Transient Receptor Potential Melastatin 6 Knockout Mice Are Lethal whereas Heterozygous Deletion Results in Mild Hypomagnesemia

Background: Hypomagnesemia with secondary hypocalcemia is due to disturbed renal and intestinal magnesium (Mg 2+ ) (re)absorption. The underlying defect is a mutation in the transient receptor potential melastatin type 6 (TRPM6), a Mg 2+ -permeable ion channel expressed in the kidney and intestine. Our aim was to characterize homozygous (–/–) and heterozygous (+/–) TRPM6 knockout mice with respect to Mg 2+ homeostasis. Methods: TRPM6 +/– mice were bred on a normal (0.19% wt/wt Mg 2+ ) and high (0.48% wt/wt Mg 2+ ) Mg 2+ diet. In the offspring, 24-hour urinary Mg 2+ and calcium excretion as well as serum concentrations of both were determined. TRPM6 mRNA expression in the kidney and colon was measured. Results: On the regular diet, 30% of the offspring were TRPM6 wild-type ( +/+ ), 70% were TRPM6 +/– , and none were TRPM6 –/– . The genotypic distribution of the litters remained the same on the 0.48% Mg 2+ diet. In TRPM6 +/– mice on both diets, serum Mg 2+ levels were significantly lower, and renal and intestinal TRPM6 mRNA expression was reduced. Urinary Mg 2+ excretion was unaffected. Conclusions: Received: February 23, 2010 Accepted: July 5, 2010 Published online: September 1, 2010 Joost Hoenderop, PhD 286 Physiology, Radboud University Nijmegen Medical Centre PO Box 9101 NL–6500 HB Nijmegen (The Netherlands) Tel. +31 24 361 0580, Fax +31 24 361 6413, E-Mail J.Hoenderop @ fysiol.umcn.nl © 2010 S. Karger AG, Basel 1660–2137/11/1172–0011$38.00/0 Accessible online at: www.karger.com/nep Woudenberg-Vrenken/Sukinta/ van der Kemp/Bindels/Hoenderop Nephron Physiol 2011;117:p11–p19 p12 loop. The minority of filtered Mg 2+ (5–10%) is reabsorbed in the distal convoluted tubule (DCT) in an active, transcellular manner [4] . Mg 2+ reabsorption in the DCT determines the final urinary excretion, because no reabsorption occurs beyond this nephron segment [2] . Various disorders are related to disturbances in Mg 2+ homeostasis. Hypomagnesemia with secondary hypocalcemia (HSH; OMIM 602014) is a rare, hereditary disorder caused by disturbed renal and intestinal Mg 2+ (re) absorption [5–8] . The disease clinically manifests during the first months after birth, and symptoms comprise generalized seizures. HSH is characterized by low-serum Mg 2+ concentrations, and hypomagnesemia results in secondary hypocalcemia in patients [9] . Therapy includes lifelong oral Mg 2+ administration to relieve clinical symptoms and to normalize calcium (Ca 2+ ) homeostasis, while serum Mg 2+ levels usually remain in the subnormal range (0.5–0.6 mmol/l, reference 0.7–1.1) [10] . Numerous patients suffer from diarrhea, a side effect of oral Mg 2+ supplementation [11] . Genetic screening of HSH patients identified mutations in transient receptor potential melastatin type 6 (TRPM6) as underlying defect [8, 12] . TRPM6 belongs to the TRPM subfamily of ion channels. TRPM6 has been characterized as an Mg 2+ permeable ion channel [13] , which contains an -kinase domain at the carboxy (C) terminus [14, 15] . TRPM6 mutations in HSH patients include point and missense mutations, premature stops, inserted splice sites, frame shifts and exon deletions [8, 11, 12, 16–18] . The mutations are spread over the entire TRPM6 gene, and most result in truncation of the TRPM6 protein. Recently, Chubanov et al. [16, 17] described 2 dominant negative TRPM6 missense mutations in HSH patients, S141L and P1017R, which directly affect the trafficking and function of this TRP channel. In humans, TRPM6 is abundantly expressed in the colon and kidney (DCT); both sites are associated with active Mg 2+ (re)absorption [12] . The expression pattern of TRPM6 in mice resembles that of humans, although TRPM6 is also highly present in the lung [19] . Colocalization with the sodium-chloride cotransporter confirmed TRPM6 presence within the luminal membrane of DCT cells [13] . Dietary Mg 2+ restriction was shown to increase TRPM6 mRNA and protein expression in the mouse kidney [19, 20] , while Mg 2+ supplementation induces TRPM6 expression in the colon [19, 20] . TRPM6 expression and Mg 2+ (re)absorption are also regulated by a variety of hormones and compounds. Epidermal growth factor (EGF) and estrogen were discovered as magnesiotropic hormones directly affecting TRPM6 expression [19, 21] . EGF enhances activation of TRPM6 via the EGF receptor and intracellular signaling cascades [22] . Recently, Glaudemans et al. [23] discovered a missense mutation (N255D) in the voltage-gated potassium channel Kv1.1 in a Brazilian family with isolated autosomal dominant hypomagnesemia. This Kv1.1 mutant depolarizes the membrane of the DCT, which reduces the driving force for Mg 2+ uptake, resulting in renal Mg 2+ wasting in these patients [23] . Another TRPM family member, TRPM7, is the second Mg 2+ permeable ion channel known. TRPM7 is the closest homologue of TRPM6, but in contrast to TRPM6, the channel is ubiquitously expressed [15, 24] . Similar to TRPM6, TRPM7 contains an -kinase domain at the C terminus [15, 24, 25] . In vitro and in vivo studies established a role for TRPM7 in cell survival [25, 26] . Jin et al. [27] described that homozygous deletion of TRPM7 in mice results in embryonic lethality. Recently, Walder et al. [28] showed that TRPM6 knockout mice suffer from embryonic lethality and neural tube defects. Their study mainly focused on the role of TRPM6 in embryogenesis and less on the role of TRPM6 and/or TRPM7 in Mg 2+ homeostasis. Therefore, the aim of our study was to generate and characterize TRPM6 +/– and TRPM6 –/– mice in order to examine the role of TRPM6 and TRPM7 in renal and intestinal Mg 2+ handling in more detail.