Genetics of Hereditary Disorders of Magnesium Homeostasis

Pediatr Nephrol (2004) 19:13–25 DOI 10.1007/s00467-003-1293-z

REVIEW

Karl P. Schlingmann · Martin Konrad · Hannsjrg W. Seyberth Genetics of hereditary disorders of magnesium homeostasis

Received: 28 April 2003 / Revised: 25 July 2003 / Accepted: 28 July 2003 / Published online: 22 November 2003 IPNA 2003

Abstract Magnesium plays an essential role in many transporters and in signal transduction. Under physiolog- biochemical and physiological processes. Homeostasis of ical conditions, serum magnesium levels are maintained magnesium is tightly regulated and depends on the at almost constant values. Homeostasis depends on the balance between intestinal absorption and renal excretion. balance between intestinal absorption and renal excretion. During the last decades, various hereditary disorders of Magnesium deficiency can result from reduced dietary magnesium handling have been clinically characterized intake, intestinal malabsorption, or renal loss. The control and genetic studies in affected individuals have led to the of body magnesium homeostasis primarily resides in the identification of some molecular components of cellular kidney tubules. magnesium transport. In addition to these hereditary A number of acquired and hereditary disorders of forms of magnesium deficiency, recent studies have magnesium handling, most of them due to renal magne- revealed a high prevalence of latent hypomagnesemia in sium loss, have been described, all of them being the general population. This finding is of special interest relatively rare. The phenotypic characterization of clin- in view of the association between hypomagnesemia and ically affected individuals and experimental studies have common chronic diseases such as diabetes, coronary heart allowed the identification of the nephron segments disease, hypertension, and asthma. However, valuable involved in these conditions. Together with the mode of methods for the diagnosis of body and tissue magnesium inheritance this has led to a classification of inherited deficiency are still lacking. This review focuses on magnesium wasting disorders into different subtypes [1, clinical and genetic aspects of hereditary disorders of 2]. During the last few years, genetic studies have resulted magnesium homeostasis. We will review primary defects in the identification of a number of genes involved in the of epithelial magnesium transport, disorders associated pathogenesis of these disorders and provided a first with defects in Ca2+/Mg2+ sensing, as well as diseases insight into the physiology of epithelial magnesium characterized by renal salt wasting and hypokalemic transport at the molecular level. Some of these genotypes alkalosis, with special emphasis on disturbed magnesium are associated with a mild or even asymptomatic clinical homeostasis. course. Consequently, the diagnosis is often delayed and the disease prevalence might be underestimated. Keywords Magnesium · Hypomagnesemia · Magnesium In contrast to hypomagnesemia of hereditary origin, deficiency · Hereditary magnesium loss latent or subclinical hypomagnesemia is relatively frequent, with a prevalence of around 14% in the general population [3]. There is growing evidence of an associ- Introduction ation of magnesium deficiency with common chronic diseases such as coronary heart disease, stroke, diabetes Magnesium is the fourth most-abundant cation in the mellitus, and asthma [4, 5]. Whether magnesium supple- body. As a cofactor for many enzymes, it is involved in mentation has a beneficial effect under these conditions energy metabolism and protein and nucleic acid synthesis. remains unknown. It also plays a critical role in the modulation of membrane

K. P. Schlingmann · M. Konrad · H. W. Seyberth ()) Magnesium physiology Department of Pediatrics, Philipps University, Deutschhausstrasse 12, 35037 Marburg, Germany Magnesium is the second most-prevalent intracellular e-mail: [email protected] cation. The normal body magnesium content is approx- Tel.: +49-6421-2862789 imately 24 g (1,000 mmol). Magnesium is distributed Fax: +49-6421-2865724 14 mainly in bone and the intracellular compartments of muscle and soft tissues; less than 1% of total body magnesium is located in the blood [6]. Serum magnesium levels are maintained in a narrow range. Circulating magnesium is present in three different states: dissociated/ ionized, bound to albumin, or complexed to phosphate, citrate, or other anions. Ionized and complexed forms account for the ultrafiltrable fraction, the biological active portion is the free, ionized magnesium ([Mg2+]). First studies on body magnesium kinetics were con- ducted in the 1960s with the radioactive isotope 28Mg. Avioli and Berman [7] proposed a multicompartmental model of exchangeable magnesium pools: a magnesium pool mass with a relatively fast turnover comprising ~15% of the estimated body content, representing primarily the extracellular fluid, and a slow-turnover intracellular pool, comprising >70% of total body magnesium. The mechanisms that regulate the exchange between the extracellular and intracellular compartments have not been clearly elucidated. Equilibration between serum magnesium and body stores occurs slowly [6]. Magnesium homeostasis depends on the balance between intestinal absorption and renal excretion. The daily dietary intake of magnesium varies substantially. In 1964, Seelig [8] analyzed a large number of balance studies and concluded that the minimum intake of Fig. 1 a Schematic model of intestinal magnesium absorption via magnesium required to maintain external balance is two independent pathways: passive absorption via the paracellular around 0.25 mmol or 6 mg/kg body weight per day in pathway and active, transcellular transport consisting of an apical entry through a putative magnesium channel and a basolateral exit adults. However, estimations from studies in the United mediated by a putative sodium-coupled exchange. b Kinetics of States and European countries indicate substantially lower human intestinal magnesium absorption. Paracellular transport mean daily magnesium intakes, around 0.16 mmol or linearly rising with intraluminal concentrations (dotted line) and 4 mg/kg body weight per day [9]. For the pediatric saturable active transcellular transport (dashed line) together yield a population, data on daily magnesium requirements are curvilinear function for net magnesium absorption (solid line) sparse. During growth, a positive magnesium balance is expected, but the desirable extent is unknown. For more detailed information on estimated average requirements in different age groups please refer to the 1997 FDA guidelines [10]. Within physiological ranges, diminished magnesium intake is balanced by enhanced magnesium absorption in the intestine and reduced renal excretion. These transport processes are regulated by metabolic and hormonal factors [11, 12]. The principal site of magnesium absorption is the small intestine with smaller amounts being absorbed in the colon. Intestinal magnesium absorption occurs via two different pathways: a saturable active transcellular pathway and a non-saturable paracellular passive transport pathway [11, 13] (Fig. 1a). Satu- ration kinetics of the transcellular transport system indicate a limited active transport capacity. At low intraluminal concentrations magnesium is absorbed primarily via the active transcellular route and with rising concentrations via the paracellular pathway, yielding a curvilinear function for total absorption (Fig. 1b). In the kidney, approximately 80% of total serum magnesium is filtered at the glomeruli, of which more than 95% is reabsorbed along the nephron. In the adult (Fig. 2), 15%–20% is reabsorbed in the proximal tubule. Interestingly, the newborn is able to reabsorb up to 70% Fig. 2 Magnesium reabsorption along the nephron 15 Around 70% of magnesium is reabsorbed in the loop of Henle, especially in the cortical thick ascending limb (TAL). Transport in this segment is passive and paracellular, driven by the lumen-positive voltage (Fig. 3a). Approximately 5%–10% of the filtered magnesium is reabsorbed in the distal convoluted tubule (DCT) via an active, transcellular process (Fig. 3b). The reabsorption rate in the DCT defines the final urinary magnesium excretion, as there is no significant reabsorption of magnesium in the collecting duct. Physiological studies indicate that apical entry into DCT cells is mediated by a specific and regulated magnesium channel driven by a favorable transmembrane voltage [16]. The mechanism of basolateral transport into the interstitium is unknown. Magnesium has to be extruded against an unfavorable electrochemical gradient. Most physiological studies favor a sodium-dependent exchange mechanism [17]. Magnesium entry into DCT cells appears to be the rate- limiting step and the site of regulation. Magnesium transport in the distal tubule has recently been reviewed in detail by Dai et al. [16]. Finally, 3%–5% of the filtered magnesium is excreted in the urine.

Diagnosis of magnesium deficiency

Magnesium deficiency and hypomagnesemia often re- main asymptomatic. Clinical symptoms are mostly non- specific and magnesium deficiency is frequently associated with additional electrolyte abnormalities, especially hypocalcemia and hypokalemia. Alternatively, the symptoms of hypomagnesemia may be minor compared with the symptoms of the primary disease causing the magnesium depletion. In addition, symptoms do not necessarily correlate with serum magnesium levels, as these do not always reflect body magnesium content. Among the first symptoms of hypomagnesemia are abdominal pain, nausea, vomiting, lethargy, and weakness. In more pronounced magnesium depletion symptoms of increased neuromuscular excitability predominate, such as tremor, carpopedal spasms, muscle cramps, tetany, and generalized seizures. In addition, irritability, attention deficit, or mental confusion can occur. Cardiac manifestations Fig. 3 a Magnesium reabsorption in the thick ascending limb of Henle0s loop. Paracellular reabsorption of magnesium and calcium include atrial/ventricular tachycardia, premature contrac- is driven by lumen-positive transcellular voltage generated by the tions, a prolonged QT interval, and torsades de pointes. transcellular reabsorption of NaCl. b Magnesium rebsorption in the Magnesium depletion is often associated with concur- distal convoluted tubule. In this nephron segment magnesium is rent disturbances in potassium homeostasis. This rela- reabsorbed actively via the transcellular pathway involving an apical entry step probably through a magnesium-selective ion tionship is in part due to underlying disorders that cause channel and a basolateral exit, presumably mediated by a sodium- both magnesium and potassium loss, such as diuretic use coupled exchange mechanism. The molecular identity of basolat- or diarrhea, but hypomagnesemia itself can induce eral exchange is unknown hypokalemia by increasing potassium secretion in the loop of Henle and in the collecting duct [18]. Hypokal- emia in the setting of magnesium depletion is largely of the filtered magnesium in this nephron segment [14]. refractory to potassium replacement alone and requires This increased magnesium reabsorption in the neonatal correction of the magnesium deficit. proximal tubule is explained by a paracellular magnesium As yet, appropriate methods for the routine testing of permeability that disappears with the maturation of the body magnesium status are lacking, rendering the iden- nephron segment [15]. tification of subclinical forms of magnesium deficiency a diagnostic challenge. Assessment of total serum magne- 16 sium levels is most widely used for the evaluation of 0.1 mmol/kg body weight of magnesium aspartate given magnesium status, although their limitations in reflecting over 1 h yielded comparable results and might represent a magnesium deficiency are well recognized [6]. The useful alternative [30]. Excretion of less than 70% of the reference range for normal total serum magnesium is a infused magnesium is considered indicative of functional subject of ongoing debate. Concentrations of 0.70– magnesium deficiency [21]. Besides its role for the 1.10 mmol/l are generally accepted. However, epidemi- identification of magnesium deficiency in normomagne- ological studies have revealed correlations between semic individuals with normal renal magnesium handling, increased cardiovascular risk and serum magnesium the parenteral MLT allows differentiation between renal concentrations within this reference interval [19]. Conse- and extrarenal magnesium losses in patients with inher- quently, values of at least 0.80 mmol/l seem to be ited forms of magnesium wasting, which are overtly desirable. hypomagnesemic. It has been suggested that measurement of ionized The substitution of magnesium in hypomagnesemia is serum magnesium or intracellular magnesium concentra- primarily aimed at the relief of clinical symptoms. tions might provide more precise information on magne- Unfortunately, especially in cases of hereditary renal sium status. Measurement of ionized magnesium in magnesium wasting, normal values for total serum addition to total serum magnesium might be particularly magnesium are hardly achieved by oral substitution useful in evaluating patients with hypoalbuminemia to without considerable side effects, mainly resulting from exclude “pseudohypomagnesemia” [20]. The relevance of the cathartic effects of magnesium salts. such measurements to body magnesium stores, however, The route of administration depends on the severity of has been questioned, as ionized magnesium levels did not the clinical findings. Acute intravenous infusion is usually correlate with the results of a parenteral loading test [21]. reserved for patients with symptomatic hypomagnesemia, Several investigators explored the value of measuring i.e., with cerebral convulsions [18]. Intravenous admin- blood cell intracellular magnesium (total and ionized), but istration should be preferred to painful intramuscular the correlation with tissue magnesium of muscle and bone injections, especially in children. was poor and correlations with magnesium retention in In neonates and children, the initial treatment usually the parenteral loading test were contradictory [22, 23]. consists of 25–50 mg magnesium sulfate (0.1–0.2 mmol The use of stable magnesium isotopes and muscle 31P- magnesium) per kilogram body weight slowly given nuclear magnetic resonance spectroscopy represent intravenously (over 20 min) (up to a maximum of 2 g promising new methods for non-invasive estimation of magnesium sulfate, which is the adult dosage). This dose body and/or tissue magnesium pools as a better indicator can be repeated every 6–8 h or can be followed by a of body magnesium status. However, they are not continuous infusion of 100–200 mg magnesium sulfate particularly suitable as a routine measurement. (0.4–0.8 mmol magnesium) per kilogram body weight Hypomagnesemia develops late in the course of given over 24 h [31, 32]. magnesium deficiency and intracellular magnesium de- In the presence of hypocalcemia, this regimen can be pletion may be present despite normal serum magnesium continued for 3–5 days. When magnesium is administered levels. Due to the kidney0s ability to sensitively adapt its intravenously, calcium gluconate (i.v.) should be avail- magnesium transport rate to imminent deficiency, urinary able as an antidote. Control of blood pressure, heart rate, magnesium excretion is of special importance in the and respiration is of special importance, as well as a close assessment of the magnesium status. In patients with monitoring of serum magnesium levels. Before adminis- hypomagnesemia, urinary magnesium excretion levels tration, normal renal function has to be ascertained. help to differentiate between renal magnesium wasting Special caution is required in cases of renal insufficiency. and extrarenal losses. In the presence of hypomagnese- In asymptomatic hypomagnesemic or magnesium- mia, the 24-h magnesium excretion is expected to be deficient patients, oral replacement represents the pre- below 1 mmol [24]. Magnesium/creatinine ratios and ferred route of administration. Exact dosages required to fractional excretion of magnesium have also been advo- correct magnesium deficiency are largely unknown. For cated as indicators of evolving magnesium deficiency in the pediatric population, 10–20 mg magnesium (0.4– normomagnesemic individuals [25, 26]. However, the 0.8 mmol) per kg body weight given three to four times a interpretation of results seems to be limited due to intra- day has been recommended to correct hypomagnesemia and inter-individual variability [27, 28]. [33]. However, in our personal experience, a continuous The parenteral magnesium loading test (MLT) remains administration of magnesium, for example dissolved in the gold standard for the evaluation of body magnesium mineral water, has proven to be of advantage, as peak status [6, 21]. The major limitation is a decreased renal magnesium blood levels are avoided. function. The aim is to calculate the retention of an Solubility, intestinal absorption, and side effects intravenous magnesium load. This is done by using the greatly differ depending on the magnesium salt used for formula 1(Mg2+ in 24-h urine Mg2+ test dose)100. oral treatment. The bioavailability and pharmacokinetics Several protocols have been proposed. In one protocol, of diverse magnesium salts have been reviewed recently 1 mmol of elemental magnesium per kilogram body [34]. Considering solubility, intestinal absorption and weight is infused over 4 h and a 24-h urine collection is bioavailability, organic magnesium salts such as magne- started simultaneously [29]. A short-term MLT with sium citrate or aspartate appear most suitable for oral 17 replacement therapy. In addition, the laxative effect of hypomagnesemia such as muscle weakness, tremor, or these preparations seems to be less pronounced compared tetany were absent. One of the patients remained asymp- with inorganic magnesium salts. tomatic until adolescence. The second patient became In addition to replacement therapy, the use of certain symptomatic in infancy and was treated with anti-epileptic diuretics has been proposed for the reduction of renal drugs until serum magnesium levels were evaluated in magnesium excretion. The aldosterone antagonist spiro- adolescence. Severe mental retardation was evident in this nolactone, as well as potassium-sparing diuretics such as patient. Serum magnesium measurements performed in amiloride, exerts magnesium-sparing effects [35, 36]. other members of both families indicated an autosomal Studies in patients with hereditary magnesium wasting dominant mode of inheritance. Interestingly, many other showed a beneficial effect of these diuretics on renal affected family members remained asymptomatic. How- magnesium excretion, serum magnesium levels, and ever, newborns of affected mothers have been found to clinical manifestations [37, 38]. have severe hypomagnesemia at birth despite moderate, subclinical maternal hypomagnesemia [40]. A low urinary excretion of calcium was found in all Hereditary disorders of magnesium handling hypomagnesemic family members, a finding that is reminiscent of patients presenting with Gitelman syn- Recent advances in the molecular genetics of hereditary drome. However, in contrast to patients with the Gitelman hypomagnesemia have substantiated the role of a variety variant of Bartter syndrome, no other associated biochem- of genes and their encoded proteins in human epithelial ical findings were reported. 28Mg retention studies indi- magnesium transport. The knowledge on underlying cated a primary renal defect. Linkage analysis mapped the genetic defects allowed identification of different clinical gene locus to chromosome 11q23 [41]. Haplotype analysis subtypes of hereditary disorders of magnesium homeo- revealed a common ancestor of both families. Within the stasis. The following entities will be discussed: isolated critical interval, Meij et al. [42] identified the gene FXYD2 renal magnesium wasting, familial hypomagnesemia with coding for a gamma subunit of the basolateral Na-K- hypercalciuria and nephrocalcinosis (FHHNC), hypomag- ATPase that is localized in the DCT segment of the nesemia with secondary hypocalcemia (HSH), calcium- nephron, the site of active transcellular magnesium sensing receptor-associated disorders, and disorders of transport. Sequence analysis showed a heterozygous renal salt wasting with hypokalemic metabolic alkalosis, mutation, Gly41Arg, in all affected individuals. Expres- comprising the Gitelman syndrome and other Bartter-like sion studies in mammalian renal tubule cells revealed a syndromes (Tables 1 and 2). dominant-negative effect of this mutation leading to a retention of the gamma subunit within the cell, resulting in failure to modulate pump kinetics. This in turn may lead to Isolated dominant hypomagnesemia a decrease in pump activity and secondary reduction in magnesium reabsorption [43]. However, the mechanism Geven et al. [39] first described two patients who by which disturbed function of Na-K-ATPase impairs presented with generalized convulsions. Other signs of magnesium reabsorption in the DCT remains unknown,

Table 1 Inherited disorders or magnesium handling (AD autosomal dominant, AR autosomal recessive) Disorder OMIM Inherit- Gene Gene Protein ance locus Isolated dominant hypomagnesemia 154020 AD 11q23 FXYD2 g-subunit of the Na+-K+-ATPase with hypocalciuria 154020 AD ? ? ? Isolated recessive hypomagnesemia 248250 AR ? ? ? with normocalciuria Familial hypomagnesemia 248250 AR 3q28 CLDN16 Paracellin-1, tight junction protein with hypercalciuria/nephrocalcinosis Hypomagnesemia with secondary hypocalcemia 602014 AR 9q22 TRPM6 TRPM6, putative ion channel Autosomal dominant hypoparathyroidism 601198 AD 3q21 CASR CaSR, Ca2+/Mg2+ sensing receptor Familial hypocalciuric hypercalcemia 145980 AD 3q21 CASR CaSR, Ca2+/Mg2+ sensing receptor Neonatal severe hyperparathyroidism 239200 AR 3q21 CASR CaSR, Ca2+/Mg2+ sensing receptor Hyperprostaglandin E syndrome/antenatal 601678 AR 15q21 SLC12A1 NKCC2, Na+K+2Cl- co-transporter, Bartter syndrome bumetanide-sensitive co-transporter 241200 AR 11q24 KCNJ1 ROMK, renal potassium channel Hyperprostaglandin E syndrome/antenatal 602522 AR 1p31 BSND Renal chloride channel b-subunit Barttin Bartter syndrome with sensorineural deafness Classic Bartter syndrome 602023 AR 1p36 CLCNKB CLC-Kb, distal tubule chloride channel Gitelman variant of Bartter syndrome 263800 AR 16q13 SLC12A3 NCCT, Na+Cl- co-transporter, thiazide-sensitive co-transporter 18 Table 2 Clinical and biochemical characteristics (N normal) Disorder Age at onset Serum Serum Serum Blood Urine Urine Nephro- Renal Mg2+ Ca2+ K+ pH Mg2+ Ca2+ calcinosis stones Isolated dominant hypomagnesemia Childhood # NNN"# No No with hypocalciuria Isolated recessive hypomagnesemia Childhood # NNN" NNoNo with normocalciuria Familial hypomagnesemia with Childhood # NNNor#"" "" Yes Yes hypercalciuria/ nephrocalcinosis Hypomagnesemia with secondary Infancy ### # NN" NNoNo hypocalcemia Autosomal dominant hypoparathyroidism Infancy ##NNor#" "to "" Yesa Yesa Familial hypocalciuric hypercalcemia Often Nto"" NN## No ? asymptomatic Neonatal severe hyperparathyroidism Infancy N to " """ NN## No ? Hyperprostaglandin E syndrome/antenatal Neonatal N N # to ## " ? "" Yes ? Bartter syndrome Hyperprostaglandin E syndrome/antenatal Neonatal ? N ## " ?Nto" No No Bartter syndrome with sensorineural deafness Classic Bartter syndrome Infancy N or # N ## " Nto" Variable Rare No Gitelman variant of Bartter syndrome Variable # N ## " " # No No a Frequent complication under therapy with calcium and vitamin D especially since there is an increase in renal calcium calcium wasting. Affected individuals had bilateral reabsorption and hypocalciuria in these patients. One nephrocalcinosis and developed progressive renal failure. possibility is that the gamma subunit is involved not only Since then, patients of at least 50 different kindreds have in Na-K-ATPase function but also in an ATP-dependent been reported, allowing a comprehensive characterization transport system specific for magnesium. of the clinical spectrum and discrimination from other Interestingly, isolated dominant hypomagnesemia magnesium-losing tubular disorders [48, 49, 50, 51, 52, seems to be genetically heterogeneous, as linkage to the 53]. FXYD2 locus has been excluded in a large Californian FHHNC patients usually present during early child- family with a phenotype very similar to that seen in the hood with recurrent urinary tract infections, polyuria/ Dutch families [44]. polydispsia, nephrolithiasis, and/or failure to thrive. Signs of severe hypomagnesemia such as cerebral convulsions and muscular tetany are less common. Extrarenal man- Isolated recessive hypomagnesemia ifestations, especially ocular involvement (including severe myopia, nystagmus, or chorioretinitis), have also Geven et al. [45] also reported a form of isolated been reported [50, 51]. Additional abnormalities include hypomagnesemia in a consanguineous family, indicating elevated serum parathyroid hormone (PTH) levels before autosomal recessive inheritance. The affected individuals the onset of chronic renal failure, incomplete distal renal presented with symptoms of hypomagnesemia early tubular acidosis, hypocitraturia, and hyperuricemia pres- during infancy. In addition to hypomagnesemia due to ent in most patients. The prognosis of FHHNC patients is increased urinary magnesium excretion, no biochemical rather poor with progression to chronic renal failure early abnormality was reported. Isolated recessive hypomag- during adolescence. In a series of 33 patients, one-third nesemia is distinguished from autosomal dominant hypo- already had a markedly reduced glomerular filtration rate magnesemia by the lack of hypocalciuria. Linkage (GFR) (<60 ml/min per 1.73 m2) at the time of diagnosis analysis excluded all gene loci involved in known forms [53]. Hypomagnesemia may completely disappear with of hereditary hypomagnesemia, indicating genetic het- the decline of GFR due to a reduction of filtered erogeneity [46]. magnesium. In addition to oral magnesium supplementation, therapeutic approaches include thiazides in order to reduce Familial hypomagnesemia with hypercalciuria calcium excretion and progression of nephrocalcinosis and nephrocalcinosis and stone formation, as the degree of renal calcification has been correlated with the impairment of renal function In 1972, Michelis et al. [47] first described patients with [51]. However, these therapeutic strategies do not seem to hypomagnesemia and excessive renal magnesium and significantly influence the progression of renal failure. 19 Nevertheless, supportive therapy is essential for the of PTH action. As reported by Anast et al. [60], in severe protection of kidney function and should include provi- hypomagnesemia there is impaired synthesis and/or sion of sufficient fluids and effective treatment of stone release of PTH. Consistently, PTH levels in HSH patients formation and bacterial colonization. As expected, renal were found to be inappropriately low at initial presenta- transplantation may be performed without evidence of tion. Furthermore, several findings point to a role of end recurrence because the primary defect resides in the organ resistance to PTH in the development of hypocal- kidney. cemia. Studies have shown that administration of PTH Based on clinical observation and clearance studies, fails to correct the hypocalcemia in the presence of Rodriguez-Soriano et al. [49] postulated that the primary hypomagnesemia [61]. In addition, PTH-induced release defect in FHHNC is related to impaired magnesium and of calcium from bone is substantially impaired in calcium reabsorption in the loop of Henle. In this part of hypomagnesemia [62], as magnesium depletion interferes the nephron magnesium and calcium are reabsorbed with the generation of cAMP in response to PTH [63]. passively through the paracellular pathway. The hypocalcemia is resistant to treatment with calcium Using a positional cloning approach, Simon et al. [54] or vitamin D. Normocalcemia, normalization of PTH identified a new gene (CLDN16, formerly PCLN-1) that is levels, and relief of clinical symptoms can be achieved by mutated in patients with FHHNC. CLDN16 codes for administration of high doses of magnesium. Daily paracellin-1, a member of the claudin family of tight requirements of more than 4 mmol/kg body weight per junction proteins. Expression studies revealed expression day (>16 times the mean daily intake) have been of CLDN16 in the medullary and cortical thick ascending described [64], although an average daily requirement limb of Henle’s loop. There, it co-localizes with occludin of 1.6 mmol/kg body weight per day was reported to be at tight junctions [54]. The selective defect of paracellular sufficient in other HSH patients [65]. reabsorption of calcium and magnesium (with intact NaCl The main side effect is chronic diarrhea, which is reabsorption ability in this nephron segment) suggests that observed in a considerable number of patients on oral paracellin-1 contributes to the formation of a calcium- replacement therapy. This prompted the evaluation of and magnesium-selective paracellular pore [55]. Recent- alternative regimens and routes of administration. Split- ly, expression of CLDN16 was also demonstrated in ting of oral doses can reduce fluctuations of serum bovine cornea and retinal pigment epithelium indicating a magnesium levels and peak urinary excretion and also direct link between defects in paracellin-1 and ocular alleviate diarrhea. Additional intramuscular magnesium abnormalities observed in some patients [56]. injections might be necessary to reduce oral intake. A In heterozygous family members of FHHNC patients, regimen consisting of daily intramuscular injections given two independent studies described a high incidence of over a 20-year period has been described [64]. Ultimately, hypercalciuria and nephrolithiasis [51, 53]. In addition, the authors used continuous nocturnal administration via mild hypomagnesemia was observed in family members nasogastric tube as a therapeutic alternative to improve with heterozygous CLDN16 mutations [57]. Therefore, quality of life. In another HSH patient, hypomagnesemic one might speculate that heterozygous CLDN16 muta- seizures only ceased after implantation of a subcutaneous tions might be involved in idiopathic hypercalciuric stone pump system providing continuous magnesium infusions formation. Recently, a homozygous mutation affecting [66]. the C-terminal PDZ domain of paracellin-1 was identified The pathophysiology of HSH was largely unknown in two families with isolated hypercalciuria and medul- until recent times, although physiological studies indicat- lary nephrocalcinosis without disturbances in renal mag- ed a primary defect in intestinal magnesium absorption nesium conservation. Interestingly, the nephrocalcinosis [67]. In some patients an additional renal leak of did not progress and the hypercalciuria disappeared with magnesium was suspected [68]. age [58]. A gene locus (HOMG1) for HSH had been mapped to chromosome 9q22 [69] and further refined to a critical interval of approximately 1 cM [70]. Recently, mutations Hypomagnesemia with secondary hypocalcemia in TRPM6 were identified as the underlying defect in patients with HSH [71, 72]. TRPM6 codes for a new HSH is an autosomal recessive disorder that manifests in member of the transient receptor potential (TRP) family early infancy with generalized convulsions refractory to of cation channels. The TRPM6 protein is highly anticonvulsant treatment or other symptoms of increased homologous to TRPM7, which was characterized as a neuromuscular excitability like muscle spasms or tetany. calcium- and magnesium-permeable ion channel regulat- It was first described by Paunier et al. in 1968 [59]. ed by Mg-ATP [73]. Expression studies detected TRPM6 Failure of early diagnosis or non-compliance with treat- along the entire gastrointestinal tract as well as in kidney. ment can be fatal or result in permanent neurological Reverse transcription/polymerase chain reaction of rat damage. nephron segments revealed TRPM6 expression in DCT Laboratory evaluation reveals extremely low serum cells [71]. magnesium and serum calcium levels. The mechanism The observation that in HSH patients the administra- leading to hypocalcemia is still not completely under- tion of high oral doses of magnesium are successful in stood. Several factors seem to contribute to an impairment achieving at least subnormal serum magnesium levels 20 supports the theory of two independent intestinal transport treatment. Urinary excretion rates for calcium and systems for magnesium. TRPM6 probably represents a magnesium are markedly reduced and serum PTH levels molecular component of the active transcellular magne- are inappropriately high. In addition, affected individuals sium transport pathway. An increased intraluminal mag- also show mild hypermagnesemia [81]. In contrast, nesium concentration (by increased oral intake) would patients with NSHPT usually present under the age of allow compensation for the defect in active transcellular 6 months with polyuria and dehydration due to severe transport by increasing absorption via the passive para- symptomatic hypercalcemia. Unrecognized and untreated, cellular pathway. The detection of TRPM6 expression in hyperparathyroidism and hypercalcemia result in skeletal the DCT confirmed the assumption of Cole and Quamme deformities, extraosseous calcifications, muscle wasting, [1] of an additional source of renal magnesium loss in and a devastating neurodevelopmental deficit. Early patients with HSH. This is also supported by the treatment with partial to total parathyroidectomy there- observation that HSH patients subjected to an intravenous fore seems to be essential for outcome [82]. MLT have a considerable renal magnesium leak while Activating mutations of the CaSR result in autosomal still being hypomagnesemic [72]. dominant hypocalcemia (ADH) [83]. Affected individuals are often given the diagnosis of primary hypoparathyroidism on the basis of inadequately low PTH levels Ca2+/Mg2+-sensing receptor-associated disorders despite their hypocalcemia. Hypocalcemia is generally mild to moderate. Clinical correlates are seizures, The extracellular Ca2+/Mg2+-sensing receptor (CaSR) predominantly during childhood, or carpopedal spasms, plays an essential role in magnesium and calcium but patients can also be asymptomatic. The laboratory homeostasis by influencing not only PTH secretion but evaluation also reveals hypomagnesemia in the majority also by directly regulating the rate of magnesium and of patients. The differentiation from primary hypopar- calcium reabsorption in the kidney. The CaSR was first athyroidism is of particular importance because treatment cloned by Brown et al. in 1993 [74]. CaSR expression was with vitamin D may result in a dramatic increase in demonstrated in PTH-secreting cells of the parathyroid hypercalciuria and the occurrence of nephrocalcinosis and gland, along the nephron predominantly in the TAL [75], impairment of renal function. Therefore, therapy with as well as in various tissues not primarily involved in vitamin D or calcium supplementation should be reserved calcium and magnesium homeostasis. The CaSR belongs for symptomatic patients with the aim of maintaining to the family of G-protein-coupled receptors and bears serum calcium levels just sufficient for the relief of several low-affinity cation binding sites in the extracel- symptoms [84]. lular domain, allowing cooperative interaction with Activating mutations lead to a lower set point of the multiple cations in a millimolar concentration range receptor or an increased affinity for extracellular calcium [76]. In mouse DCT cells a sensitive and equipotent CaSR and magnesium. In ADH patients, this inadequate CaSR response to either magnesium or calcium at physiological activation by physiological extracellular calcium and concentrations has been demonstrated [75]. magnesium levels then results in diminished PTH secre- In the kidney, CaSR activation results in a diminished tion and decreased reabsorption of both divalent cations reabsorption of divalent cations in the TAL, as well as a mainly in the cTAL. For magnesium, the inhibition of diminished water reabsorption in the collecting duct. In hormone-stimulated reabsorption in the DCT may signif- the TAL, passive paracellular reabsorption of magnesium icantly contribute to an increased renal loss, in addition to and calcium is inhibited by a reduction in positive the effects observed in the TAL [16]. Recently, a Bartter- transcellular voltage following diminished transcellular like phenotype in patients with activating CaSR mutations NaCl transport [77]. Inhibition of ROMK channels and/or has been described [85, 86]. In addition to hypocalcemia NKCC2 transporters by CaSR activation via phospholi- and deficient PTH secretion, these patients developed pase A2 has been postulated [78]. In the collecting duct, renal salt and water loss associated with hypokalemic CaSR activation mediates a decrease in water reabsorp- alkalosis. Furthermore, all patients were found to be tion by a reduction of vasopressin-stimulated incorpora- hypomagnesemic. Functional expression of the underly- tion of aquaporin-2 water channels into the apical ing mutations revealed a complete CaSR activation under membrane. This leads to an increase in urinary water physiological serum calcium concentrations. This proba- flow that minimizes the risk of stone formation in the face bly leads to a reduction in NaCl reabsorption in the cTAL of an increase in calcium and magnesium excretion [76]. possibly via inhibition of ROMK and/or NKCC2, result- Several diseases associated with both activating and ing in renal salt loss with secondary hyperaldosteronism inactivating mutations in the CaSR gene have been and hypokalemia. described. Familial hypocalciuric hypercalcemia (FHH) and neonatal severe hyperparathyroidism (NSHPT) result from inactivating mutations present in either the hetero- Salt-losing tubular disorders zygous or homozygous (or compound heterozygous) state, respectively [79, 80]. FHH patients normally This comprises a group of clinically and genetically present with mild-to-moderate hypercalcemia, accompa- distinct hereditary disorders characterized by the cardinal nied by few if any symptoms, and often do not require symptoms of renal salt wasting, hypokalemic metabolic 21 alkalosis, and elevated plasma renin and aldosterone detected in up to 50% of affected individuals, calcium levels with normal blood pressure. The associated distur- excretion is variable, but hypocalciuria is not an unusual bances in renal magnesium and calcium handling help to finding [94, 96]. distinguish clinical phenotypes and provide clues about A possible explanation for the greater variability in the the affected nephron segments. The elucidation of the clinical picture of cBS compared with HPS/aBS might be underlying genetic defects supported a new clinical the broad expression pattern of CLC-Kb, which appears to classification but also disclosed a marked variability in be particularly critical for electrolyte handling in the the phenotypic spectrum of the classic Bartter syndrome DCT. Some cBS patients present with combined hypo- (cBS). magnesemia and hypocalciuria, a phenotypic characteris- tic of the Gitelman syndrome also found in autosomal dominant hypomagnesemia, both caused by defects in the Hyperprostaglandin E syndrome DCT. Thus one can assume that in cBS disturbed or antenatal Bartter syndrome reabsorption of NaCl in distal nephron segments impairs the renal conservation of magnesium. Hyperprostaglandin E syndrome or antenatal Bartter Therefore, next to an adequate salt and fluid replace- syndrome (HPS/aBS) is caused by mutations either in ment, potassium supplementation, and inhibition of the furosemide-sensitive Na+/K+/2Cl co-transporter prostaglandin formation, magnesium substitution is nec- (NKCC2) or in the potassium channel ROMK, which essary in some patients. co-operate in the apical uptake of sodium chloride in the TAL [87, 88, 89]. The apical potassium channel ROMK mediates potassium recirculation into the tubular lumen, Hyperprostaglandin E syndrome which is essential for NaCl reabsorption driven by the or antenatal Bartter syndrome with sensorineural deafness NKCC2 transporter. The defect in NaCl reabsorption caused by mutations in either protein reduces the lumen- Recently, mutations in a new gene named Barttin have positive transepithelial voltage and therefore also impairs been identified in a subgroup of patients presenting with a passive paracellular reabsorption of calcium and magne- variant of HPS/aBS associated with sensorineural deaf- sium in this part of the nephron [90]. HPS/aBS is ness (SND) [99]. Barttin has been functionally character- characterized by massive hyposthenuric polyuria that ized as an activating b-subunit of renal chloride channels manifests in utero with the development of polyhydram- CLC-Ka and CLC-Kb [100, 101]. nios, which in turn results in premature birth in most The clinical phenotype of BSND patients was first patients. Postnatally, affected children rapidly develop described by Landau et al. [102]. Renal salt and water massive salt wasting and subsequent hypokalemic meta- losses can be even more severe than in HPS/aBS patients, bolic alkalosis. In addition, hypercalciuria and nephro- often requiring long-term parenteral fluid replacement. calcinosis occur in all affected individuals [91]. However, hypercalciuria and nephrocalcinosis are un- In contrast, disturbances in magnesium homeostasis common, but patients often show progression to renal are not a common finding in HPS/aBS. The lack of failure of unknown origin [103]. magnesium loss despite pronounced hypercalciuria might In contrast to cBS due to impaired ClC-Kb function, be explained by a compensatory mechanism that is more hypomagnesemia does not appear to be a common efficient for magnesium in the distal tubule. Possibly the finding. The reason for this discrepancy is unknown. increased prostaglandin E synthesis observed in HPS/aBS The impairment of renal function with decreased GFR contributes to an increased magnesium reabsorption in the might prevent the development of magnesium wasting. DCT, as demonstrated in a mouse DCT cell line [92]. Alternatively, high prostaglandin levels, as in HPS/aBS, could promote active magnesium reabsorption in the DCT. Classic Bartter syndrome cBS is caused by mutations in the CLCNKB gene coding Gitelman variant of Bartter syndrome for the chloride channel CLC-Kb [93, 94]. CLC-Kb is expressed in the distal tubule where it mediates chloride Another disorder affecting the transport of sodium efflux from the tubular epithelial cell to the interstitium. chloride in the DCT represents the Gitelman syndrome The clinical picture of cBS patients varies widely, with a [104]. Clinical features of Gitelman syndrome, in addition clinical spectrum ranging from a phenotype similar to to persistent hypokalemia and metabolic alkalosis, in- HPS/aBS, in rare cases, to a phenotype almost indistin- clude the combination of hypomagnesemia and hypocal- guishable from that of Gitelman syndrome [95, 96, 97]. ciuria, a finding pathognomonic for disturbed DCT However, the majority of patients develop hypokalemia, function [105]. Salt and water losses in these patients hypochloremia, and failure to thrive during the first are less pronounced than in HPS/aBS, the urinary 2 years of life, as described by Bartter et al. [98] in their concentrating ability is conserved to a greater extent. initial report. Prenatal onset and nephrocalcinosis, as seen Patients usually present during childhood or adolescence in the antenatal variant, is unusual. Hypomagnesemia is with symptoms of muscle weakness or tetanic episodes 22 that are related to profound hypomagnesemia. Additional Recent advances in molecular genetics led to the iden- symptoms are fatigue or joint pain due to chondrocalci- tification of a variety of genes and their encoded proteins nosis. Many “asymptomatic” patients have been reported involved in human magnesium homeostasis. This infor- and Gitelman syndrome is frequently diagnosed following mation allows the development of a classification into measurement of electrolytes for other reasons (e.g., different clinical subtypes and provides insight into the preoperative assessment). However, Cruz et al. [106] physiology of epithelial magnesium transport. demonstrated that Gitelman syndrome affects the quality Magnesium deficiency has been associated with of life to the same degree as hypertension or diabetes, for diverse chronic diseases such as coronary heart disease, example. None of their 50 patients was truly asymptom- hypertension, diabetes, and asthma. This together with atic. Salt craving, nocturia, and paresthesia were among clinical evidence of a high percentage of latent or the most frequent symptoms. subclinical hypomagnesemia in the general population The dissociation of renal magnesium and calcium have generated further studies on magnesium balance and handling together with the observed unresponsiveness to clinical trials to evaluate the benefits of magnesium thiazides suggested a primary defect in the DCT. Simon et replacement for prevention and treatment of these con- al. [107] identified mutations in the SLC12A3 gene coding ditions. Furthermore, the clinical manifestations, progres- for the NaCl co-transporter NCCT in patients with sion, and outcome of these chronic conditions might be Gitelman syndrome. The NCCT is expressed exclusively influenced by genetic variations in genes underlying the at the apical membrane of the DCT where it reabsorbs hereditary forms of hypomagnesemia. It is reasonable to approximately 7% of the filtered NaCl [108]. The assume that carriers of polymorphisms have a predispo- hypocalciuria is thought to result from a reduction in sition to develop one of these diseases. NaCl entry into DCT cells, leading to hyperpolarization, Studies in patients with hereditary disorders of mag- which in turn increases passive apical calcium entry and nesium homeostasis point to the existence of yet uniden- basolateral Na+/Ca2+ exchange [109]. tified genetic defects of both recessive and dominant The mechanism of impaired magnesium reabsorption patterns of inheritance. The identification of new genes in the DCT, as present in Gitelman syndrome and cBS, involved in magnesium transport will add to our current remains unknown. Reilly and Ellison [109] hypothesized knowledge of the physiology of magnesium metabolism that magnesium may backflux through a paracellular and to the treatment of disturbances associated with these pathway involving paracellin-1. 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