Iwan C. Meij

Dominant isolated renal magnesium loss is caused by misrouting of the Na(+),K(+)-ATPase gamma-subunit.

Primary hypomagnesaemia is composed of a heterogeneous group of disorders characterized by renal or intestinal Mg2+ wasting, often associated with disturbances in Ca2+ excretion. We identified a putative dominant-negative mutation in the gene encoding the Na+,K+-ATPase γ-subunit (FXYD2), leading to defective routing of the protein in a family with dominant renal hypomagnesaemia.

A Novel Claudin 16 Mutation Associated with Childhood Hypercalciuria Abolishes Binding to ZO-1 and Results in Lysosomal Mistargeting

Mutations in the gene coding for the renal tight junction protein claudin 16 cause familial hypomagnesemia with hypercalciuria and nephrocalcinosis, an autosomal recessive disorder of renal Ca(2+) and Mg(2+) handling that progressively leads to chronic renal failure, with nephrolithiasis having been reported in heterozygous carriers. Screening a cohort of 11 families with idiopathic hypercalciuria identified a novel homozygous mutation in the claudin 16 gene in two families. In contrast to classical symptoms of familial hypomagnesemia with hypercalciuria and nephrocalcinosis, the patients displayed serious but self-limiting childhood hypercalciuria with preserved glomerular filtration rate. The mutation results in inactivation of a PDZ-domain binding motif, thereby disabling the association of the tight junction scaffolding protein ZO-1 with claudin 16. In contrast to wild-type claudin 16, the mutant no longer localizes to tight junctions in kidney epithelial cells but instead accumulates in lysosomes. Thus, mutations at different intragenic sites in the claudin 16 gene may lead to particular clinical phenotypes with a distinct prognosis. Mutations in claudin 16 that affect interaction with ZO-1 lead to lysosomal mistargeting, providing-for the first time, to our knowledge-insight into the molecular mechanism of a disease-associated mutation in the claudin 16 gene.

Claudin-10 exists in six alternatively spliced isoforms that exhibit distinct localization and function

The tight junction protein claudin-10 is known to exist in two isoforms, resulting from two alternative exons, 1a and 1b (Cldn10a, Cldn10b). Here, we identified and characterized another four claudin-10 splice variants in mouse and human. One (Cldn10a_v1) results from an alternative splice donor site, causing a deletion of the last 57 nucleotides of exon 1a. For each of these three variants one further splice variant was identified (Cldn10a_v2, Cldn10a_v3, Cldn10b_v1), lacking exon 4. When transfected into MDCK cells, Cldn10a, Cldn10a_v1 and Cldn10b were inserted into the tight junction, whereas isoforms of splice variants lacking exon 4 were retained in the endoplasmic reticulum. Cldn10a transfection into MDCK cells confirmed the previously described increase in paracellular anion permeability. Cldn10a_v1 transfection had no direct effect, but modulated Cldn10a-induced organic anion permeability. At variance with previous reports in MDCK-II cells, transfection of high-resistance MDCK-C7 cells with Cldn10b dramatically decreased transepithelial resistance, increased cation permeability, and changed monovalent cation selectivity from Eisenman sequence IV to X, indicating the presence of a high field-strength binding site that almost completely removes the hydration shell of the permeating cations. The extent of all these effects strongly depended on the endogenous claudins of the transfected cells.

CNNM2, encoding a basolateral protein required for renal Mg2+ handling, is mutated in dominant hypomagnesemia

Familial hypomagnesemia is a rare human disorder caused by renal or intestinal magnesium (Mg(2+)) wasting, which may lead to symptoms of Mg(2+) depletion such as tetany, seizures, and cardiac arrhythmias. Our knowledge of the physiology of Mg(2+) (re)absorption, particularly the luminal uptake of Mg(2+) along the nephron, has benefitted from positional cloning approaches in families with Mg(2+) reabsorption disorders; however, basolateral Mg(2+) transport and its regulation are still poorly understood. Here, by using a candidate screening approach, we identified CNNM2 as a gene involved in renal Mg(2+) handling in patients of two unrelated families with unexplained dominant hypomagnesemia. In the kidney, CNNM2 was predominantly found along the basolateral membrane of distal tubular segments involved in Mg(2+) reabsorption. The basolateral localization of endogenous and recombinant CNNM2 was confirmed in epithelial kidney cell lines. Electrophysiological analysis showed that CNNM2 mediated Mg(2+)-sensitive Na(+) currents that were significantly diminished in mutant protein and were blocked by increased extracellular Mg(2+) concentrations. Our data support the findings of a recent genome-wide association study showing the CNNM2 locus to be associated with serum Mg(2+) concentrations. The mutations found in CNNM2, its observed sensitivity to extracellular Mg(2+), and its basolateral localization signify a critical role for CNNM2 in epithelial Mg(2+) transport.

Targeted deletion of murine Cldn16 identifies extra- and intrarenal compensatory mechanisms of Ca2+ and Mg2+ wasting

Claudin-16 (CLDN16) is critical for renal paracellular epithelial transport of Ca(2+) and Mg(2+) in the thick ascending loop of Henle. To gain novel insights into the role of CLDN16 in renal Ca(2+) and Mg(2+) homeostasis and the pathological mechanisms underlying a human disease associated with CLDN16 dysfunction [familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC), OMIM 248250], we generated a mouse model of CLDN16 deficiency. Similar to patients, CLDN16-deficient mice displayed hypercalciuria and hypomagnesemia. Contrary to FHHNC patients, nephrocalcinosis was absent in our model, indicating the existence of compensatory pathways in ion handling in this model. In line with the renal loss of Ca(2+), compensatory mechanisms like parathyroid hormone and 1,25(OH)(2)D(3) were significantly elevated. Also, gene expression profiling revealed transcriptional upregulation of several Ca(2+) and Mg(2+) transport systems including Trpv5, Trpm6, and calbindin-D9k. Induced gene expression was also seen for the transcripts of two putative Mg(2+) transport proteins, Cnnm2 and Atp13a4. Moreover, urinary pH was significantly lower when compared with wild-type mice. Taken together, our findings demonstrate that loss of CLDN16 activity leads to specific alterations in Ca(2+) and Mg(2+) homeostasis and that CLDN16-deficient mice represent a useful model to further elucidate pathways involved in renal Ca(2+) and Mg(2+) handling.

Deletion of claudin-10 (Cldn10) in the thick ascending limb impairs paracellular sodium permeability and leads to hypermagnesemia and nephrocalcinosis

In the kidney, tight junction proteins contribute to segment specific selectivity and permeability of paracellular ion transport. In the thick ascending limb (TAL) of Henles loop, chloride is reabsorbed transcellularly, whereas sodium reabsorption takes transcellular and paracellular routes. TAL salt transport maintains the concentrating ability of the kidney and generates a transepithelial voltage that drives the reabsorption of calcium and magnesium. Thus, functionality of TAL ion transport depends strongly on the properties of the paracellular pathway. To elucidate the role of the tight junction protein claudin-10 in TAL function, we generated mice with a deletion of Cldn10 in this segment. We show that claudin-10 determines paracellular sodium permeability, and that its loss leads to hypermagnesemia and nephrocalcinosis. In isolated perfused TAL tubules of claudin-10–deficient mice, paracellular permeability of sodium is decreased, and the relative permeability of calcium and magnesium is increased. Moreover, furosemide-inhibitable transepithelial voltage is increased, leading to a shift from paracellular sodium transport to paracellular hyperabsorption of calcium and magnesium. These data identify claudin-10 as a key factor in control of cation selectivity and transport in the TAL, and deficiency in this pathway as a cause of nephrocalcinosis.

Dominant Isolated Renal Magnesium Loss Is Caused by Misrouting of the Na+,K+‐ATPase γ‐Subunit

Abstract: Hereditary primary hypomagnesemia comprises a clinically and genetically heterogeneous group of disorders in which hypomagnesemia is due to either renal or intestinal Mg2+ wasting. These disorders share the general symptoms of hypomagnesemia, tetany and epileptiformic convulsions, and often include secondary or associated disturbances in calcium excretion. In a large Dutch family with autosomal dominant renal hypomagnesemia, associated with hypocalciuria, we mapped the disease locus to a 5.6‐cM region on chromosome 11q23. After candidate screening, we identified a heterozygous mutation in the FXYD2 gene, encoding the Na+,K+‐ATPase γ‐subunit, cosegregating with the patients of this family, which was not found in 132 control chromosomes. The mutation leads to a G41R substitution, introducing a charged amino acid residue in the predicted transmembrane region of the γ‐subunit protein. Expression studies in insect Sf9 and COS‐1 cells showed that the mutant γ‐subunit protein was incorrectly routed and accumulated in perinuclear structures. In addition to disturbed routing of the G41R mutant, Western blot analysis of Xenopus oocytes expressing wild‐type or mutant γ‐subunit showed mutant γ‐subunit lacking a posttranslational modification. Finally, we investigated two individuals lacking one copy of the FXYD2 gene and found their serum Mg2+ levels to be within the normal range. We conclude that the arrest of mutant γ‐subunit in distinct intracellular structures is associated with aberrant posttranslational processing and that the G41R mutation causes dominant renal hypomagnesemia associated with hypocalciuria through a dominant negative mechanism.

Hereditary isolated renal magnesium loss maps to chromosome 11q23

Hypomagnesemia due to isolated renal magnesium loss has previously been demonstrated in two presumably unrelated Dutch families with autosomal dominant mode of inheritance. Patients with magnesium deficiency may suffer from tetany and convulsions, but the patients with hereditary renal magnesium wasting can also be clinically nonsymptomatic. In a genomewide linkage study, we first excluded a possible candidate region, on chromosome 9q, that encompasses the gene for intestinal hypomagnesemia with secondary hypocalcemia and, subsequently, found linkage to markers on chromosome 11q23. Detailed haplotype analyses identified a common haplotype segregating in both families, suggesting both their relationship through a common ancestor and the existence of a single, hypomagnesemia-causing mutation within them. The maximum two-point LOD score (Zmax) was found for marker D11S4127 (Zmax=6.41 at a recombination fraction of. 00), whereas a multipoint analysis gave a Zmax of 8.24 between markers D11S4142 and D11S4171. Key recombination events define a 5. 6-cM region between these two markers on chromosome 11q23. We conclude that this region encompasses a gene, involved in renal magnesium handling, that is mutated in our patients and is different from the gene involved in intestinal magnesium handling.

Membrane Topology and Intracellular Processing of Cyclin M2 (CNNM2)

Background: Mutations in CNNM2 cause severe dominant hypomagnesemia. Results: Structure of CNNM2 consists of an extracellular N terminus and intracellular C terminus containing CBS domains, which are affected by the identified mutations. Conclusion: CNNM2 is intensively processed before being expressed in its final structure at the plasma membrane. Significance: CNNM2 structure analysis will aid to elucidate CNNM2 function in renal magnesium transport. Recently, mutations in the cyclin M2 (CNNM2) gene were identified to be causative for severe hypomagnesemia. In kidney, CNNM2 is a basolaterally expressed protein with predominant expression in the distal convoluted tubule. Transcellular magnesium (Mg2+) reabsorption in the distal convoluted tubule represents the final step before Mg2+ is excreted into the urine, thus fine-tuning its final excretion via a tightly regulated mechanism. The present study aims to get insight in the structure of CNNM2 and to characterize its post-translational modifications. Here, membrane topology studies using intramolecular epitopes and immunocytochemistry showed that CNNM2 has an extracellular N terminus and an intracellular C terminus. This suggests that one of the predicted transmembrane regions might be re-entrant. By homology modeling, we demonstrated that the loss-of-function mutation as found in patients disturbs the potential ATP binding by the intracellular cystathionine β-synthase domains. In addition, the cellular processing pathway of CNNM2 was exposed in detail. In the endoplasmic reticulum, the signal peptidase complex cleaves off a large N-terminal signal peptide of about 64 amino acids. Mutagenesis screening showed that CNNM2 is glycosylated at residue Asn-112, stabilizing CNNM2 on the plasma membrane. Interestingly, co-immunoprecipitation studies evidenced that CNNM2a forms heterodimers with the smaller isoform CNNM2b. These new findings on CNNM2 structure and processing may aid to elucidate the physiological role of CNNM2 in Mg2+ reabsorption in the kidney.

Disturbed function of the blood–cerebrospinal fluid barrier aggravates neuro-inflammation

Multiple sclerosis (MS) is a chronic neuro-inflammatory disorder, which is marked by the invasion of the central nervous system by monocyte-derived macrophages and autoreactive T cells across the brain vasculature. Data from experimental animal models recently implied that the passage of leukocytes across the brain vasculature is preceded by their traversal across the blood–cerebrospinal fluid barrier (BCSFB) of the choroid plexus. The correlation between the presence of leukocytes in the CSF of patients suffering from MS and the number of inflammatory lesions as detected by magnetic resonance imaging suggests that inflammation at the choroid plexus contributes to the disease, although in a yet unknown fashion. We here provide first insights into the involvement of the choroid plexus in the onset and severity of the disease and in particular address the role of the tight junction protein claudin-3 (CLDN3) in this process. Detailed analysis of human post-mortem brain tissue revealed a selective loss of CLDN3 at the choroid plexus in MS patients compared to control tissues. Importantly, mice that lack CLDN3 have an impaired BCSFB and experience a more rapid onset and exacerbated clinical signs of experimental autoimmune encephalomyelitis, which coincides with enhanced levels of infiltrated leukocytes in their CSF. Together, this study highlights a profound role for the choroid plexus in the pathogenesis of multiple sclerosis, and implies that CLDN3 may be regarded as a crucial and novel determinant of BCSFB integrity.