REVIEW J Am Soc Nephrol 14: 249–260, 2003

Recent Advances in Molecular Genetics of Hereditary Magnesium-Losing Disorders

MARTIN KONRAD and STEFANIE WEBER University Children’s Hospital, Marburg, Germany.

Abstract. Recent advances in molecular genetics in hereditary disorders associated with magnesium wasting and focuses on hypomagnesemia substantiated the role of a variety of genes the recent progress that has been made in molecular genetics. and their encoded proteins in human magnesium transport Besides isolated renal forms of hereditary hypomagnesemia, mechanisms. This knowledge on underlying genetic defects the following disorders will also be presented: familial hypo- helps to distinguish different clinical subtypes and gives first magnesemia with hypercalciuria and nephrocalcinosis, hypo- insight into molecular components involved in magnesium magnesemia with secondary hypocalcemia, Ca2ϩ/Mg2ϩ-sens- transport. By mutation analysis and functional protein studies, ing receptor–associated disorders, and disorders associated novel pathophysiologic aspects were elucidated. For some of with renal salt-wasting and hypokalemic metabolic alkalosis, these disorders, transgenic animal models were generated to comprising the Gitelman syndrome and the Bartter-like study genotype-phenotype relations and disease pathology. syndromes. This review will discuss genetic and clinical aspects of familial

Magnesium is the second most abundant intracellular cation literature. Depending on the genotype, the clinical course is and plays an important role for protein synthesis, nucleic acid sometimes mild or even asymptomatic. Therefore, the disease stability, neuromuscular excitability, and oxidative phosphor- prevalence might be underestimated for some of these syn- ylation. Under normal conditions, extracellular magnesium dromes. Several reports have demonstrated latent hypomag- concentration is maintained at nearly constant values. Hypo- nesemia in a high percentage in the general population (2), e.g., magnesemia results from decreased dietary intake, intestinal 14.5% in a recent German study (3). Furthermore, associations malabsorption, or renal loss. of hypomagnesemia with common chronic diseases have been During the last four decades, numerous reports concerning reported (4,5). Hypomagnesemia is frequently detected in pa- inherited magnesium-losing disorders have been published and tients with diabetes mellitus type II, arterial hypertension, their distinctive phenotypic features have been intensively dis- coronary heart disease, or asthma bronchiale, and diminished cussed. Whereas intestinal magnesium absorption is still magnesium is commonly related to an aggravation of these poorly understood, phenotypic characterization of clinically diseases. Hypomagnesemia predisposes to hyperglycemia in affected patients and experimental studies of appropriate ani- diabetes mellitus type II, and magnesium supplementation mal models have contributed to a growing knowledge of renal seems to retard the induction of insulin resistance (6). Reports magnesium transport mechanisms. The identification of the have linked hypomagnesemia to mortality in coronary heart most likely affected nephron segments in the kidney, the pre- disease, and administration of magnesium has a cardioprotec- sentation of different modes of inheritance and the observation tive effect in patients with acute myocardial infarction (7). The of additional characteristic symptoms promoted a classification BP in patients with arterial hypertension can be lowered by into different subtypes of inherited magnesium-losing magnesium supplementation (7). Magnesium treatment of sub- disorders. jects with chronic asthma is currently receiving attention, be- In general, primary magnesium-wasting disorders are rela- cause of a role for magnesium in relaxation of the arterial and tively rare. The prevalence of the more frequent entities, for bronchial smooth muscle cells (8). This obvious coherence has example Gitelman and Bartter syndrome, has been estimated to provoked further interest in understanding magnesium trans- be only approximately 1:50,000 (1). For most of the other port mechanisms and elucidating possible roles of inherited disease entities, relatively few cases have been reported in the predispositions to hypomagnesemia. The question arises whether genetic variations of genes underlying hereditary hy- pomagnesemia might contribute to the pathophysiology of Correspondence to Dr. Martin Konrad, University Children’s Hospital, Deut- schhausstr. 12, D-35037 Marburg, Germany. Phone: 49-6421-2862649; Fax: these widespread chronic diseases. 49-6421-2865724; E-mail: [email protected] Magnesium transport has been intensively studied in humans 1046-6673/1401-0249 and various animal models, leading to accepted concepts un- Journal of the American Society of Nephrology derlying the pathophysiology of the different forms of hypo- Copyright © 2003 by the American Society of Nephrology magnesemia (9). However, the electrophysiologic characteriza- DOI: 10.1097/01.ASN.0000049161.60740.CE tion of magnesium pathways has been complicated by 250 Journal of the American Society of Nephrology J Am Soc Nephrol 14: 249–260, 2003 unintentional simultaneous measurement of other cations so A that the molecular correlates mediating mammalian magne- lumen blood 2+ sium transport components remained undefined. The first mag- Mg + nesium transporter genes have been cloned in bacteria and 3 Na + 2 K plants (10,11). Meanwhile, a human homologue of the yeast Mg2+ MRS2 gene, characterized as a mitochondrial magnesium HSH Mg2+ transporter has been identified (12). Its relevance for magne- Na+ sium homeostasis outside mitochondria remains to be deter- mined, but it may be a candidate for genetic disturbances in Mg2+ cellular magnesium homeostasis. A different approach to study components of magnesium transport arises from genetic analysis of families affected with B magnesium-wasting diseases. Linkage disequilibrium studies bined on affected kindreds have enabled the chromosomal localiza- com tion of several genes involved in hereditary hypomagnesemia; in the last decade, a number of genes have been identified by positional cloning. These genes have provided first insight into paracellular mammalian magnesium transport molecules. A detailed anal- Net Mg absorption ysis of hereditary magnesium-losing disorders was given by transcellular Cole and Quamme (13) in this Journal only three years ago. Remarkable progress has been made in this field since that time. In this review, we would like to focus on the most recent Mg intake research as it adds considerably to our understanding of renal magnesium conservation. Figure 1. (A) Schematic model of magnesium absorption in small intestine. Passive absorption occurs via the paracellular pathway and Physiology of Magnesium Homeostasis active magnesium transport by apical entry through a putative mag- nesium channel and basolateral exit through a putative Naϩ/Mg2ϩ In healthy humans, serum magnesium concentration is main- exchanger. (B) Increase of magnesium absorption with increasing tained in a narrow range of 0.7 to 1.1 mmol/L. This extracel- magnesium intake. The curvilinear absorption of magnesium in hu- lular magnesium is in continuous exchange with magnesium mans (solid line) results from the saturable transcellular transport stores in bone and muscle tissue. A balanced Western diet (dotted line) and a paracellular passive transport (dashed line) linearly offers approximately 12 mmol (400 mg) magnesium per day; 6 rising with elevated luminal magnesium concentrations. mmol is absorbed, and 2 mmol is secreted in the intestine with a net gain of 4 mmol that is excreted in the urine. Diminished magnesium intake is balanced by enhanced magnesium absorp- to twenty percent of the ultrafiltrable magnesium is reabsorbed tion in the intestine and reduced renal excretion. These trans- in the proximal tubules of the adult kidney. In the premature port processes are regulated by metabolic and hormonal influ- kidney of the newborn, proximal tubules can absorb up to 70% ences (9). A magnesium deficit is frequently associated with (16). In the mature kidney, the majority of magnesium is other electrolyte disturbances, including hypokalemia, hy- reabsorbed in the loop of Henle, especially in the cortical thick pophosphatemia, and hypocalcemia, adding to the presenting ascending limb (cTAL). Here, 70% of the ultrafiltrable mag- symptoms. Prolonged insufficiency of magnesium supply may nesium is normally reabsorbed. With the help of animal mod- result in the manifestation of anorexia, nausea, vomiting, and els, it was demonstrated that this transport is passive and weakness within weeks, and in paraesthesia, muscle weakness, paracellular, driven by the lumen-positive electrochemical gra- cerebral cramps, and cardiac manifestations within months. dient characteristic of this nephron segment (Figure 3A). On The principal site of dietary magnesium absorption is the the contrary, magnesium reabsorption in the distal convoluted small bowel with smaller amounts absorbed in the colon. tubule (DCT) is of transcellular and active nature (Figure 3B). Intestinal magnesium absorption occurs via (1) a saturable The absorption rate in the DCT (5 to 10%) is much lower than transcellular active pathway and (2) a nonsaturable paracellular in the cTAL, but it defines the final urinary excretion, as there passive transport, the latter dependent on the electrochemical is no significant magnesium reabsorption in the collecting duct. gradient (14,15). Saturation of the transcellular absorption at Three to five percent of the filtered magnesium is finally high luminal magnesium concentrations is best explained by excreted in the urine. Distal tubule magnesium transport has the limited transport capacity of active transport (Figure 1A). been recently reviewed in detail by Dai et al. (17). At low intraluminal magnesium concentrations, magnesium is In the following, we will discuss the clinical and genetic transported mainly by the active transcellular pathway and with aspects of hereditary magnesium-losing disorders. To facilitate rising concentrations by the paracellular pathway (Figure 1B), this task, we will deal with the following groups of disorders: yielding a curvilinear function for total absorption. (1) primary isolated magnesium loss; (2) familial hypomag- In the kidney, magnesium transport differs in quantity and nesemia with hypercalciuria and nephrocalcinosis (FHHNC); kinetics depending on the nephron segment (Figure 2). Fifteen (3) hypomagnesemia with secondary hypocalcemia (HSH); (4) J Am Soc Nephrol 14: 249–260, 2003 Hereditary Magnesium-Losing Disorders 251

distal convoluted tubule A 5-10 % thick ascending limb urine blood 0.25 mM Mg2+ 0.50 mM Mg2+ 0.75 mM Mg2+ 2+ proximal tubule Mg 2+ Ca 15-20 % + aBS/HPS ROMK 3 Na ATPase 2 K+ K+ aBS/HPS 65-75 % ADH + CaSR K - NKCC2 cBS loop of 2 Cl+ Na Cl- ClC-Kb barttin BSND collecting duct Henle 2+ Mg 2+ Ca paracellin-1 FHHNC

+ 8 mV

B distal convoluted tubule

3-5 % urine blood

2+ 2+ 2+ Figure 2. Magnesium reabsorption along the nephron. 0.25 mM Mg 0.50 mM Mg 0.75 mM Mg

2+ 2+ Mg ϩ ϩ Mg Na+ 2 2 TR HSH P Ca /Mg -sensing receptor (CASR)–associated disorders; M6 3 Na+ and (5) disorders associated with renal salt-wasting and hy- ATPase 2 K+ pokalemic metabolic alkalosis, comprising the Gitelman syn- γ-subunit IDH Na+ NCCT CaSR ADH drome and the Bartter-like syndromes. An overview on the GS Cl- genetic aspects of these disorders is given in Table 1, an cBS + ENaC Cl- ClC-Kb overview on clinical aspects in Table 2. Na barttin BSND Isolated Magnesium Loss Isolated Dominant Hypomagnesemia (IDH). IDH fol- lows an autosomal dominant mode of inheritance. It was first -10 mV described by Geven et al. (18) in 1987 in two Dutch families. The index cases of these two families presented with general- ized seizures that led to the detection of primary hypomag- Figure 3. (A) Magnesium reabsorption in the thick ascending limb of nesemia. Interestingly, many other affected members of these the loop of Henle. Driving force for the paracellular reabsorption of families remained asymptomatic. Typically, hypomagnesemia magnesium and calcium is the lumen-positive electrochemical gradi- in IDH is associated with hypocalciuria that is phenotypically ent generated by the transcellular reabsorption of NaCl. See text for reminiscent of patients presenting with Gitelman syndrome details and abbreviations. (B) Magnesium reabsorption in the distal convoluted tubule. In this segment, magnesium is reabsorbed by an (GS). But GS patients also have hypokalemic alkalosis as a active transcellular pathway involving an apical entry step probably consequence of renal salt wasting, whereas IDH subjects have via a magnesium permeable ion channel and a basolateral exchange no other biochemical abnormalities. Newborns of affected mechanism, presumably a Naϩ/Mg2ϩ exchanger. The molecular iden- mothers may have severe hypomagnesemia at birth, even tity of this exchanger is still unknown. See text for details and though maternal hypomagnesemia is less pronounced and de- abbreviations. void of clinical symptoms (19). Thus, immediate control in the neonatal period with appropriate magnesium supplementation is necessary to avoid the potential risks related to sorption indicated that the primary molecular defect resides in hypomagnesemia. the kidney (18). As promising candidate genes for IDH were The results of retention studies after oral administration of not available, linkage analysis was performed in the two large 28Mg2ϩ and the effects of magnesium infusions on renal reab- families, and a gene locus was mapped to chromosome 11q23 252 Journal of the American Society of Nephrology J Am Soc Nephrol 14: 249–260, 2003

Table 1. Inherited disorders or magnesium handling

OMIM Disorder # Inheritance Gene Locus Gene Protein

Isolated dominant hypomagnesemia 154020 AD 11q23 FXYD2 ␥-subunit of the Naϩ-Kϩ-ATPase with hypocalciuria 154020 AD ? ? ? Isolated recessive hypomagnesemia 248250 AR ? ? ? with normocalciuria Familial hypomagnesemia with 603959 AR 3q27-29 CLDN16 paracellin-1, tight junction protein hypercalciuria/nephrocalcinosis Hypomagnesemia with secondary 602014 AR 9q22 TRPM6 TRPM6, putative ion channel hypocalcemia Autosomal dominant 601189 AD 3q13.3-21 CASR CASR, Ca2ϩ/Mg2ϩ sensing receptor hypoparathyreoidism Antenatal Bartter syndrome/ 241200 AR 15q15-21 SLC12A1 NKCC2, NaϩKϩ2ClϪ cotransporter hyperprostaglandin E syndrome 600359 AR 11q24 KCNJ1 ROMK, renal potassium channel Classic Bartter syndrome 602023 AR 1p36 CLCNKB CLC-Kb, distal tubule chloride channel Antenatal Bartter syndrome with 602522 AR 1p31 BSND Barttin, chloride channel ␤ subunit sensorineural deafness Gitelman syndrome 263800 AR 16q SLC12A3 NCCT, NaϩClϪ cotransporter

Table 2. Clinical and biochemical characteristics

Age at Serum Serum Serum Blood Urine Urine Renal Disorder Onset Mg2ϩ Ca2ϩ Kϩ pH Mg2ϩ Ca2ϩ Nephrocalcinosis Stones

Isolated dominant hypomagnesemia Childhood 2 NN N12 No No with hypocalciuria Isolated recessive hypomagnesemia Childhood 2 NN N1 NNoNo with normocalciuria Familial hypomagnesemia with Childhood 2 NNNor21111 Yes Yes hypercalciuria/nephrocalcinosis Hypomagnesemia with secondary Infancy 22 2 NN1 NNoNo hypocalcemia Autosomal dominant Infancy 22 NNor211–11 Yesa Yesa hypoparathyreoidism Antenatal Bartter syndrome/ Neonatal N N 2–22 1 N 11 Yes ? hyperprostaglandin E syndrome Classic Bartter syndrome Infancy N or 2 N 22 1 Nto1 Variable Rare No Antenatal Bartter syndrome with Neonatal N N 22 1 NNto1 No No sensorineural deafness Gitelman syndrome Variable 2 N 22 1 1 2 No No

a Frequent complication under therapy with calcium and vitamin D.

(20). Extended haplotype analysis strongly suggested a com- that two individuals with large heterozygous 11q23.3-ter mon ancestor in the two families. Within the critical interval, genomic deletions, which comprise the FXYD2 gene locus, did Meij et al. identified the gene FXYD2 coding for the ␥-subunit not present with hypomagnesemia (22). This points to a dom- of the basolateral Naϩ-Kϩ-ATPase. This subunit is localized to inant-negative effect of the Gly41Arg mutation rather than to the DCT segment of the nephron, the site of active magnesium simple haploinsufficiency. It was demonstrated by expression transport (21). Sequence analysis showed a heterozygous studies in mammalian renal tubule cells (COS-1) that this Gly41Arg mutation in all affected individuals, replacing a amino acid exchange results in a misrouting of the ␥-subunit. highly conserved amino acid residue within the single trans- Additional studies showed that the mutant ␥-subunit also af- membrane domain of the ␥-subunit (22). Meij et al. reported fects the trafficking of the ␣-subunit in Sf9 cells, indicating J Am Soc Nephrol 14: 249–260, 2003 Hereditary Magnesium-Losing Disorders 253 that the complete ATPase complex might be affected (22). tend to have increased parathyroid hormone (PTH) levels pre- However, subsequent studies in mammalian cells demonstrated ceding the impairment of GFR. A substantial percentage of that the Gly41Arg mutation only disturbs trafficking of the patients show incomplete distal renal tubular acidosis, hypoci- ␥-subunit gamma but not ␣␤-subunits to the cell surface (23). traturia, and hyperuricemia. Extrarenal manifestations, such as Thus, the impaired trafficking of the mutant ␥-subunit seems to ocular involvement (including severe myopia, nystagmus, and abrogate its functional effects on the ␣␤-subunits rather than to chorioretinitis), have been repeatedly reported (30,31). impair trafficking of the multimeric Naϩ-Kϩ-ATPase. More In addition to continuous magnesium supplementation, ther- recently, Meij et al. demonstrated that the Gly 41Arg mutant apy aims at the reduction of calcium excretion by using thia- FXYD2 is retarded in the Golgi complex, pointing to a dis- zides to prevent the progression of nephrocalcinosis and stone turbed posttranslational processing (24). Why a mutation in the formation, because the degree of renal calcification has been ␥-subunit would result in selective renal magnesium wasting is correlated with progression of chronic renal failure (CRF) (31). unknown. The normal ␥-subunit decreases sodium affinity and However, it seems that current therapeutic approaches do not increases ATP affinity to the Naϩ-Kϩ-ATPase enzyme that significantly influence the progression of renal failure. In a should increase intracellular sodium concentration and lead to large series of 33 patients, one third had already developed more energy efficient sodium transport (23). Accordingly, di- end-stage renal disease early during adolescence (33). Hypo- minished basolateral Naϩ/Mg2ϩ exchange with malfunction- magnesemia may completely disappear with the decline of ing ␥-subunit cannot be invoked for a decrease in magnesium GFR due to the reduction of the filtered magnesium load. transport (17). Meij et al. have suggested that diminished Renal transplantation corrects the abnormal magnesium and intracellular potassium may depolarize the apical membrane, calcium handling clearly demonstrating that the primary defect resulting in a decrease in magnesium uptake (24). Further resides in the kidney. studies are needed to clarify this issue. It is also unknown why Based on clinical observation and clearance studies, Rodri- there is an increase in distal calcium reabsorption and hypocal- guez-Soriano et al. postulated that the primary defect in FH- ciuria in these patients. HNC was related to impaired magnesium and calcium reab- Interestingly, IDH seems to be genetically heterogenous, sorption in the loop of Henle. Using a positional cloning because FXYD2 has been excluded in a large Californian approach, Simon et al. identified ten different mutations in a family with a phenotype very similar to that seen in the Dutch novel gene (CLDN16, formerly PCLN-1) on chromosome families (25). This may suggest a different genetic disease 3q27–29 in the affected individuals of ten FHHNC families converging on the same transport function. (34). CLDN16 mutations as the underlying cause of FHHNC Isolated Recessive Hypomagnesemia (IRH). Isolated au- have subsequently been confirmed in additional patients tosomal recessive primary hypomagnesemia was originally (33,35). In addition, a common mutation due to a founder described in a consanguinous family (26). The affected indi- effect was characterized in FHHNC patients originating from viduals presented with symptoms of hypomagnesemia early Germany and Eastern European countries (33). As this muta- during infancy. Hypomagnesemia due to increased urinary tion is present in approximately 50% of the mutant alleles, magnesium excretion appears to be the only abnormal bio- molecular diagnosis is greatly facilitated in patients originating chemical finding. IRH is distinguished from the autosomal from these countries. dominant form by the lack of hypocalciuria. Linkage analyses CLDN16 codes for paracellin-1 (claudin-16), a member of have excluded thus far all established gene loci involved in the claudin family of tight junction proteins. The claudin hereditary hypomagnesemia indicating a different genetic dis- family comprises a set of structurally related proteins involved ease (24). in the formation of tight junction strands in various tissues (36). RT-PCR studies on microdissected nephron segments and Familial Hypomagnesemia with Hypercalciuria and immunohistochemical studies demonstrated that CLDN16 is Nephrocalcinosis (FHHNC) strongly expressed both in the medullary and cortical segments In 1972, Michelis et al. (27) first described a hypomag- of the loop of Henle in human and rodent kidneys. It was nesemic disorder characterized by excessive renal magnesium shown by immunohistochemistry that paracellin-1 colocalized and calcium wasting. In addition, affected individuals had with occludin at tight junctions (34). These data indicate that bilateral nephrocalcinosis and developed progressive renal fail- paracellin-1 is an important component of tight junction for- ure. Subsequent clinical descriptions allowed the characteriza- mation in the thick ascending limb. Impaired expression of tion of the whole clinical spectrum of this disease and enabled paracellin-1 is associated with severe renal calcium and mag- this entity to be discerned from other magnesium-losing tubu- nesium loss without loss of other electrolytes; it is therefore lar disorders (28–32). FHHNC patients usually present during speculated that paracellin-1 contributes to the formation of early childhood with recurrent urinary tract infections, poly- calcium and magnesium selective paracellular pathways. This uria/polydipsia, isosthenuria, and renal stones. Some children hypothesis is supported by the recent observation that two come to the attention of physicians with failure to thrive, other claudins (CLDN4 and CLND15) influence ion selectivity vomiting, abdominal pain, tetanic episodes, or generalized by creating charge-selective channels through the tight-junc- seizures. Besides hypomagnesemia, biochemical abnormalities tion barrier (37,38). Hydrophobicity analysis predicts that para- include hypermagnesiuria and hypercalciuria, and impaired cellin-1 is a four-transmembrane domain protein with intracel- GFR is often detected at the time of diagnosis. Patients also lular N- and C-termini. The C-terminus has a C-terminal PDZ 254 Journal of the American Society of Nephrology J Am Soc Nephrol 14: 249–260, 2003 domain, which is possibly involved in protein-protein interac- of mutant paracellin-1 in the apical membrane, whereas wild- tions. The first extracellular loop is highly negatively charged, type paracellin-1 was correctly targeted to the basolateral which distinguishes paracellin-1 from other members of the membrane (44). It still remains to be determined why this type claudin family. It is proposed that the pathway formed by of misrouting is associated with transient isolated hypercalci- paracellin-1 involves this first extracellular loop and that its uria without increased magnesium excretion. negative charge contributes to the observed cation sensitivity. The identification of the molecular components involved in The longest open reading frame of the human cDNA encodes transport across renal epithelial cells via the paracellular path- a protein of 305 amino acids with a cytoplasmatic N-terminus way has already considerably increased our current knowledge of 73 amino acids. This structure is in contrast to all other of these processes. It might be expected that the analysis of claudins that share a very short N-terminus of only six or seven other components, such as occludins or other claudins ex- amino acids. Interestingly, there is a second inframe start pressed in tubular epithelia, will further contribute to the un- codon within a suitable Kozak consensus sequence at position derstanding of the physiologic functions but also of disease Met71, which is analogous to all other claudins. Sequence states related to impaired paracellular reabsorption. comparison of the human cDNA with other species and the results of mutation analysis suggest that the second translation Hypomagnesemia with Secondary Hypocalciuria (HSH) initiation start site is used in vivo (33,39,40). HSH or primary intestinal hypomagnesemia is an autosomal A large deletion of CLDN16 has also been shown to underlie recessive disorder that is characterized by very low serum the development of a chronic interstitial nephritis in Japanese magnesium and low calcium levels. It was first described by cattle that rapidly develop CRF shortly after birth (39,41). Paunier et al. in 1968 (45). Patients usually present within the Interestingly, the typical electrolyte abnormalities seen in FH- first 3 mo of life with neurologic symptoms of hypomag- HNC were not detected in affected animals. They typically nesemic hypocalcemia, including seizures, tetany, and muscle show hypocalcemia but no hypomagnesemia. This is probably spasms. Untreated, the disorder may result in permanent neu- due to the advanced CRF present at the time of examination. In rologic damage or may be fatal. Hypocalcemia is secondary to this context, it is important to note that the majority of muta- parathyroid failure and peripheral parathyroid hormone resis- tions reported so far in FHHNC are simple missense mutations tance as a result of sustained magnesium deficiency (46). affecting the transmembrane domains and the extracellular Usually, the hypocalcemia is resistant to calcium or vitamin D loops. Thus, the disease phenotype resulting from a large therapy. Normocalcemia and relief of clinical symptoms can be deletion in CLDN16 might be expected to be more pronounced attained by administration of high oral doses of magnesium, up with early onset renal failure, whereas point mutations might to 20 times the normal intake (47). As large oral amounts of primarily affect calcium and magnesium reabsorption with magnesium may induce severe diarrhea and noncompliance in progressive renal failure as a secondary event. However, some patients, parenteral magnesium administration has some- Cldn16 knockout mice did not show renal failure during the times to be considered. Alternatively, continuous nocturnal first months of life (42). Referring to the ocular abnormalities nasogastric magnesium infusions have been proven to effi- observed in some FHHNC patients, it is interesting to note, that ciently reduce gastrointestinal side effects (48). The patho- Meij et al. found CLDN16 expression in bovine cornea and physiology of HSH was largely unknown, but physiologic retinal pigment epithelia (43). Examination of the eyes of studies pointed to a primary defect in saturable intestinal mag- affected Japanese cattle and of the Cldn16 knockout mice nesium transport (49). In some patients, an additional renal might give an answer to the question whether myopia, nystag- leak of magnesium, probably caused by altered magnesium mus, and chorioretinitis observed in humans are directly linked entry into epithelial cells of the DCT was suspected (13,50). to CLDN16 mutations. Using a DNA pooling strategy, a gene locus (HOMG1) for There is evidence from family analyses that carriers of HSH was mapped to chromosome 9q22 in a large inbred heterozygous CLDN16 mutations may also present with clini- Bedouin kindred (51). It was further refined to a critical inter- cal symptoms. Two independent studies describe a high inci- val of less than 1 centimorgan (52). Recently, Schlingmann et dence of hypercalciuria, nephrolithiasis, and nephrocalcinosis al. (53) and Walder et al. (54) identified mutations in a novel in first-degree relatives of FHHNC patients (31,33). In a sub- gene, TRPM6, in several unrelated HSH families as the under- sequent study, Blanchard et al. (35) also found a tendency lying cause of HSH. TRPM6 encodes a new putative ion toward hypercalciuria or mild hypomagnesemia in family channel belonging to the transient receptor potential (TRP) members with heterozygous CLDN16 mutations. Thus, one channel family. TRP ion channels are characterized by six might speculate that CLDN16 mutations are involved in idio- transmembrane segments, a highly conserved putative pore- pathic hypercalciuric stone formation. Very recently, a ho- forming region, and a Pro-Pro-Pro motif following the last mozygous CLDN16 mutation (Thr303Gly) affecting the C- transmembrane segment (55). Within the TRP family, TRPM6 terminal PDZ domain has been identified in two families with belongs to the TRPM subclass, whose members share a long isolated hypercalciuria and nephrocalcinosis without distur- conserved specific N-terminal domain of unknown function. It bances in renal magnesium handling (44). Interestingly, hyper- shows highest homology with TRPM7, which has recently calciuria disappeared during follow-up and reached normal been identified as a magnesium and calcium permeable ion values beyond puberty. Transient transfection of MDCK cells channel regulated by Mg2ϩATP (56). In addition to the ion with the Thr303Gly mutant CLDN16 revealed the localization channel domain, TRPM6 and TRPM7 have a C-terminal re- J Am Soc Nephrol 14: 249–260, 2003 Hereditary Magnesium-Losing Disorders 255 gion with sequence similarity to serine-threonine protein ki- seizures are typical symptoms but ADH is also sometimes nases of the atypical ␣-kinase family, therefore representing a asymptomatic. new class of bifunctional proteins (57). Whether this kinase Activating mutations shift the setpoint of the receptor to a domain is necessary for the channel function is still unclear. By level of enhanced sensitivity by increasing the apparent affinity ϩ ϩ RT-PCR and in situ hybridization, TRPM6 expression has of the mutant receptor for extracellular Ca2 and Mg2 . This been demonstrated along the entire small and large intestinal results in diminished PTH secretion and decreased reabsorp- tract and in distinct distal tubule segments representing DCT tion of divalent cations in the cTAL and DCT, which leads to cells (53). loss of urinary calcium and magnesium (68). The inhibition of The observation that HSH patients may achieve normal calcium and magnesium reabsorption in the loop of Henle is serum magnesium levels by high oral magnesium intake sup- thought to be due to a selective reduction of the paracellular ports the theory of two independent pathways for intestinal permeability and/or to the reduction of the lumen-positive magnesium absorption. TRPM6 probably represents a molec- transepithelial voltage by inhibiting the transcellular NaCl re- ular component of the active transcellular pathway so that high absorption (68). Hormone-stimulated magnesium reabsorption oral doses of magnesium are sufficient to compensate the is also inhibited in the DCT that probably contributes to a defect of TRPM6-mediated transport by increasing magnesium greater extent to renal magnesium loss than does its action in absorption through the paracellular pathway. the loop (17). The detection of TRPM6 expression in the DCT substanti- After vitamin D or calcium therapy, ADH patients dramat- ated the hypothesis advanced by Cole and Quamme of an ically increase urinary calcium excretion even though serum additional role of impaired renal magnesium reabsorption in calcium levels may be still in the low-normal range (66). This HSH pathophysiology (13). This was confirmed by intrave- may lead to polyuria, nephrocalcinosis, nephrolithiasis, and nous magnesium loading studies in HSH patients, who clearly even irreversible reduction of renal function. Therefore, the showed a considerable renal magnesium leak, even under treatment of hypocalcemia in ADH with vitamin D and cal- hypomagnesemic conditions (54). cium supplementation should be restricted to symptomatic patients.

2ϩ 2ϩ Very recently, three additional patients with ADH due to Ca /Mg -Sensing Receptor–Associated Disorders activating CASR mutations have been described (69,70). Dur- An important regulator of the magnesium homeostasis is the ing follow-up, the clinical course in these patients was com- 2ϩ 2ϩ Ca /Mg -sensing receptor (CASR), which was identified in plicated by a Bartter-like syndrome, i.e., the patients developed 1993 by Brown, Hebert, and colleagues (58). The CASR is a renal salt and water loss associated with hypokalemic meta- member of the G protein-coupled receptor family, which forms bolic alkalosis. In addition, all three patients had hypomag- dimers through interactions of the cysteine residues of the nesemia (69,70). Heterologous expression of the mutant CASR extracellular domain (59). CASR is located in the apical mem- revealed that the underlying mutations (L125P, C131W, brane of PTH-secreting cells of the parathyroid glands and A843E) figure among the most potent gain-of-function CASR basolaterally in the nephron segments, i.e., TAL and DCT mutations reported so far. These mutations appear to be fully involved in renal calcium and magnesium reabsorption (60,61). activated under normal serum calcium concentrations and are It also has been detected in tissues not primarly involved in able to induce a significant loss of NaCl by inhibiting the calcium homeostasis, among others in the apical membrane of reabsorption of NaCl in the TAL and thus patients present with the inner medullary collecting duct, where it probably contrib- a Bartter-like syndrome. utes to the inhibitory effect of hypercalcemia on vasopressin- stimulated water reabsorption. Salt-Losing Tubular Disorders An important function of the CASR is to sense ionized Several disorders have been described that have in common serum calcium and magnesium concentrations and to regulate the cardinal symptoms of renal salt wasting, hypokalemic these levels by controlling PTH secretion. In the past decade, metabolic alkalosis, and elevated plasma renin and aldosterone both activating and inactivating mutations of the CASR have levels but normal BP. On the basis of clinical presentation, been described, resulting in different clinical entities. Familial additional symptoms, and the biochemical profile, especially hypocalciuric hypercalcemia (FHH) and neonatal severe hy- with respect to calcium and magnesium handling, at least four perparathyroidism result from inactivation mutations present in different disease entities can be discerned (71,72). Over the last a either heterozygous or homozygous (or compound heterozy- few years, this clinical differentiation has been substantiated by gous) state (62,63). In addition to hypercalcemia, FHH patients the characterization of the underlying molecular defects in five also have a tendency to elevated serum magnesium levels (64). different genes in patients suffering from these diseases. Activating mutations of the CASR gene were first described Antenatal Bartter Syndrome or Hyperprostaglandin E in families affected with autosomal dominant hypocalcemia Syndrome (aBS/HPS). Antenatal Bartter Syndrome or hy- (ADH) (65,66). Affected individuals present with hypocalce- perprostaglandin E syndrome (aBS/HPS) is characterized by mia, hypercalciuria, and polyuria, and about 50% of these massive polyuria that manifests in utero with the development patients have hypomagnesemia (66,67). Hypocalcemia in ADH of polyhydramnios that results in premature birth in almost all is generally mild to moderate; severe hypocalcemia has rarely cases. Postnatally affected infants rapidly develop salt wasting been reported in this syndrome. Carpopedal spasms and/or and hypokalemic metabolic alkalosis. In addition, hypercalci- 256 Journal of the American Society of Nephrology J Am Soc Nephrol 14: 249–260, 2003 uria and pronounced medullary nephrocalcinosis occur in all supplementation. Hypercalciuria and nephrocalcinosis are un- affected individuals (1). Magnesium wasting is not a common common, and the response to indomethacin therapy is poor. In finding in aBS/HPS (73). As these patients fail to respond to addition, many patients develop progressive renal failure of furosemide, a defective NaCl reabsorption in the thick ascend- unknown etiology (83). By homozygosity mapping in a large ing limb was suspected (74). By combining a candidate gene Bedouin kindred, Brennan et al. mapped this disease to chro- approach with linkage analysis, Simon et al. demonstrated that mosome 1p31 (84). Within the critical interval, a new gene ϩ ϩ Ϫ aBS/HPS is either due to mutations in the Na K 2Cl co- (BSND) coding for a protein named Barttin was identified (85). transporter (NKCC2) or to the apical potassium channel Barttin has no similarity with any known protein and does not ROMK, which is necessary for proper NKCC2 function in the share any similarities with any ion channel. Consistent with the TAL (75,76). characteristic phenotypic presentation, Barttin is expressed Mutations in NKCC2 or ROMK are thought to reduce salt along the kidney tubule and in the stria vascularis (marginal reabsorption in the TAL and thus should impair not only cells) of the inner ear. Functional expression studies revealed, transepithelial calcium but also magnesium reabsorption. How- that Barttin is a beta-subunit of the renal chloride channels ever, renal magnesium wasting and overt hypomagnesemia do (ClC-Ka and -Kb in humans, ClC-K1 and -K2 in mice) (86,87). not reliably segregate with the aBS/HPS phenotype. This is Interestingly, in contrast to cBS due to CLCNKB mutations, consistent with the observation of normal magnesium ho- hypomagnesemia has not been reported in BSND individuals. meostasis in NKCC2 knockout mice (77). Also chronic furo- The reason for this is unknown, but the decreased GFR in semide treatment is not generally associated with hypomag- BSND patients might, at least in part, prevent the development nesemia. Possibly, the loop of Henle or more distal parts of the of magnesium wasting. Alternatively, as in aBS/HPS, prosta- nephron may adapt and compensate more efficiently for mag- glandin formation is also highly stimulated in BSND patients nesium relative to calcium explaining the presence of hyper- so that increases in magnesium reabsorption in the DCT might calciuria without overt magnesium loss in this syndrome. In- mitigate magnesium losses. creased renal prostaglandin synthesis, which is generally Gitelman Syndrome (GS). GS was first described by observed in aBS/HPS, may contribute to increased magnesium Gitelman et al. in 1966 (88). Salt and water losses in patients reabsorption in the DCT, as has been demonstrated for PGE in 2 with GS are less pronounced than in aBS/HPS or cBS because a mouse DCT cell line (78). Alternatively, chronic volume their urinary-concentrating ability is preserved to a greater depletion could stimulate passive magnesium reabsorption in extend. They usually present during childhood or adolescence, the proximal tubule and TAL. In TAL, this adaptation might even if the biochemical abnormalities may be detected earlier. involve paracellin-1 so that more magnesium is absorbed at a Cardinal symptoms include muscle weakness or tetanic epi- lowered transepithelial potential difference. sodes that are related to profound hypomagnesemia. In addi- Classic Bartter Syndrome (cBS). Classic Bartter Syn- tion, GS patients virtually always have hypocalciuria. The drome (cBS) usually presents during infancy or early child- presence of both hypomagnesemia and hypocalciuria is highly hood, and the phenotype of these patients best fits with the original description given by Bartter et al. (79), i.e., without predictive for the diagnosis of GS (89) even if in rare cases this prenatal onset and without nephrocalcinosis seen in the ante- combination is also detected in cBS patients (90). GS is often natal variant. cBS is caused by mutations in CLCNKB encod- described as the mild variant of the salt-losing tubular disor- ing the basolaterally located renal chloride channel ClC-Kb, ders, and many asymptomatic patients have been reported. which mediates chloride efflux from the tubular epithelial cell However, this notion is probably not true. Recently, Cruz et al. to the interstitium along the TAL and DCT (80,81). The broad demonstrated that GS affects quality-of-life to the same degree expression pattern of ClC-Kb might explain the wide clinical as hypertension or diabetes for example (91). None of the 50 spectrum of cBS phenotypes ranging from that similar to GS patients of this study were truly asymptomatic. Salt crav- aBS/HPS in rare cases to those almost identical to Gitelman ing, nocturia, and paresthesia were among the most frequent syndrome (73) (see below). An alternative explanation could symptoms. The authors did not find any correlation between be an inter-individual difference in the ability to compensate the symptoms reported and either potassium or magnesium for a defect in basolateral chloride transport, e.g., by activation levels. of alternative chloride efflux pathways such as the KϩCl- The dissociation of renal magnesium and calcium handling cotransporter (82). Hypomagnesemia is detected in up to 50% together with the observed unresponsiveness to thiazides of patients with mutations in CLCNKB (81) and calcium ex- pointed to a primary defect in the DCT. Again, using a posi- cretion is variable, but hypocalciuria is not an unusual finding tional-candidate gene approach, Simon et al. identified putative (73). loss of function mutations in the gene coding for the NaCl Antenatal Bartter Syndrome with Sensorineural Deaf- cotransporter (NCCT) of the DCT (92). GS appears to be a ness (BSND). Landau et al. (72) first described a subtype of genetically homogenous disorder, and more than 100 different antenatal BS that is characterized by a similar clinical pheno- mutations have been reported to date. Results from heterolo- type but which is associated in all cases with sensorineural gous expression of naturally occurring GS mutations in Xeno- deafness. The renal phenotypic presentation is even more se- pus oocytes revealed that the majority of the mutations are vere than in aBS/HPS individuals with massive salt and fluid retained in the endoplasmic reticulum, resulting in impaired loss from birth, that often needs long-term parenteral fluid trafficking to the plasma membrane (93). Other mutant pro- J Am Soc Nephrol 14: 249–260, 2003 Hereditary Magnesium-Losing Disorders 257 teins were shown to be inserted in the plasma membrane, but • TRPM6, a novel channel with magnesium-conducting sodium transport was considerably reduced (94). properties Schultheis et al. created a transgenic mouse model that • The CASR, a member of the G protein-coupled receptor completely lacked the NCCT by disrupting the SLC12A3 gene family (95). These mice display all the cardinal features of GS, espe- • NCCT, a sodium chloride cotransporter cially hypomagnesemia and hypocalciuria, but hypokalemia • The chloride channel ClC-Kb was not observed. Hypocalciuria in GS is explained by the The characterization of corresponding transgenic animal reduced entry of NaCl into the DCT cell, leading to hyperpo- models has proven to be extremely helpful to study the phys- larization. Cellular hyperpolarization increases calcium reab- iology of the newly identified proteins. Further investigations sorption mediated by an apical entry via an epithelial calcium ϩ ϩ on these animal models will hopefully give more insight into channel and basolateral extrusion through the Na /Ca2 ex- regulatory mechanisms, new therapeutic approaches, and the changer that could explain the hypocalciuria. The reason for consequences of long-term follow-up. Advances in molecular the often-pronounced hypomagnesemia in GS is still unknown. modeling will possibly promote the development of new drugs Chronic thiazide therapy, a model for GS, does not uniformly acting specifically on the novel components of magnesium lead to magnesium wasting and hypomagnesemia. The NCCT metabolism, for example inhibitors of the CASR, activating knockout model does not favor a common hypothesis that agents for TRPM6, NKCC2, and NCCT, or even chaperone- hypomagnesemia is secondary to the hypokalemia observed in like drugs, directing the trafficking of misrouted proteins. GS. Reilly and Ellison speculated that magnesium may back- The association of hypomagnesemia with diabetes mellitus flux through a paracellular pathway in the DCT (96,97). This type II, arterial hypertension, coronary heart disease, and hypothesis is based on the assumption that in GS the DCT cells asthma bronchiale challenges further research on genetic vari- are converted from mainly electroneutral cells to cells that ants in genes underlying primary hypomagnesemia. reabsorb NaCl in an electrogenic manner via the epithelial Genetic variations of these genes might influence disease sodium channel ENaC, and the action of aldosterone, which manifestation, progression, and clinical outcome. It could be normally stimulates NaCl reabsorption by NCCT, would in- imagined, that carriers of polymorphisms or constellations of crease the magnitude of the lumen-negative voltage. This, in polymorphisms have a predisposition to develop one of the turn would favor paracellular secretion of magnesium and mentioned diseases. As these chronic diseases affect a high potassium. Reilly and Ellison proposed that paracellin-1 could percentage of the population, this is of great socioeconomic be involved in this paracellular pathway, because in the initial impact. report, RT-PCR showed paracellin-1 expression in the DCT The results of mutation analysis in familial hypomagnesemia (34). Recent data analyzing the expression profile of several demonstrate that there is still a number of patients with an claudins along the nephron by immunofluorescence micros- unidentified genetic defect both of recessive and dominant copy could not detect paracellin-1 in the DCT (98). In addition, pattern of inheritance (24,25). Thus, it seems likely that new earlier microperfusion studies failed to detect any magnesium genes involved in magnesium transport mechanisms will be secretion in the distal tubule (99). An alternative explanation identified in the near future adding to our current knowledge on comes from studies in rats receiving chronic thiazide treatment. magnesium metabolism. Loffing et al. showed that a complete block of NCCT results in an increased rate of apoptosis in DCT cells (100). This is Acknowledgments consistent with the observation of Schultheis et al. in the We thank Gary Quamme, Nikola Jeck, Melanie Peters, and Sieg- NCCT knockout model, who also demonstrated a decrease in fried Waldegger for their critical reading of the manuscript and the number, height, and basolateral infolding of DCT cells (95). helpful discussions during its preparation. This work was supported Perhaps diminished absorptive surface area of the DCT in GS by the Deutsche Forschungsgemeinschaft (Ko1480–3/2). compromises magnesium reabsorption to such an extent that magnesium wasting ensues. The reason for hypomagnesemia References in GS is an ongoing debate that needs further research 1. Seyberth H, Soergel M, Koeckerling A: Hypokalaemic tubular activities. disorders: The hyperprostaglandin E syndrome and Gitelman- In summary, inherited disorders of magnesium metabolism Bartter syndrome. In: Oxford Textbook of Clinical Nephrology, represent a heterogenous group of diseases with complex phe- 2nd ed., edited by Davison AM, Cameron JS, Gru¨nfeld JP, Kerr notypes. This is partly due to the simultaneous disturbance of DN, Ritz E, Oxford, Oxford University Press, 1998, pp 1085– other electrolyte systems, especially of calcium and potassium 1094 homeostasis. 2. Iannello S, Belfiore F: Hypomagnesemia. A review of patho- The proteins underlying the pathogenesis of these disorders physiological, clinical and therapeutical aspects. Panminerva Med 43: 177–209, 2001 demonstrate a surprising variety: 3. Schimatschek HF, Rempis R: Prevalence of hypomagnesemia in

ϩ ϩ an unselected German population of 16,000 individuals. Magnes • The ␥-subunit of the enzyme Na -K -ATPase Res 14: 283–290, 2001 • Paracellin-1, a tight-junction protein mediating paracellular 4. Innerarity S: Hypomagnesemia in acute and chronic illness. Crit transport Care Nurs Q 23: 1–19, 2000 258 Journal of the American Society of Nephrology J Am Soc Nephrol 14: 249–260, 2003

5. Sanders GT, Huijgen HJ, Sanders R: Magnesium in disease: A 25. Kantorovich V, Adams JS, Gaines JE, Guo X, Pandian MR, review with special emphasis on the serum ionized magnesium. Cohn DH, Rude RK: Genetic heterogeneity in familial renal Clin Chem Lab Med 37: 1011–1033, 1999 magnesium wasting. J Clin Endocrinol Metab 87: 612–617, 6. Balon TW, Jasman A, Scott S, Meehan WP, Rude RK, Nadler 2002 JL: Dietary magnesium prevents fructose-induced insulin insen- 26. Geven WB, Monnens LA, Willems JL, Buijs W, Hamel CJ: sitivity in rats. Hypertension 23: 1036–1039, 1994 Isolated autosomal recessive renal magnesium loss in two sisters. 7. Chakraborti S, Chakraborti T, Mandal M, Mandal A, Das S, Clin Genet 32: 398–402, 1987 Ghosh S: Protective role of magnesium in cardiovascular dis- 27. Michelis MF, Drash AL, Linarelli LG, De Rubertis FR, Davis eases: A review. Mol Cell Biochem 238: 163–179, 2002 BB: Decreased bicarbonate threshold and renal magnesium wast- 8. Alamoudi OS: Hypomagnesaemia in chronic, stable asthmatics: ing in a sibship with distal renal tubular acidosis. (Evaluation of Prevalence, correlation with severity and hospitalization. Eur the pathophysiological role of parathyroid hormone). Metabo- Respir J 16: 427–431, 2000 lism 21: 905–920, 1972 9. Quamme GA, de Rouffignac C: Epithelial magnesium transport 28. Manz F, Scharer K, Janka P, Lombeck J: Renal magnesium and regulation by the kidney. Front Biosci 5: D694–D711, 2000 wasting, incomplete tubular acidosis, hypercalciuria and nephro- 10. Smith RL, Maguire ME: Microbial magnesium transport: Un- calcinosis in siblings. Eur J Pediatr 128: 67–79, 1978 usual transporters searching for identity. Mol Microbiol 28: 217– 29. Rodriguez-Soriano J, Vallo A, Garcia-Fuentes M: Hypomagne- 226, 1998 saemia of hereditary renal origin. Pediatr Nephrol 1: 465–472, 11. Li L, Tutone AF, Drummond RS, Gardner RC, Luan S: A novel 1987 family of magnesium transport genes in Arabidopsis. Plant Cell 30. Nicholson JC, Jones CL, Powell HR, Walker RG, McCredie DA: 13: 2761–2775, 2001 Familial hypomagnesaemia–hypercalciuria leading to end-stage 12. Zsurka G, Gregan J, Schweyen RJ: The human mitochondrial renal failure. Pediatr Nephrol 9: 74–76, 1995 Mrs2 protein functionally substitutes for its yeast homologue, a 31. Praga M, Vara J, Gonzalez-Parra E, Andres A, Alamo C, Araque candidate magnesium transporter. Genomics 72: 158–168, 2001 A, Ortiz A, Rodicio JL: Familial hypomagnesemia with hyper- 13. Cole DE, Quamme GA: Inherited disorders of renal magnesium calciuria and nephrocalcinosis. Kidney Int 47: 1419–1425, 1995 handling. J Am Soc Nephrol 11: 1937–1947, 2000 32. Benigno V, Canonica CS, Bettinelli A, von Vigier RO, Trutt- 14. Fine KD, Santa Ana CA, Porter JL, Fordtran JS: Intestinal mann AC, Bianchetti MG: Hypomagnesaemia-hypercalciuria- absorption of magnesium from food and supplements. J Clin nephrocalcinosis: A report of nine cases and a review. Nephrol Invest 88: 396–402, 1991 Dial Transplant 15: 605–610, 2000 15. Kerstan D, Quamme G: Physiology and pathophysiology of 33. Weber S, Schneider L, Peters M, Misselwitz J, Ronnefarth G, intestinal absorption of magnesium. In: Calcium in Internal Boswald M, Bonzel KE, Seeman T, Sulakova T, Kuwertz- Medicine, edited by Massry SG, Morii H, Nishizawa Y, London, Broking E, Gregoric A, Palcoux JB, Tasic V, Manz F, Scharer K, Springer-Verlag, 2002, pp 171–183 Seyberth HW, Konrad M: Novel paracellin-1 mutations in 25 16. de Rouffignac C, Quamme G: Renal magnesium handling and its families with familial hypomagnesemia with hypercalciuria and hormonal control. Physiol Rev 74: 305–322, 1994 nephrocalcinosis. J Am Soc Nephrol 12: 1872–1881, 2001 17. Dai LJ, Ritchie G, Kerstan D, Kang HS, Cole DE, Quamme GA: 34. Simon DB, Lu Y, Choate KA, Velazquez H, Al-Sabban E, Praga Magnesium transport in the renal distal convoluted tubule. M, Casari G, Bettinelli A, Colussi G, Rodriguez-Soriano J, Physiol Rev 81: 51–84, 2001 McCredie D, Milford D, Sanjad S, Lifton RP: Paracellin-1, a 18. Geven WB, Monnens LA, Willems HL, Buijs WC, ter Haar BG: renal tight junction protein required for paracellular Mg2ϩ re- Renal magnesium wasting in two families with autosomal dom- sorption. Science 285: 103–106, 1999 inant inheritance. Kidney Int 31: 1140–1144, 1987 35. Blanchard A, Jeunemaitre X, Coudol P, Dechaux M, Froissart M, 19. Meij I, Illy KE, Monnens L: Severe hypomagnesemia in a May A, Demontis R, Fournier A, Paillard M, Houillier P: Para- neonate with isolated renal magnesium loss. Nephron 84: 198, cellin-1 is critical for magnesium and calcium reabsorption in the 2000 human thick ascending limb of Henle. Kidney Int 59: 2206– 20. Meij IC, Saar K, van den Heuvel LP, Nuernberg G, Vollmer M, 2215, 2001 Hildebrandt F, Reis A, Monnens LA, Knoers NV: Hereditary 36. Morita K, Furuse M, Fujimoto K, Tsukita S: Claudin multigene isolated renal magnesium loss maps to chromosome 11q23. Am J family encoding four-transmembrane domain protein compo- Hum Genet 64: 180–188, 1999 nents of tight junction strands. Proc Natl Acad Sci USA 96: 21. Wetzel RK, Sweadner KJ: Immunocytochemical localization of 511–516, 1999 Na-K-ATPase alpha- and gamma-subunits in rat kidney. Am J 37. Van Itallie C, Rahner C, Anderson JM: Regulated expression of Physiol Renal Physiol 281: F531–F545, 2001 claudin-4 decreases paracellular conductance through a selective 22. Meij IC, Koenderink JB, van Bokhoven H, Assink KF, Groene- decrease in sodium permeability. J Clin Invest 107: 1319–1327, stege WT, de Pont JJ, Bindels RJ, Monnens LA, van den Heuvel 2001 LP, Knoers NV: Dominant isolated renal magnesium loss is 38. Colegio OR, Van Itallie CM, McCrea HJ, Rahner C, Anderson caused by misrouting of the Na(ϩ), K(ϩ)-ATPase gamma-sub- JM: Claudins create charge-selective channels in the paracellular unit. Nat Genet 26: 265–266, 2000 pathway between epithelial cells. Am J Physiol Cell Physiol 283: 23. Pu HX, Scanzano R, Blostein R: Distinct regulatory effects of the C142–C147, 2002 Na, K-ATPase gamma subunit. J Biol Chem 277: 20270–20276, 39. Ohba Y, Kitagawa H, Kitoh K, Sasaki Y, Takami M, Shinkai Y, 2002 Kunieda T: A deletion of the paracellin-1 gene is responsible for 24. Meij IC: Gaining insights in renal magnesium handling. In: renal tubular dysplasia in cattle. Genomics 68: 229–236, 2000 Thesis, Department of Pediatrics and Human Genetics. Nijme- 40. Weber S, Schlingmann KP, Peters M, Nejsum LN, Nielsen S, gen, Catholic University Nijmegen, 2002 Engel H, Grzeschik KH, Seyberth HW, Grone HJ, Nusing R, J Am Soc Nephrol 14: 249–260, 2003 Hereditary Magnesium-Losing Disorders 259

Konrad M: Primary gene structure and expression studies of LTRPC7 is a Mg.ATP-regulated divalent cation channel required rodent paracellin-1. J Am Soc Nephrol 12: 2664–2672, 2001 for cell viability. Nature 411: 590–595, 2001 41. Hirano T, Kobayashi N, Itoh T, Takasuga A, Nakamaru T, 57. Ryazanov AG, Ward MD, Mendola CE, Pavur KS, Dorovkov Hirotsune S, Sugimoto Y: Null mutation of PCLN-1/Claudin-16 MV, Wiedmann M, Erdjument-Bromage H, Tempst P, Parmer results in bovine chronic interstitial nephritis. Genome Res 10: TG, Prostko CR, Germino FJ, Hait WN: Identification of a new 659–663, 2000 class of protein kinases represented by eukaryotic elongation 42. Lu Y, Choate KA, Wang T, Lifton RP: Paracellin-1 kock-out factor-2 kinase. Proc Natl Acad Sci USA 94: 4884–4889, 1997 mouse model of recessive renal hypomagnesemia, hypercalciuria 58. Brown EM, Gamba G, Riccardi D, Lombardi M, Butters R, Kifor and nephrocalcinosis [Abstract]. J Am Soc Nephrol 12: 2001 O, Sun A, Hediger MA, Lytton J, Hebert SC: Cloning and 43. Meij IC, van den Heuvel LP, Knoers NV: Genetic disorders of characterization of an extracellular Ca(2ϩ)-sensing receptor magnesium homeostasis. Biometals 15: 297–307, 2002 from bovine parathyroid. Nature 366: 575–580, 1993 44. Mu¨ller D, Claverie-Martin F, Eggert P, Garcia-Nieto V: Muta- 59. Bai M, Trivedi S, Kifor O, Quinn SJ, Brown EM: Intermolecular tionen im PDZ-Motif von Paracellin-1 als Ursache der Hyperkal- interactions between dimeric calcium-sensing receptor mono- ziurie im Kindesalter [Abstract]. Nieren-und Hochdruck- mers are important for its normal function. Proc Natl Acad Sci krankheiten 31: 52, 2002 USA 96: 2834–2839, 1999 45. Paunier L, Radde IC, Kooh SW, Conen PE, Fraser D: Primary 60. Bapty BW, Dai LJ, Ritchie G, Canaff L, Hendy GN, Quamme ϩ ϩ hypomagnesemia with secondary hypocalcemia in an infant. GA: Activation of Mg2 /Ca2 sensing inhibits hormone-stim- ϩ Pediatrics 41: 385–402, 1968 ulated Mg2 uptake in mouse distal convoluted tubule cells. 46. Anast CS, Mohs JM, Kaplan SL, Burns TW: Evidence for Am J Physiol 275: F353–F360, 1998 parathyroid failure in magnesium deficiency. Science 177: 606– 61. Hebert SC: Extracellular calcium-sensing receptor: Implications 608, 1972 for calcium and magnesium handling in the kidney. Kidney Int 47. Shalev H, Phillip M, Galil A, Carmi R, Landau D: Clinical 50: 2129–2139, 1996 presentation and outcome in primary familial hypomagnesaemia. 62. Pollak MR, Brown EM, Chou YH, Hebert SC, Marx SJ, Stein- Arch Dis Child 78: 127–130, 1998 mann B, Levi T, Seidman CE, Seidman JG: Mutations in the ϩ 48. Cole DE, Kooh SW, Vieth R: Primary infantile hypomagne- human Ca(2 )-sensing receptor gene cause familial hypocalci- uric hypercalcemia and neonatal severe hyperparathyroidism. saemia: Outcome after 21 years and treatment with continuous Cell 75: 1297–1303, 1993 nocturnal nasogastric magnesium infusion. Eur J Pediatr 159: 63. Pollak MR, Chou YH, Marx SJ, Steinmann B, Cole DE, Brandi 38–43, 2000 ML, Papapoulos SE, Menko FH, Hendy GN, Brown EM, et al: 49. Milla PJ, Aggett PJ, Wolff OH, Harries JT: Studies in primary Familial hypocalciuric hypercalcemia and neonatal severe hyper- hypomagnesaemia: Evidence for defective carrier-mediated parathyroidism. Effects of mutant gene dosage on phenotype. small intestinal transport of magnesium. Gut 20: 1028–1033, J Clin Invest 93: 1108–1112, 1994 1979 64. Marx SJ, Attie MF, Levine MA, Spiegel AM, Downs RW Jr, 50. Matzkin H, Lotan D, Boichis H: Primary hypomagnesemia with Lasker RD: The hypocalciuric or benign variant of familial a probable double magnesium transport defect. Nephron 52: hypercalcemia: Clinical and biochemical features in fifteen kin- 83–86, 1989 dreds. Medicine (Baltimore) 60: 397–412, 1981 51. Walder RY, Shalev H, Brennan TM, Carmi R, Elbedour K, Scott 65. Pollak MR, Brown EM, Estep HL, McLaine PN, Kifor O, Park DA, Hanauer A, Mark AL, Patil S, Stone EM, Sheffield VC: J, Hebert SC, Seidman CE, Seidman JG: Autosomal dominant Familial hypomagnesemia maps to chromosome 9q, not to the X hypocalcaemia caused by a Ca(2ϩ)-sensing receptor gene mu- chromosome: Genetic linkage mapping and analysis of a bal- tation. Nat Genet 8: 303–307, 1994 anced translocation breakpoint. Hum Mol Genet 6: 1491–1497, 66. Pearce SH, Williamson C, Kifor O, Bai M, Coulthard MG, 1997 Davies M, Lewis-Barned N, McCredie D, Powell H, Kendall- 52. Walder RY, Borochowitz Z, Shalev H, Carmi R, Elbedour K, Taylor P, Brown EM, Thakker RV: A familial syndrome of Scott DA, Stone EM, Sheffield VC: Hypomagnesemia with hypocalcemia with hypercalciuria due to mutations in the calci- secondary hypocalcemia (HSH): Narrowing the disease region um-sensing receptor [see comments]. N Engl J Med 335: 1115– on chromosome 9 [Abstract]. Am J Hum Genet 65: A451, 1999 1122, 1996 53. Schlingmann KP, Weber S, Peters M, Niemann Nejsum L, 67. Okazaki R, Chikatsu N, Nakatsu M, Takeuchi Y, Ajima M, Miki Vitzthum H, Klingel K, Kratz M, Haddad E, Ristoff E, Dinour D, J, Fujita T, Arai M, Totsuka Y, Tanaka K, Fukumoto S: A novel Syrrou M, Nielsen S, Sassen M, Waldegger S, Seyberth HW, activating mutation in calcium-sensing receptor gene associated Konrad M: Hypomagnesemia with secondary hypocalcemia is with a family of autosomal dominant hypocalcemia. J Clin caused by mutations in TRPM6, a new member of the TRPM Endocrinol Metab 84: 363–366, 1999 gene family. Nat Genet 31: 166–170, 2002 68. Brown EM, MacLeod RJ: Extracellular calcium sensing and 54. Walder RY, Landau D, Meyer P, Shalev H, Tsolia M, Boro- extracellular calcium signaling. Physiol Rev 81: 239–297, 2001 chowitz Z, Boettger MB, Beck GE, Englehardt RK, Carmi R, 69. Vargas-Poussou R, Huang C, Hulin P, Houillier P, Jeunemaitre Sheffield VC: Mutation of TRPM6 causes familial hypomag- X, Paillard M, Planelles G, Dechaux M, Miller RT, Antignac C: nesemia with secondary hypocalcemia. Nat Genet 31: 171–174, Functional characterization of a calcium-sensing receptor muta- 2002 tion in severe autosomal dominant hypocalcemia with a Bartter- 55. Harteneck C, Plant TD, Schultz G: From worm to man: Three like syndrome. J Am Soc Nephrol 13: 2259–2266, 2002 subfamilies of TRP channels. Trends Neurosci 23: 159–166, 70. Watanabe S, Fukumoto S, Chang H, Takeuchi Y, Hasegawa Y, 2000 Okazaki R, Chikatsu N, Fujita T: Association between activating 56. Nadler MJ, Hermosura MC, Inabe K, Perraud AL, Zhu Q, Stokes mutations of calcium-sensing receptor and Bartter’s syndrome. AJ, Kurosaki T, Kinet JP, Penner R, Scharenberg AM, Fleig A: Lancet 360: 692–694, 2002 260 Journal of the American Society of Nephrology J Am Soc Nephrol 14: 249–260, 2003

71. Rodriguez-Soriano J: Bartter and related syndromes: The puzzle 86. Estevez R, Boettger T, Stein V, Birkenhager R, Otto E, Hilde- is almost solved. Pediatr Nephrol 12: 315–327, 1998 brandt F, Jentsch TJ: Barttin is a Cl- channel beta-subunit crucial 72. Landau D, Shalev H, Ohaly M, Carmi R: Infantile variant of for renal Cl- reabsorption and inner ear Kϩ secretion. Nature Bartter syndrome and sensorineural deafness: A new autosomal 414: 558–561, 2001 recessive disorder. Am J Med Genet 59: 454–459, 1995 87. Waldegger S, Jeck N, Barth P, Peters M, Vitzthum H, Wolf K, 73. Peters M, Jeck N, Reinalter S, Leonhardt A, Tonshoff B, Klaus Kurtz A, Konrad M, Seyberth HW: Barttin increases expression G, Konrad M, Seyberth HW: Clinical presentation of genetically and changes current properties of ClC-K channels. Pflugers Arch defined patients with hypokalemic salt-losing tubulopathies. 444: 411–418, 2002 Am J Med 112: 183–190, 2002 88. Gitelman HJ, Graham JB, Welt LG: A new familial disorder 74. Kockerling A, Reinalter SC, Seyberth HW: Impaired response to characterized by hypokalemia and hypomagnesemia. Trans As- furosemide in hyperprostaglandin E syndrome: Evidence for a soc Am Physicians 79: 221–235, 1966 tubular defect in the loop of Henle. J Pediatr 129: 519–528, 89. Bettinelli A, Bianchetti MG, Girardin E, Caringella A, Cecconi 1996 M, Appiani AC, Pavanello L, Gastaldi R, Isimbaldi C, Lama G, 75. Simon DB, Karet FE, Hamdan JM, DiPietro A, Sanjad SA, et al: Use of calcium excretion values to distinguish two forms of Lifton RP: Bartter’s syndrome, hypokalaemic alkalosis with hy- primary renal tubular hypokalemic alkalosis: Bartter and Gitel- percalciuria, is caused by mutations in the Na-K-2Cl cotrans- man syndromes. J Pediatr 120: 38–43, 1992 porter NKCC2. Nat Genet 13: 183–188, 1996 90. Jeck N, Konrad M, Peters M, Weber S, Bonzel KE, Seyberth 76. Simon DB, Karet FE, Rodriguez-Soriano J, Hamdan JH, DiPi- etro A, Trachtman H, Sanjad SA, Lifton RP: Genetic heteroge- HW: Mutations in the chloride channel gene, CLCNKB, leading neity of Bartter’s syndrome revealed by mutations in the Kϩ to a mixed Bartter-Gitelman phenotype. Pediatr Res 48: 754– channel, ROMK. Nat Genet 14: 152–156, 1996 758, 2000 77. Takahashi N, Chernavvsky DR, Gomez RA, Igarashi P, Gitelman 91. Cruz DN, Shaer AJ, Bia MJ, Lifton RP, Simon DB: Gitelman’s HJ, Smithies O: Uncompensated polyuria in a mouse model of syndrome revisited: An evaluation of symptoms and health- Bartter’s syndrome. Proc Natl Acad Sci USA 97: 5434–5439, 2000 related quality of life. Kidney Int 59: 710–717, 2001 78. Dai LJ, Bapty B, Ritchie G, Quamme GA: PGE2 stimulates 92. Simon DB, Nelson-Williams C, Bia MJ, Ellison D, Karet FE, Mg2ϩ uptake in mouse distal convoluted tubule cells. Am J Molina AM, Vaara I, Iwata F, Cushner HM, Koolen M, Gainza Physiol 275: F833–F839, 1998 FJ, Gitleman HJ, Lifton RP: Gitelman’s variant of Bartter’s 79. Bartter F, Pronove P, Gill J Jr, MacCardle R: Hyperplasia of the syndrome, inherited hypokalaemic alkalosis, is caused by muta- juxtaglomerular complex with hyperaldosteronism and hy- tions in the thiazide-sensitive Na-Cl cotransporter. Nat Genet 12: pokalemic alkalosis. A new syndrome. Am J Med 33: 811–828, 24–30, 1996 1962 93. Kunchaparty S, Palcso M, Berkman J, Velazquez H, Desir GV, 80. Simon DB, Bindra RS, Mansfield TA, Nelson-Williams C, Men- Bernstein P, Reilly RF, Ellison DH: Defective processing and donca E, Stone R, Schurman S, Nayir A, Alpay H, Bakkaloglu A, expression of thiazide-sensitive Na-Cl cotransporter as a cause of Rodriguez-Soriano J, Morales JM, Sanjad SA, Taylor CM, Pilz Gitelman’s syndrome. Am J Physiol 277: F643–F649, 1999 D, Brem A, Trachtman H, Griswold W, Richard GA, John E, 94. De Jong JC, Van Der Vliet WA, Van Den Heuvel LP, Willems Lifton RP: Mutations in the chloride channel gene, CLCNKB, PH, Knoers NV, Bindels RJ: Functional expression of mutations cause Bartter’s syndrome type III. Nat Genet 17: 171–178, 1997 in the human NaCl cotransporter: Evidence for impaired routing 81. Konrad M, Vollmer M, Lemmink HH, Van Den Heuvel LP, Jeck mechanisms in Gitelman’s syndrome. J Am Soc Nephrol 13: N, Vargas-Poussou R, Lakings A, Ruf R, Deschenes G, Antignac 1442–1448, 2002 C, Guay-Woodford L, Knoers NV, Seyberth HW, Feldmann D, 95. Schultheis PJ, Lorenz JN, Meneton P, Nieman ML, Riddle TM, Hildebrandt F: Mutations in the chloride channel gene CLCNKB Flagella M, Duffy JJ, Doetschman T, Miller ML, Shull GE: as a cause of classic Bartter syndrome. J Am Soc Nephrol 11: Phenotype resembling Gitelman’s syndrome in mice lacking the 1449–1459, 2000 apical Naϩ-Cl- cotransporter of the distal convoluted tubule. 82. Mount DB, Delpire E, Gamba G, Hall AE, Poch E, Hoover RS, J Biol Chem 273: 29150–29155, 1998 Hebert SC: The electroneutral cation-chloride cotransporters. J 96. Reilly RF, Ellison DH: Mammalian distal tubule: Physiology, Exp Biol 201: 2091–2102, 1998 pathophysiology, and molecular anatomy. Physiol Rev 80: 277– 83. Jeck N, Reinalter SC, Henne T, Marg W, Mallmann R, Pasel K, 313, 2000 Vollmer M, Klaus G, Leonhardt A, Seyberth HW, Konrad M: Hypokalemic salt-losing tubulopathy with chronic renal failure 97. Ellison DH: Divalent cation transport by the distal nephron: and sensorineural deafness. Pediatrics 108: E5, 2001 Insights from Bartter’s and Gitelman’s syndromes. Am J Physiol 84. Brennan TM, Landau D, Shalev H, Lamb F, Schutte BC, Walder Renal Physiol 279: F616–F625, 2000 RY, Mark AL, Carmi R, Sheffield VC: Linkage of infantile 98. Kiuchi-Saishin Y, Gotoh S, Furuse M, Takasuga A, Tano Y, Bartter syndrome with sensorineural deafness to chromosome 1p. Tsukita S: Differential expression patterns of claudins, tight Am J Hum Genet 62: 355–361, 1998 junction membrane proteins, in mouse nephron segments. JAm 85. Birkenhager R, Otto E, Schurmann MJ, Vollmer M, Ruf EM, Soc Nephrol 13: 875–886, 2002 Maier-Lutz I, Beekmann F, Fekete A, Omran H, Feldmann D, 99. Quamme GA: Renal magnesium handling: new insights in un- Milford DV, Jeck N, Konrad M, Landau D, Knoers NV, Antig- derstanding old problems. Kidney Int 52: 1180–1195, 1997 nac C, Sudbrak R, Kispert A, Hildebrandt F: Mutation of BSND 100. Loffing J, Loffing-Cueni D, Hegyi I, Kaplan MR, Hebert SC, Le causes Bartter syndrome with sensorineural deafness and kidney Hir M, Kaissling B: Thiazide treatment of rats provokes apopto- failure. Nat Genet 29: 310–314, 2001 sis in distal tubule cells. Kidney Int 50: 1180–1190, 1996