hypocalcemia if treated with calcimimetics, since they have metabolic alkalosis, which decreases the proportion of ionized calcium. It is AC K NOW L E D G E M E N T S unknown whether hypophosphatemia, especially if associated This project has been supported by the European Reference with bone changes, should be treated with phosphate supple- Network for Rare Kidney Diseases (ERKNet), which is partly mentation. co-funded by the European Union within the framework of the The main limitations of our study relate to the cross- Third Health Programme ‘ERN-2016-Framework Partnership sectional design and the relatively large group of Bartter NOS Agreement 2017–2021’. This work is generated within the patients in the analysis. The cross-sectional design allowed European Society for Paediatric Nephrology working group us to assemble data on a very large number of patients to on inherited renal disorders. This work was supported by an estimate the prevalence of PTH and phosphate abnormalities Innovation Grant 19OI06 from the Dutch Kidney Foundation in these diseases. However, longitudinal follow-up of patients (to T.N.). would provide additional information about the course of PTH and other variables throughout the years, which could have helped identify possible causal effects. Future studies should C ON F L IC T OF I N T E R E ST STAT E M E N T collect data at the initial presentation, thus pre-treatment The authors have no conflicts of interest to declare. and should also evaluate follow-up data of patients. An important opportunity to prospectively capture such data is by adding PTH and phosphate as variables to the ERKNet DATA AVA I L A B I L I T Y STAT E M E N T patient registry (ERKReg). The second main limitation is the The data underlying this article will be shared on reasonable relatively large group of patients with Bartter syndrome NOS, request to the corresponding author. who lacked a genetic diagnosis and were diagnosed based on their phenotype. 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