Inhibition of pannexin-1 does not restore electrolyte balance in precystic Pkd1 knockout mice.

Mutations in PKD1 and PKD2 cause autosomal dominant polycystic kidney disease (ADPKD), which is characterized by the formation of fluid-filled cysts in the kidney. In a subset of ADPKD patients, reduced blood calcium (Ca2+) and magnesium (Mg2+) concentrations are observed. As cystic fluid contains increased ATP concentrations and purinergic signaling reduces electrolyte reabsorption, we hypothesized that inhibiting ATP release could normalize blood Ca2+ and Mg2+ levels in ADPKD. Inducible kidney-specific Pkd1 knockout mice (iKsp-Pkd1-/-) exhibit hypocalcemia and hypomagnesemia in a precystic stage and show increased expression of the ATP-release channel pannexin-1. Therefore, we administered the pannexin-1 inhibitor brilliant blue-FCF (BB-FCF) every other day from Day 3 to 28 post-induction of Pkd1 gene inactivation. On Day 29, both serum Ca2+ and Mg2+ concentrations were reduced in iKsp-Pkd1-/- mice, while urinary Ca2+ and Mg2+ excretion was similar between the genotypes. However, serum and urinary levels of Ca2+ and Mg2+ were unaltered by BB-FCF treatment, regardless of genotype. BB-FCF did significantly decrease gene expression of the ion channels Trpm6 and Trpv5 in both control and iKsp-Pkd1-/- mice. Finally, no renoprotective effects of BB-FCF treatment were observed in iKsp-Pkd1-/- mice. Thus, administration of BB-FCF failed to normalize serum Ca2+ and Mg2+ levels.

Liver and spleen predominantly mediate calciprotein particle clearance in a rat model of chronic kidney disease.

Calciprotein particles (CPPs) provide an efficient mineral buffering system to prevent the complexation of phosphate and calcium in the circulation. However, in chronic kidney disease (CKD), the phosphate load exceeds the mineral buffering capacity, resulting in the formation of crystalline CPP2 particles. CPP2 have been associated with cardiovascular events and mortality. Moreover, CPP2 have been demonstrated to induce calcification in vitro. In this study, we examined the fate of CPP2 in a rat model of CKD. Calcification was induced in Sprague-Dawley rats by 5/6 nephrectomy (5/6-Nx) combined with a high-phosphate diet. Control rats received sham surgery and high-phosphate diet. Twelve weeks after surgery, kidney failure was significantly induced in 5/6-Nx rats as determined by enhanced creatinine and urea plasma levels and abnormal kidney histological architecture. Subsequently, radioactive and fluorescent (FITC)-labeled CPP2 ([89Zr]Zr-CPP2-FITC) were injected intravenously to determine clearance in vivo. Using positron emission tomography scans and radioactive biodistribution measurements, it was demonstrated that [89Zr]Zr-CPP2-FITC are mainly present in the liver and spleen in both 5/6-Nx and sham rats. Immunohistochemistry showed that [89Zr]Zr-CPP2-FITC are predominantly taken up by Kupffer cells and macrophages. However, [89Zr]Zr-CPP2-FITC could also be detected in hepatocytes. In the different parts of the aorta and in the blood, low values of [89Zr]Zr-CPP2-FITC were detectable, independent of the presence of calcification. CPP2 are cleared rapidly from the circulation by the liver and spleen in a rat model of CKD. In the liver, Kupffer cells, macrophages, and hepatocytes contribute to CPP2 clearance.NEW & NOTEWORTHY Calciprotein particles (CPPs) buffer calcium and phosphate in the blood to prevent formation of crystals. In CKD, increased phosphate levels may exceed the buffering capacity of CPPs, resulting in crystalline CPPs that induce calcification. This study demonstrates that labeled CPPs are predominantly cleared from the circulation in the liver by Kupffer cells, macrophages, and hepatocytes. Our results suggest that targeting liver CPP clearance may reduce the burden of crystalline CPP in the development of vascular calcification.

HNF1ß-associated cyst development and electrolyte disturbances are not explained by BAIAP2L2 expression.

Mutations or deletions in transcription factor hepatocyte nuclear factor 1 homeobox ß (HNF1ß) cause renal cysts and/or malformation, maturity-onset diabetes of the young and electrolyte disturbances. Here, we applied a comprehensive bioinformatic approach on ChIP-seq, RNA-seq, and gene expression array studies to identify novel transcriptional targets of HNF1ß explaining the kidney phenotype of HNF1ß patients. We identified BAR/IMD Domain Containing Adaptor Protein 2 Like 2 (BAIAP2L2), as a novel transcriptional target of HNF1ß and validated direct transcriptional activation of the BAIAP2L2 promoter by a reporter luciferase assay. Using mass spectrometry analysis, we show that BAIAP2L2 binds to other members of the I-BAR domain-containing family: BAIAP2 and BAIAP2L1. Subsequently, the role of BAIAP2L2 in maintaining epithelial cell integrity in the kidney was assessed using Baiap2l2 knockout cell and mouse models. Kidney epithelial cells lacking functional BAIAP2L2 displayed normal F-actin distribution at cell-cell contacts and formed polarized three-dimensional spheroids with a lumen. In vivo, Baiap2l2 knockout mice displayed normal kidney and colon tissue morphology and serum and urine electrolyte concentrations were not affected. Altogether, our study is the first to characterize the function of BAIAP2L2 in the kidney in vivo and we report that mice lacking BAIAP2L2 exhibit normal electrolyte homeostasis and tissue morphology under physiological conditions.

Possible role for rare TRPM7 variants in patients with hypomagnesaemia with secondary hypocalcaemia.

BACKGROUND: Hypomagnesaemia with secondary hypocal-caemia (HSH) is a rare autosomal recessive disorder caused by pathogenic variants in TRPM6, encoding the channel-kinase transient receptor potential melastatin type 6. Patients have very low serum magnesium (Mg2+) levels and suffer from muscle cramps and seizures. Despite genetic testing, a subgroup of HSH patients remains without a diagnosis. METHODS: In this study, two families with an HSH phenotype but negative for TRPM6 pathogenic variants were subjected to whole exome sequencing. Using a complementary combination of biochemical and functional analyses in overexpression systems and patient-derived fibroblasts, the effect of the TRPM7-identified variants on Mg2+ transport was examined. RESULTS: For the first time, variants in TRPM7 were identified in two families as a potential cause for hereditary HSH. Patients suffer from seizures and muscle cramps due to magnesium deficiency and episodes of hypocalcaemia. In the first family, a splice site variant caused the incorporation of intron 1 sequences into the TRPM7 messenger RNA and generated a premature stop codon. As a consequence, patient-derived fibroblasts exhibit decreased cell growth. In the second family, a heterozygous missense variant in the pore domain resulted in decreased TRPM7 channel activity. CONCLUSIONS: We establish TRPM7 as a prime candidate gene for autosomal dominant hypomagnesaemia and secondary hypocalcaemia. Screening of unresolved patients with hypocalcaemia and secondary hypocalcaemia may further establish TRPM7 pathogenic variants as a novel Mendelian disorder.

SLC41A1 knockout mice display normal magnesium homeostasis.

Transcellular Mg2+ reabsorption in the distal convoluted tubule (DCT) of the kidneys plays an important role in maintaining systemic Mg2+ homeostasis. SLC41A1 is a Na+/Mg2+ exchanger that mediates Mg2+ efflux from cells and is hypothesized to facilitate basolateral extrusion of Mg2+ in the DCT. In this study, we generated a SLC41A1 knockout mouse model to examine the role of SLC41A1 in Mg2+ homeostasis. Slc41a1-/- mice exhibited similar serum and urine Mg2+ levels as their wild-type littermates. Dietary restriction of Mg2+ resulted in reduced serum Mg2+ concentration and urinary Mg2+ excretion, which was similar in the wild-type and knockout groups. Expression of genes encoding Mg2+ channels and transporters such as transient receptor potential melastatin 6 (Trpm6), transient receptor potential melastatin 7 (Trpm7), cyclin and CBS domain divalent metal cation transport mediator 2 (Cnnm2), and Slc41a3 were unchanged based on genotype. We investigated the potential redundancy of SLC41A1 and its homolog SLC41A3 by generating a double knockout mouse. Although Slc41a3-/- knockout mice showed significantly reduced serum Mg2+ compared with wild-type and Slc41a1-/- knockout groups, double knockout mice displayed similar serum Mg2+ levels as Slc41a3-/- knockout mice. In conclusion, our data show that SLC41A1 is not involved in the regulation of systemic Mg2+ homeostasis in mice. Our data also demonstrate that SLC41A1 does not compensate for the loss of SLC41A3, suggesting different functions of these SLC41 proteins in vivo.NEW & NOTEWORTHY SLC41A1 has been hypothesized to mediate Mg2+ extrusion in the distal convoluted tubule and thus regulate Mg2+ homeostasis. This study investigated the role of SLC41A1 in Mg2+ homeostasis in vivo using a transgenic mouse model. Our results demonstrate that SLC41A1 is not required to maintain normal Mg2+ balance in mice. We also show that SLC41A3 is more important than SLC41A1 in regulating systemic Mg2+ levels.

FAM111A is dispensable for electrolyte homeostasis in mice.

Autosomal dominant mutations in FAM111A are causative for Kenny-Caffey syndrome type 2. Patients with Kenny-Caffey syndrome suffer from severe growth retardation, skeletal dysplasia, hypoparathyroidism, hypocalcaemia, hyperphosphataemia and hypomagnesaemia. While recent studies have reported FAM111A to function in antiviral response and DNA replication, its role in regulating electrolyte homeostasis remains unknown. In this study, we assessed the role of FAM111A in the regulation of serum electrolyte balance using a Fam111a knockout (Fam111a-/-) C57BL/6 N mouse model. Fam111a-/- mice displayed normal weight and serum parathyroid hormone (PTH) concentration and exhibited unaltered magnesium, calcium and phosphate levels in serum and 24-hour urine. Expression of calciotropic (including Cabp28k, Trpv5, Klotho and Cyp24a1), magnesiotropic (including Trpm6, Trpm7, Cnnm2 and Cnnm4) and phosphotropic (Slc20a1, Slc20a2, Slc34a1 and Slc34a3) genes in the kidneys, duodenum and colon were not affected by Fam111a depletion. Only Slc34a2 expression was significantly upregulated in the duodenum, but not in the colon. Analysis of femurs showed unaffected bone morphology and density in Fam111a-/- mice. Kidney and parathyroid histology were also normal in Fam111a-/- mice. In conclusion, our study is the first to characterise the function of FAM111A in vivo and we report that mice lacking FAM111A exhibit normal electrolyte homeostasis on a standard diet.

Dietary magnesium supplementation inhibits abdominal vascular calcification in an experimental animal model of chronic kidney disease.

BACKGROUND: Vascular calcification is a key process involved in cardiovascular morbidity and mortality in patients with chronic kidney disease (CKD). Magnesium supplementation may counteract vascular calcification. In this study we aimed to determine whether increased dietary magnesium intake inhibits vascular calcification in CKD in vivo and explore the mechanisms underlying these effects. METHODS: Sprague Dawley rats were partially nephrectomized and fed a diet with high phosphate and either high or normal magnesium content for 16 weeks. The primary outcome was the tissue calcium content of the aorta in the high versus normal dietary magnesium group. In addition, we analysed plasma mineral concentrations, aortic vascular calcification identified with von Kossa staining, calcium apposition time and aortic expression of genes related to vascular calcification. RESULTS: The number of animals in the highest tissue calcium content tertile was significantly lower in the abdominal aorta [1 (10%) versus 6 (55%); P = .03] in the high versus normal dietary magnesium group, but did not differ in the aortic arch and thoracic aorta. Von Kossa staining and calcium apposition time corresponded to these results. The median tissue calcium content was not significantly different between the groups. Serum phosphate concentrations and expression of osteogenic markers in the aorta did not differ between the groups. CONCLUSIONS: This study demonstrates that increased dietary magnesium inhibits abdominal vascular calcification in an experimental animal model of CKD in vivo. These are promising results for CKD patients and further study is needed to identify the mechanisms involved and to determine the clinical relevance in patients.

Colonic expression of calcium transporter TRPV6 is regulated by dietary sodium butyrate.

Dietary fibers have been shown to increase the intestinal absorption of calcium (Ca2+) and magnesium (Mg2+). However, the mechanisms that explain the enhanced electrolyte absorption remain unknown. Therefore, this study aims to investigate the short-term and long-term effects of 5% (w/w) sodium butyrate (Na-butyrate), an important end-metabolite of bacterial fermentation of dietary fibers, on Ca2+ and Mg2+ homeostasis in mice. Serum Ca2+ levels were only significantly increased in mice treated with Na-butyrate for 1 day. This was associated with a twofold increase in the mRNA expression levels of Trpv6 in the proximal and distal colon. Contrary, Na-butyrate did not affect serum Mg2+ concentrations at either of the intervention periods. However, we observed a reduction in urinary Mg2+ excretion, although not significantly, after 1 day of treatment. A significant reduction of 2.5-fold in urinary Mg2+ excretion was observed after 14 days of treatment. Indeed, 14-day Na-butyrate supplementation increased colonic Trpm7 expression by 1.2-fold compared to control mice. In conclusion, short-term Na-butyrate supplementation increases serum Ca2+ levels in mice. This was associated with increased mRNA expression levels of Trpv6 in the colon, suggesting that Na-butyrate regulates the expression of genes involved in active intestinal Ca2+ absorption.

mTOR-Activating Mutations in RRAGD Are Causative for Kidney Tubulopathy and Cardiomyopathy.

BACKGROUND: Over the last decade, advances in genetic techniques have resulted in the identification of rare hereditary disorders of renal magnesium and salt handling. Nevertheless, approximately 20% of all patients with tubulopathy lack a genetic diagnosis. METHODS: We performed whole-exome and -genome sequencing of a patient cohort with a novel, inherited, salt-losing tubulopathy; hypomagnesemia; and dilated cardiomyopathy. We also conducted subsequent in vitro functional analyses of identified variants of RRAGD, a gene that encodes a small Rag guanosine triphosphatase (GTPase). RESULTS: In eight children from unrelated families with a tubulopathy characterized by hypomagnesemia, hypokalemia, salt wasting, and nephrocalcinosis, we identified heterozygous missense variants in RRAGD that mostly occurred de novo. Six of these patients also had dilated cardiomyopathy and three underwent heart transplantation. We identified a heterozygous variant in RRAGD that segregated with the phenotype in eight members of a large family with similar kidney manifestations. The GTPase RagD, encoded by RRAGD, plays a role in mediating amino acid signaling to the mechanistic target of rapamycin complex 1 (mTORC1). RagD expression along the mammalian nephron included the thick ascending limb and the distal convoluted tubule. The identified RRAGD variants were shown to induce a constitutive activation of mTOR signaling in vitro. CONCLUSIONS: Our findings establish a novel disease, which we call autosomal dominant kidney hypomagnesemia (ADKH-RRAGD), that combines an electrolyte-losing tubulopathy and dilated cardiomyopathy. The condition is caused by variants in the RRAGD gene, which encodes Rag GTPase D; these variants lead to an activation of mTOR signaling, suggesting a critical role of Rag GTPase D for renal electrolyte handling and cardiac function.

Serum Magnesium Is Inversely Associated With Heart Failure, Atrial Fibrillation, and Microvascular Complications in Type 2 Diabetes.

OBJECTIVE: We investigated whether serum magnesium (Mg2+) was prospectively associated with macro- or microvascular complications and mediated by glycemic control (hemoglobin A1c [HbA1c]), in type 2 diabetes (T2D). RESEARCH DESIGN AND METHODS: We analyzed in 4,348 participants the association of serum Mg2+ with macrovascular disease and mortality (acute myocardial infarction [AMI], coronary heart disease [CHD], heart failure [HF], cerebrovascular accident [CVA], and peripheral arterial disease [PAD]), atrial fibrillation (AF), and microvascular complications (chronic kidney disease [CKD], diabetic retinopathy, and diabetic foot) using Cox regression, adjusted for confounders. Mediation analysis was performed to assess whether HbA1c mediated these associations. RESULTS: The average baseline serum Mg2+ concentration was 0.80 ± 0.08 mmol/L. During 6.1 years of follow-up, serum Mg2+ was inversely associated with major macrovascular, 0.87 (95% CI 0.76; 1.00); HF, 0.76 (95% CI 0.62; 0.93); and AF, 0.59 (95% CI 0.49; 0.72). Serum Mg2+ was not associated with AMI, CHD, CVA, and PAD. During 5.1 years of follow-up, serum Mg2+ was inversely associated with overall microvascular events, 0.85 (95% CI 0.78; 0.91); 0.89 (95% CI 0.82; 0.96) for CKD, 0.77 (95% CI 0.61; 0.98) for diabetic retinopathy, and 0.85 (95% CI 0.78; 0.92) for diabetic foot. HbA1c mediated the associations of serum Mg2+ with HF, overall microvascular events, diabetic retinopathy, and diabetic foot. CONCLUSIONS: Serum Mg2+ concentration is inversely associated with the risk to develop HF and AF and with the occurrence of CKD, diabetic retinopathy, and foot complications in T2D. Glycemic control partially mediated the association of serum Mg2+ with HF and microvascular complications.

Cyclin M2 (CNNM2) knockout mice show mild hypomagnesaemia and developmental defects.

Patients with mutations in Cyclin M2 (CNNM2) suffer from hypomagnesaemia, seizures, and intellectual disability. Although the molecular function of CNNM2 is under debate, the protein is considered essential for renal Mg2+ reabsorption. Here, we used a Cnnm2 knock out mouse model, generated by CRISPR/Cas9 technology, to assess the role of CNNM2 in Mg2+ homeostasis. Breeding Cnnm2+/- mice resulted in a Mendelian distribution at embryonic day 18. Nevertheless, only four Cnnm2-/- pups were born alive. The Cnnm2-/- pups had a significantly lower serum Mg2+ concentration compared to wildtype littermates. Subsequently, adult Cnnm2+/- mice were fed with low, control, or high Mg2+ diets for two weeks. Adult Cnnm2+/- mice showed mild hypomagnesaemia compared to Cnnm2+/+ mice and increased serum Ca2+ levels, independent of dietary Mg2+ intake. Faecal analysis displayed increased Mg2+ and Ca2+ excretion in the Cnnm2+/- mice. Transcriptional profiling of Trpm6, Trpm7, and Slc41a1 in kidneys and colon did not reveal effects based on genotype. Microcomputed tomography analysis of the femurs demonstrated equal bone morphology and density. In conclusion, CNNM2 is vital for embryonic development and Mg2+ homeostasis. Our data suggest a previously undescribed role of CNNM2 in the intestine, which may contribute to the Mg2+ deficiency in mice and patients.

Bifunctional protein PCBD2 operates as a co-factor for hepatocyte nuclear factor 1ß and modulates gene transcription.

Hepatocyte nuclear factor 1ß (HNF1ß) is an essential transcription factor in development of the kidney, liver, and pancreas. HNF1ß-mediated transcription of target genes is dependent on the cell type and the development stage. Nevertheless, the regulation of HNF1ß function by enhancers and co-factors that allow this cell-specific transcription is largely unknown. To map the HNF1ß interactome we performed mass spectrometry in a mouse kidney inner medullary collecting duct cell line. Pterin-4a-carbinolamine dehydratase 2 (PCBD2) was identified as a novel interaction partner of HNF1ß. PCBD2 and its close homolog PCBD1 shuttle between the cytoplasm and nucleus to exert their enzymatic and transcriptional activities. Although both PCBD proteins share high sequence identity (48% and 88% in HNF1 recognition helix), their tissue expression patterns are unique. PCBD1 is most abundant in kidney and liver while PCBD2 is also abundant in lung, spleen, and adipose tissue. Using immunolocalization studies and biochemical analysis we show that in presence of HNF1ß the nuclear localization of PCBD1 and PCBD2 increases significantly. Promoter luciferase assays demonstrate that co-factors PCBD1 and PCBD2 differentially regulate the ability of HNF1ß to activate the promoters of transcriptional targets important in renal electrolyte homeostasis. Deleting the N-terminal sequence of PCBD2, not found in PCBD1, diminished the differential effects of the co-factors on HNF1ß activity. All together these results indicate that PCBD1 and PCBD2 can exert different effects on HNF1ß-mediated transcription. Future studies should confirm whether these unique co-factor activities also apply to HNF1ß-target genes involved in additional processes besides ion transport in the kidney.

Dietary Mg2+ Intake and the Na+/Mg2+ Exchanger SLC41A1 Influence Components of Mitochondrial Energetics in Murine Cardiomyocytes.

Cardiomyocytes are among the most energy-intensive cell types. Interplay between the components of cellular magnesium (Mg) homeostasis and energy metabolism in cardiomyocytes is poorly understood. We have investigated the effects of dietary Mg content and presence/functionality of the Na+/Mg2+ exchanger SLC41A1 on enzymatic functions of selected constituents of the Krebs cycle and complexes of the electron transport chain (ETC). The activities of aconitate hydratase (ACON), isocitrate dehydrogenase (ICDH), α-ketoglutarate dehydrogenase (KGDH), and ETC complexes CI-CV have been determined in vitro in mitochondria isolated from hearts of wild-type (WT) and Slc41a1-/- mice fed a diet with either normal or low Mg content. Our data demonstrate that both, the type of Mg diet and the Slc41a1 genotype largely impact on the activities of enzymes of the Krebs cycle and ETC. Moreover, a compensatory effect of Slc41a1-/- genotype on the effect of low Mg diet on activities of the tested Krebs cycle enzymes has been identified. A machine-learning analysis identified activities of ICDH, CI, CIV, and CV as common predictors of the type of Mg diet and of CII as suitable predictor of Slc41a1 genotype. Thus, our data delineate the effect of dietary Mg content and of SLC41A1 functionality on the energy-production in cardiac mitochondria.

Magnesium prevents vascular calcification in Klotho deficiency.

Klotho knock-out mice are an important model for vascular calcification, which is associated with chronic kidney disease. In chronic kidney disease, serum magnesium inversely correlates with vascular calcification. Here we determine the effects of serum magnesium on aortic calcification in Klotho knock-out mice treated with a minimal or a high magnesium diet from birth. After eight weeks, serum biochemistry and aorta and bone tissues were studied. Protective effects of magnesium were characterized by RNA-sequencing of the aorta and micro-CT analysis was performed to study bone integrity. A high magnesium diet prevented vascular calcification and aortic gene expression of Runx2 and matrix Gla protein found in such mice on the minimal magnesium diet. Differential expression of inflammation and extracellular matrix remodeling genes accompanied the beneficial effects of magnesium on calcification. High dietary magnesium did not affect serum parathyroid hormone, 1,25-dihydroxyvitamin D3 or calcium. High magnesium intake prevented vascular calcification despite increased fibroblast growth factor-23 and phosphate concentration in the knock-out mice. Compared to mice on the minimal magnesium diet, the high magnesium diet reduced femoral bone mineral density by 20% and caused excessive osteoid formation indicating osteomalacia. Osteoclast activity was unaffected by the high magnesium diet. In Saos-2 osteoblasts, magnesium supplementation reduced mineralization independent of osteoblast function. Thus, high dietary magnesium prevents calcification in Klotho knock-out mice. These effects are potentially mediated by reduction of inflammatory and extracellular matrix remodeling pathways within the aorta. Hence magnesium treatment may be promising to prevent vascular calcification, but the risk for osteomalacia should be considered.

Low gut microbiota diversity and dietary magnesium intake are associated with the development of PPI-induced hypomagnesemia.

Proton pump inhibitors (PPIs) are used by millions of patients for the treatment of stomach acid-reflux diseases. Although PPIs are generally considered safe, about 13% of the users develop hypomagnesemia. Despite rising attention for this issue, the underlying mechanism is still unknown. Here, we examine whether the gut microbiome is involved in the development of PPI-induced hypomagnesemia in wild-type C57BL/6J mice. After 4 wk of treatment under normal or low dietary Mg2+ availability, omeprazole significantly reduced serum Mg2+ levels only in mice on a low-Mg2+ diet without affecting the mRNA expression of colonic or renal Mg2+ transporters. Overall, 16S rRNA gene sequencing revealed a lower gut microbial diversity in omeprazole-treated mice. Omeprazole induced a shift in microbial composition, which was associated with a 3- and 2-fold increase in the abundance of Lactobacillus and Bifidobacterium, respectively. To examine the metabolic consequences of these microbial alterations, the colonic composition of organic acids was evaluated. Low dietary Mg2+ intake, independent of omeprazole treatment, resulted in a 10-fold increase in formate levels. Together, these results imply that both omeprazole treatment and low dietary Mg2+ intake disturb the gut internal milieu and may pose a risk for the malabsorption of Mg2+ in the colon.-Gommers, L. M. M., Ederveen, T. H. A., van der Wijst, J., Overmars-Bos, C., Kortman, G. A. M., Boekhorst, J., Bindels, R. J. M., de Baaij, J. H. F., Hoenderop, J. G. J. Low gut microbiota diversity and dietary magnesium intake are associated with the development of PPI-induced hypomagnesemia.

Renal phospholipidosis and impaired magnesium handling in high-fat-diet-fed mice.

Hypomagnesemia (blood Mg2+ concentration <0.7 mM) is a common electrolyte disorder in patients with type 2 diabetes (T2D), but the etiology remains largely unknown. In patients with T2D, reduced blood Mg2+ levels are associated with an increased decline in renal function, independent of glycemic control and hypertension. To study the underlying mechanism of this phenomenon, we investigated the renal effects of hypomagnesemia in high-fat-diet (HFD)-fed mice. In mice fed a low dietary Mg2+, the HFD resulted in severe hypomagnesemia within 4 wk. Renal or intestinal Mg2+ wasting was not observed after 16 wk on the diets. Despite the absence of urinary or fecal Mg2+ loss, the HFD induced a reduction in the mRNA expression transient receptor potential melastatin type 6 in both the kidney and colon. mRNA expression of distal convoluted tubule (DCT)-specific genes was down-regulated by the LowMg-HFD, indicating atrophy of the DCT. The low dietary Mg2+ resulted in severe HFD-induced proximal tubule phospholipidosis, which was absent in mice on a NormalMg-HFD. This was accompanied by albuminuria, moderate renal damage, and alterations in renal energy metabolism, including enhanced gluconeogenesis and cholesterol synthesis. In conclusion, this study shows that hypomagnesemia is a consequence of diet-induced obesity and insulin resistance. Moreover, hypomagnesemia induces major structural changes in the diabetic kidney, including proximal tubular phospholipidosis, providing a novel mechanism for the increased renal decline in patients with hypomagnesemic T2D.-Kurstjens, S., Smeets, B., Overmars-Bos, C., Dijkman, H. B., den Braanker, D. J. W., de Bel, T., Bindels, R. J. M., Tack, C. J. J., Hoenderop, J. G. J., de Baaij, J. H. F. Renal phospholipidosis and impaired magnesium handling in high-fat-diet-fed mice.

Diabetes-induced hypomagnesemia is not modulated by metformin treatment in mice.

Approximately 30% of patients with type 2 diabetes mellitus (T2D) have hypomagnesemia (blood magnesium (Mg2+) concentration <0.7 mmol/L). In T2D patients, treatment with metformin is associated with reduced blood Mg2+ levels. To investigate how T2D and metformin affect Mg2+ homeostasis db/m and db/db mice were treated with metformin or placebo. Mice were housed in metabolic cages to measure food and water intake, and to collect urine and feces. Serum and urinary Mg2+ concentrations were determined and mRNA expression of magnesiotropic genes was determined in kidney and distal colon using RT-qPCR. Db/db mice had significantly lower serum Mg2+ levels than db/m mice. Mild hypermagnesuria was observed in the db/db mice at two weeks, but not at four weeks. Metformin-treatment had no effect on the serum Mg2+ concentration and on the urinary Mg2+ excretion. Both in kidney and distal colon of db/db mice, there was a compensatory upregulation in the mRNA expression of magnesiotropic genes, such as transient receptor potential melastatin 6 (Trpm6), whereas metformin treatment did not affect gene expression levels. In conclusion, we show that T2D causes hypomagnesemia and that metformin treatment has no effect on Mg2+ homeostasis in mice.

Increased NEFA levels reduce blood Mg2+ in hypertriacylglycerolaemic states via direct binding of NEFA to Mg2.

AIMS/HYPOTHESIS: The blood triacylglycerol level is one of the main determinants of blood Mg2+ concentration in individuals with type 2 diabetes. Hypomagnesaemia (blood Mg2+ concentration <0.7 mmol/l) has serious consequences as it increases the risk of developing type 2 diabetes and accelerates progression of the disease. This study aimed to determine the mechanism by which triacylglycerol levels affect blood Mg2+ concentrations. METHODS: Using samples from 285 overweight individuals (BMI >27 kg/m2) who participated in the 300-Obesity study (an observational cross-sectional cohort study, as part of the Human Functional Genetics Projects), we investigated the association between serum Mg2+ with laboratory variables, including an extensive lipid profile. In a separate set of studies, hyperlipidaemia was induced in mice and in healthy humans via an oral lipid load, and blood Mg2+, triacylglycerol and NEFA concentrations were measured using colourimetric assays. In vitro, NEFAs harvested from albumin were added in increasing concentrations to several Mg2+-containing solutions to study the direct interaction between Mg2+ and NEFAs. RESULTS: In the cohort of overweight individuals, serum Mg2+ levels were inversely correlated with triacylglycerols incorporated in large VLDL particles (r = -0.159, p ≤ 0.01). After lipid loading, we observed a postprandial increase in plasma triacylglycerol and NEFA levels and a reciprocal reduction in blood Mg2+ concentration both in mice (Δ plasma Mg2+ -0.31 mmol/l at 4 h post oral gavage) and in healthy humans (Δ plasma Mg2+ -0.07 mmol/l at 6 h post lipid intake). Further, in vitro experiments revealed that the decrease in plasma Mg2+ may be explained by direct binding of Mg2+ to NEFAs. Moreover, Mg2+ was found to bind to albumin in a NEFA-dependent manner, evidenced by the fact that Mg2+ did not bind to fatty-acid-free albumin. The NEFA-dependent reduction in the free Mg2+ concentration was not affected by the presence of physiological concentrations of other cations. CONCLUSIONS/INTERPRETATION: This study shows that elevated NEFA and triacylglycerol levels directly reduce blood Mg2+ levels, in part explaining the high prevalence of hypomagnesaemia in metabolic disorders. We show that blood NEFA level affects the free Mg2+ concentration, and therefore, our data challenge how the fractional excretion of Mg2+ is calculated and interpreted in the clinic.

Magnesium deficiency prevents high-fat-diet-induced obesity in mice.

AIMS/HYPOTHESIS: Hypomagnesaemia (blood Mg2+ <0.7 mmol/l) is a common phenomenon in individuals with type 2 diabetes. However, it remains unknown how a low blood Mg2+ concentration affects lipid and energy metabolism. Therefore, the importance of Mg2+ in obesity and type 2 diabetes has been largely neglected to date. This study aims to determine the effects of hypomagnesaemia on energy homeostasis and lipid metabolism. METHODS: Mice (n = 12/group) were fed either a low-fat diet (LFD) or a high-fat diet (HFD) (10% or 60% of total energy) in combination with a normal- or low-Mg2+ content (0.21% or 0.03% wt/wt) for 17 weeks. Metabolic cages were used to investigate food intake, energy expenditure and respiration. Blood and tissues were taken to study metabolic parameters and mRNA expression profiles, respectively. RESULTS: We show that low dietary Mg2+ intake ameliorates HFD-induced obesity in mice (47.00 ± 1.53 g vs 38.62 ± 1.51 g in mice given a normal Mg2+-HFD and low Mg2+-HFD, respectively, p < 0.05). Consequently, fasting serum glucose levels decreased and insulin sensitivity improved in low Mg2+-HFD-fed mice. Moreover, HFD-induced liver steatosis was absent in the low Mg2+ group. In hypomagnesaemic HFD-fed mice, mRNA expression of key lipolysis genes was increased in epididymal white adipose tissue (eWAT), corresponding to reduced lipid storage and high blood lipid levels. Low Mg2+-HFD-fed mice had increased brown adipose tissue (BAT) Ucp1 mRNA expression and a higher body temperature. No difference was observed in energy expenditure between the two HFD groups. CONCLUSIONS/INTERPRETATION: Mg2+-deficiency abrogates HFD-induced obesity in mice through enhanced eWAT lipolysis and BAT activity.

Polycystin-1 dysfunction impairs electrolyte and water handling in a renal precystic mouse model for ADPKD.

The PKD1 gene encodes polycystin-1 (PC1), a mechanosensor triggering intracellular responses upon urinary flow sensing in kidney tubular cells. Mutations in PKD1 lead to autosomal dominant polycystic kidney disease (ADPKD). The involvement of PC1 in renal electrolyte handling remains unknown since renal electrolyte physiology in ADPKD patients has only been characterized in cystic ADPKD. We thus studied the renal electrolyte handling in inducible kidney-specific Pkd1 knockout (iKsp- Pkd1-/-) mice manifesting a precystic phenotype. Serum and urinary electrolyte determinations indicated that iKsp- Pkd1-/- mice display reduced serum levels of magnesium (Mg2+), calcium (Ca2+), sodium (Na+), and phosphate (Pi) compared with control ( Pkd1+/+) mice and renal Mg2+, Ca2+, and Pi wasting. In agreement with these electrolyte disturbances, downregulation of key genes for electrolyte reabsorption in the thick ascending limb of Henle's loop (TA;, Cldn16, Kcnj1, and Slc12a1), distal convoluted tubule (DCT; Trpm6 and Slc12a3) and connecting tubule (CNT; Calb1, Slc8a1, and Atp2b4) was observed in kidneys of iKsp- Pkd1-/- mice compared with controls. Similarly, decreased renal gene expression of markers for TAL ( Umod) and DCT ( Pvalb) was observed in iKsp- Pkd1-/- mice. Conversely, mRNA expression levels in kidney of genes encoding solute and water transporters in the proximal tubule ( Abcg2 and Slc34a1) and collecting duct ( Aqp2, Scnn1a, and Scnn1b) remained comparable between control and iKsp- Pkd1-/- mice, although a water reabsorption defect was observed in iKsp- Pkd1-/- mice. In conclusion, our data indicate that PC1 is involved in renal Mg2+, Ca2+, and water handling and its dysfunction, resulting in a systemic electrolyte imbalance characterized by low serum electrolyte concentrations.