Klotho is highly expressed in the chief sites of regulated potassium secretion, and it is stimulated by potassium intake.

Klotho regulates many pathways in the aging process, but it remains unclear how it is physiologically regulated. Because Klotho is synthesized, cleaved, and released from the kidney; activates the chief urinary K+ secretion channel (ROMK) and stimulates urinary K+ secretion, we explored if Klotho protein is regulated by dietary K+ and the potassium-regulatory hormone, Aldosterone. Klotho protein along the nephron was evaluated in humans and in wild-type (WT) mice; and in mice lacking components of Aldosterone signaling, including the Aldosterone-Synthase KO (AS-KO) and the Mineralocorticoid-Receptor KO (MR-KO) mice. We found the specific cells of the distal nephron in humans and mice that are chief sites of regulated K+ secretion have the highest Klotho protein expression along the nephron. WT mice fed K+-rich diets increased Klotho expression in these cells. AS-KO mice exhibit normal Klotho under basal conditions but could not upregulate Klotho in response to high-K+ intake in the K+-secreting cells. Similarly, MR-KO mice exhibit decreased Klotho protein expression. Together, i) Klotho is highly expressed in the key sites of regulated K+ secretion in humans and mice, ii) In mice, K+-rich diets increase Klotho expression specifically in the potassium secretory cells of the distal nephron, iii) Aldosterone signaling is required for Klotho response to high K+ intake.

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.

Hypomagnesaemia with varying degrees of extrarenal symptoms as a consequence of heterozygous CNNM2 variants.

Variants in the CNNM2 gene are causative for hypomagnesaemia, seizures and intellectual disability, although the phenotypes can be variable. This study aims to understand the genotype-phenotype relationship in affected individuals with CNNM2 variants by phenotypic, functional and structural analysis of new as well as previously reported variants. This results in the identification of seven variants that significantly affect CNNM2-mediated Mg2+ transport. Pathogenicity of these variants is further supported by structural modelling, which predicts CNNM2 structure to be affected by all of them. Strikingly, seizures and intellectual disability are absent in 4 out of 7 cases, indicating these phenotypes are caused either by specific CNNM2 variant only or by additional risk factors. Moreover, in line with sporadic observations from previous reports, CNNM2 variants might be associated with disturbances in parathyroid hormone and Ca2+ homeostasis.

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.

Genome-wide association study of serum magnesium in type 2 diabetes.

People with type 2 diabetes have a tenfold higher prevalence of hypomagnesemia, which is suggested to be caused by low dietary magnesium intake, medication use, and genetics. This study aims to identify the genetic loci that influence serum magnesium concentration in 3466 people with type 2 diabetes. The GWAS models were adjusted for age, sex, eGFR, and HbA1c. Associated traits were identified using publicly available data from GTEx consortium, a human kidney eQTL atlas, and the Open GWAS database. The GWAS identified a genome-wide significant locus in TAF3 (p = 2.9 × 10-9) in people with type 2 diabetes. In skeletal muscle, loci located in TAF3 demonstrate an eQTL link to ATP5F1C, a gene that is involved in the formation of Mg2+-ATP. Serum Mg2+ levels were associated with MUC1/TRIM46 (p = 2.9 × 10-7), SHROOM3 (p = 4.0 × 10-7), and SLC22A7 (p = 1.0 × 10-6) at nominal significance, which is in combination with the eQTL data suggesting that they are possible candidates for renal failure. Several genetic loci were in agreement with previous genomic studies which identified MUC1/TRIM46 (Pmeta = 6.9 × 10-29, PQ = 0.81) and SHROOM3 (Pmeta = 2.9 × 10-27, PQ = 0.04) to be associated with serum Mg2+ in the general population. In conclusion, serum magnesium concentrations are associated with genetic variability around the regions of TAF3, MUC1/TRIM46, SHROOM3, and SLC22A7 in type 2 diabetes.

Urinary biomarkers in kidney disease.

BACKGROUND: Chronic kidney disease (CKD) affects many people worldwide and early diagnosis is essential for successful treatment and improved outcome. Unfortunately, current methods are insufficient especially for early disease detection. However, advances in the analytical methods for urinary biomarkers may provide a unique opportunity for diagnosis and management of CKD. This review explores evolving technology and highlights the importance of early marker detection in these patients. APPROACH: A search strategy was set up using the terms CKD, biomarkers, and urine. The search included 53 studies comprising 37 biomarkers. The value of these biomarkers for CKD are based on their ability to diagnose CKD, monitor progression, assess mortality and nephrotoxicity. RESULTS: KIM-1 was the best marker for diagnosis as it increased with the development of incident CKD. DKK3 increased in patients with declining eGFR, whereas UMOD decreased in those with declining kidney function. Unfortunately, none fulfilled all criteria to adequately assess mortality and nephrotoxicity. CONCLUSION: New developments in the field of urinalysis using smart toilets may open several possibilities for urinary biomarkers. This review explored which biomarkers could be used for CKD disease detection and management.

Mitochondrial Dysfunction in Kidney Tubulopathies.

Mitochondria play a key role in kidney physiology and pathology. They produce ATP to fuel energy-demanding water and solute reabsorption processes along the nephron. Moreover, mitochondria contribute to cellular health by the regulation of autophagy, (oxidative) stress responses, and apoptosis. Mitochondrial abundance is particularly high in cortical segments, including proximal and distal convoluted tubules. Dysfunction of the mitochondria has been described for tubulopathies such as Fanconi, Gitelman, and Bartter-like syndromes and renal tubular acidosis. In addition, mitochondrial cytopathies often affect renal (tubular) tissues, such as in Kearns-Sayre and Leigh syndromes. Nevertheless, the mechanisms by which mitochondrial dysfunction results in renal tubular diseases are only scarcely being explored. This review provides an overview of mitochondrial dysfunction in the development and progression of kidney tubulopathies. Furthermore, it emphasizes the need for further mechanistic investigations to identify links between mitochondrial function and renal electrolyte reabsorption.

Tubuloid differentiation to model the human distal nephron and collecting duct in health and disease.

Organoid technology is rapidly gaining ground for studies on organ (patho)physiology. Tubuloids are long-term expanding organoids grown from adult kidney tissue or urine. The progenitor state of expanding tubuloids comes at the expense of differentiation. Here, we differentiate tubuloids to model the distal nephron and collecting ducts, essential functional parts of the kidney. Differentiation suppresses progenitor traits and upregulates genes required for function. A single-cell atlas reveals that differentiation predominantly generates thick ascending limb and principal cells. Differentiated human tubuloids express luminal NKCC2 and ENaC capable of diuretic-inhibitable electrolyte uptake and enable disease modeling as demonstrated by a lithium-induced tubulopathy model. Lithium causes hallmark AQP2 loss, induces proliferation, and upregulates inflammatory mediators, as seen in vivo. Lithium also suppresses electrolyte transport in multiple segments. In conclusion, this tubuloid model enables modeling of the human distal nephron and collecting duct in health and disease and provides opportunities to develop improved therapies.

Fluid shear stress stimulates ATP release without regulating purinergic gene expression in the renal inner medullary collecting duct.

In the kidney, the flow rate of the pro-urine through the renal tubules is highly variable. The tubular epithelial cells sense these variations in pro-urinary flow rate in order to regulate various physiological processes, including electrolyte reabsorption. One of the mechanosensitive pathways activated by flow is the release of ATP, which can then act as a autocrine or paracrine factor. Increased ATP release is observed in various kidney diseases, among others autosomal dominant polycystic kidney disease (ADPKD). However, the mechanisms underlying flow-induced ATP release in the collecting duct, especially in the inner medullary collecting duct, remain understudied. Using inner medullary collecting duct 3 (IMCD3) cells in a microfluidic setup, we show here that administration of a high flow rate for 1 min results in an increased ATP release compared to a lower flow rate. Although the ATP release channel pannexin-1 contributed to flow-induced ATP release in Pkd1-/- IMCD3 cells, it did not in wildtype IMCD3 cells. In addition, flow application increased the expression of the putative ATP release channel connexin-30.3 (CX30.3) in wildtype and Pkd1-/- IMCD3 cells. However, CX30.3 knockout IMCD3 cells exhibited a similar flow-induced ATP release as wildtype IMCD3 cells, suggesting that CX30.3 does not drive flow-induced ATP release in wildtype IMDC3 cells. Collectively, our results show differential mechanisms underlying flow-induced ATP release in wildtype and Pkd1-/- IMCD3 cells and further strengthen the link between ADPKD and pannexin-1-dependent ATP release.

Extracellular vesicles contribute to early cyst development in autosomal dominant polycystic kidney disease by cell-to-cell communication.

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the formation of fluid-filled cysts within the kidney due to mutations in PKD1 or PKD2. Although the disease remains incompletely understood, one of the factors associated with ADPKD progression is the release of nucleotides (including ATP), which can initiate autocrine or paracrine purinergic signaling by binding to their receptors. Recently, we and others have shown that increased extracellular vesicle (EVs) release from PKD1 knockout cells can stimulate cyst growth through effects on recipient cells. Given that EVs are an important communicator between different nephron segments, we hypothesize that EVs released from PKD1 knockout distal convoluted tubule (DCT) cells can stimulate cyst growth in the downstream collecting duct (CD). Here, we show that administration of EVs derived from Pkd1-/- mouse distal convoluted tubule (mDCT15) cells result in a significant increase in extracellular ATP release from Pkd1-/- mouse inner medullary collecting duct (iMCD3) cells. In addition, exposure of Pkd1-/- iMCD3 cells to EVs derived from Pkd1-/- mDCT15 cells led to an increase in the phosphorylation of the serine/threonine-specific protein Akt, suggesting activation of proliferative pathways. Finally, the exposure of iMCD3 Pkd1-/- cells to mDCT15 Pkd1-/- EVs increased cyst size in Matrigel. These findings indicate that EVs could be involved in intersegmental communication between the distal convoluted tubule and the collecting duct and potentially stimulate cyst growth.

Expanding the Phenotypic Spectrum of Kenny-Caffey Syndrome.

CONTEXT: Kenny-Caffey syndrome (KCS) is a rare hereditary disorder characterized by short stature, hypoparathyroidism, and electrolyte disturbances. KCS1 and KCS2 are caused by pathogenic variants in TBCE and FAM111A, respectively. Clinically the phenotypes are difficult to distinguish. OBJECTIVE: The objective was to determine and expand the phenotypic spectrum of KCS1 and KCS2 in order to anticipate complications that may arise in these disorders. METHODS: We clinically and genetically analyzed 10 KCS2 patients from 7 families. Because we found unusual phenotypes in our cohort, we performed a systematic review of genetically confirmed KCS cases using PubMed and Scopus. Evaluation by 3 researchers led to the inclusion of 26 papers for KCS1 and 16 for KCS2, totaling 205 patients. Data were extracted following the Cochrane guidelines and assessed by 2 independent researchers. RESULTS: Several patients in our KCS2 cohort presented with intellectual disability (3/10) and chronic kidney disease (6/10), which are not considered common findings in KCS2. Systematic review of all reported KCS cases showed that the phenotypes of KCS1 and KCS2 overlap for postnatal growth retardation (KCS1: 52/52, KCS2: 23/23), low parathyroid hormone levels (121/121, 16/20), electrolyte disturbances (139/139, 24/27), dental abnormalities (47/50, 15/16), ocular abnormalities (57/60, 22/23), and seizures/spasms (103/115, 13/16). Symptoms more prevalent in KCS1 included intellectual disability (74/80, 5/24), whereas in KCS2 bone cortical thickening (1/18, 16/20) and medullary stenosis (7/46, 27/28) were more common. CONCLUSION: Our case series established chronic kidney disease as a new feature of KCS2. In the literature, we found substantial overlap in the phenotypic spectra of KCS1 and KCS2, but identified intellectual disability and the abnormal bone phenotype as the most distinguishing features.

Withdrawn as duplicate: Expanding the phenotypic spectrum of Kenny-Caffey syndrome: a case series and systematic literature review.

This article has been withdrawn due to a publisher error that caused it to be duplicated. The definitive version of this article is published under https://doi.org/10.1210/clinem/dgad147.

Tubuloid culture enables long-term expansion of functional human kidney tubule epithelium from iPSC-derived organoids.

Kidney organoids generated from induced pluripotent stem cells (iPSC) have proven valuable for studies of kidney development, disease, and therapeutic screening. However, specific applications have been hampered by limited expansion capacity, immaturity, off-target cells, and inability to access the apical side. Here, we apply recently developed tubuloid protocols to purify and propagate kidney epithelium from d7+18 (post nephrogenesis) iPSC-derived organoids. The resulting 'iPSC organoid-derived (iPSCod)' tubuloids can be exponentially expanded for at least 2.5 mo, while retaining expression of important tubular transporters and segment-specific markers. This approach allows for selective propagation of the mature tubular epithelium, as immature cells, stroma, and undesirable off-target cells rapidly disappeared. iPSCod tubuloids provide easy apical access, which enabled functional evaluation and demonstration of essential secretion and electrolyte reabsorption processes. In conclusion, iPSCod tubuloids provide a different, complementary human kidney model that unlocks opportunities for functional characterization, disease modeling, and regenerative nephrology.

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.

Transcription factor HNF1ß controls a transcriptional network regulating kidney cell structure and tight junction integrity.

Mutations in the hepatocyte nuclear factor (HNF)1ß gene (HNF1B) cause autosomal dominant tubulointerstitial kidney disease, a rare and heterogeneous disease characterized by renal cysts and/or malformation, maturity-onset diabetes of the young, hypomagnesemia, and hypokalemia. The electrolyte disturbances may develop in the distal part of the nephron, which is important for fine-tuning of Mg2+ and Ca2+ reabsorption. Therefore, we aimed to study the transcriptional network directed by HNF1ß in the distal part of the nephron. We combined HNF1ß chromatin immunoprecipitation-sequencing and mRNA expression data to identify direct targets of HNF1ß in a renal distal convoluted tubule cell line (mpkDCT). Gene Ontology term pathway analysis demonstrated enrichment of cell polarity, cell-cell junction, and cytoskeleton pathways in the dataset. Genes directly and indirectly regulated by HNF1ß within these pathways included members of the apical and basolateral polarity complexes including Crumbs protein homolog 3 (Crb3), partitioning defective 6 homolog-ß (Pard6b), and LLGL Scribble cell polarity complex component 2 (Llgl2). In monolayers of mouse inner medullary collecting duct 3 cells expressing dominant negative Hnf1b, tight junction integrity was compromised, as observed by reduced transepithelial electrical resistance values and increased permeability for fluorescein (0.4 kDa) compared with wild-type cells. Expression of dominant negative Hnf1b also led to a decrease in height (30%) and an increase in surface (58.5%) of cells grown on membranes. Moreover, three-dimensional spheroids formed by cells expressing dominant negative Hnf1b were reduced in size compared with wild-type spheroids (30%). Together, these findings demonstrate that HNF1ß directs a transcriptional network regulating tight junction integrity and cell structure in the distal part of the nephron.NEW & NOTEWORTHY Genetic defects in transcription factor hepatocyte nuclear factor (HNF)1ß cause a heterogeneous disease characterized by electrolyte disturbances, kidney cysts, and diabetes. By combining RNA-sequencing and HNF1ß chromatin immunoprecipitation-sequencing data, we identified new HNF1ß targets that were enriched for cell polarity pathways. Newly discovered targets included members of polarity complexes Crb3, Pard6b, and Llgl2. Functional assays in kidney epithelial cells demonstrated decreased tight junction integrity and a loss of typical cuboidal morphology in mutant Hnf1b cells.

Functional basis for calmodulation of the TRPV5 calcium channel.

Within the transient receptor potential (TRP) superfamily of ion channels, TRPV5 is a highly Ca2+ -selective channel important for active reabsorption of Ca2+ in the kidney. Its channel activity is controlled by a negative feedback mechanism involving calmodulin (CaM) binding. Combining advanced microscopy techniques and biochemical assays, this study characterized the dynamic lobe-specific CaM regulation. We demonstrate for the first time that functional (full-length) TRPV5 interacts with CaM in the absence of Ca2+ , and this interaction is intensified at increasing Ca2+ concentrations sensed by the CaM C-lobe that achieves channel pore blocking. Channel inactivation occurs without requiring CaM N-lobe calcification. Moreover, we show a Ca2+ -dependent binding stoichiometry at the single channel level. In conclusion, our study proposes a new model for CaM-dependent regulation - calmodulation - of this uniquely Ca2+ -selective TRP channel TRPV5 that involves apoCaM interaction and lobe-specific actions, which may be of significant physiological relevance given its role as gatekeeper of Ca2+ transport in the kidney. KEY POINTS: The renal Ca2+ channel TRPV5 is an important player in maintenance of the body's Ca2+ homeostasis. Activity of TRPV5 is controlled by a negative feedback loop that involves calmodulin (CaM), a protein with two Ca2+ -binding lobes. We investigated the dynamics of the interaction between TRPV5 and CaM with advanced fluorescence microscopy techniques. Our data support a new model for CaM-dependent regulation of TRPV5 channel activity with CaM lobe-specific actions and demonstrates Ca2+ -dependent binding stoichiometries. This study improves our understanding of the mechanism underlying fast channel inactivation, which is physiologically relevant given the gatekeeper function of TRPV5 in Ca2+ reabsorption in the kidney.

Calciprotein Particle Synthesis Strategy Determines In Vitro Calcification Potential.

Circulating calciprotein particles (CPP), colloids of calcium, phosphate and proteins, were identified as potential drivers of the calcification process in chronic kidney disease. The present study compared CPP produced using different protocols with respect to particle morphology, composition, particle number and in vitro calcification potency. CPP were synthesized with 4.4 mM (CPP-A and B) or 6 mM (CPP-C and D) phosphate and 2.8 mM (CPP-A and B) or 10 mM (CPP-C and D) calcium, with either bovine fetuin-A (CPP-C) or fetal bovine serum (CPP-A, B and D) as a source of protein, and incubated for 7 (CPP-A2) or 14 days (CPP-B2), 12 h (CPP-C2, D2 and B1) or 30 min (CPP-D1). Particle number was determined with nanoparticle tracking and calcium content was measured in CPP preparations and to determine human vascular smooth muscle cell (hVSMC) calcification. Morphologically, CPP-C2 were the largest. Particle number did not correspond to the calcium content of CPP. Both methods of quantification resulted in variable potencies of CPP2 to calcify VSMC, with CPP-B2 as most stable inducer of hVSMC calcification. In contrast, CPP-B1 and D1 were unable to induce calcification of hVSMC, and endogenous CPP derived from pooled serum of dialysis patients were only able to calcify hVSMC to a small extent compared to CPP2.CPP synthesized using different protocols appear morphologically similar, but in vitro calcification potency is dependent on composition and how the CPP are quantified. Synthetic CPP are not comparable to endogenous CPP in terms of the calcification propensity.

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.

Magnesium in chronic haemodialysis (MAGIC-HD): a study protocol for a randomised controlled trial to determine feasibility and safety of using increased dialysate magnesium concentrations to increase plasma magnesium concentrations in people treated with haemodialysis.

INTRODUCTION: People treated with haemodialysis are at increased risk for all-cause and cardiovascular mortality. Plasma magnesium concentration has been inversely associated with these risks. Therefore, plasma magnesium may be a new modifiable risk factor and an increase of dialysate magnesium concentration may be an easy, safe and effective way to increase plasma magnesium concentrations. Detailed information on modulating dialysate magnesium concentrations is limited in literature. Primary objective of this study is to determine the safety and feasibility to increase plasma magnesium concentrations in people treated with haemodialysis by means of sequentially increasing concentration of magnesium in the dialysate. METHODS AND ANALYSIS: In this randomised double-blinded standard of care controlled trial, 53 persons treated with haemodialysis will be randomly allocated 2:1 to either a stepwise individually titrated increase of dialysate magnesium concentration from 0.50 to 0.75 to 1.00 mmol/L during 8 weeks, or a standard dialysate magnesium concentration of 0.50 mmol/L. Other study measurements include dietary records, questionnaires, ECG, Holter registration and pulse wave velocity. The primary endpoint is predialysis plasma magnesium after the long interdialytic interval at the end of week 8. In addition, the predictive effect of dialysate magnesium concentration and other baseline parameters and dialysis characteristics on plasma magnesium concentration will be explored using linear mixed models. Safety endpoint is defined by the occurrence of hypermagnesemia above 1.25 mmol/L, or bradycardia or prolonged QTc interval detected on the ECG. ETHICS AND DISSEMINATION: The study is conducted in accordance with the declaration of Helsinki as revised in 2013 and was approved by the Ethical Committee of the VU University Medical Centre. The results of the study will be disseminated by publication in peer-reviewed scientific journals and presentation at national or international conferences in the field of interest. TRIAL REGISTRATION NUMBER: NTR6568/NL6393.

Butyrate reduces cellular magnesium absorption independently of metabolic regulation in Caco-2 human colon cells.

Digestion of dietary fibers by gut bacteria has been shown to stimulate intestinal mineral absorption [e.g., calcium (Ca2+) and magnesium (Mg2+)]. Although it has been suggested that local pH and short-chain fatty acid (SCFA) concentrations determine divalent cation absorption, the exact molecular mechanisms are still unknown. Therefore, this study aimed to determine the effects of SCFAs on intestinal Mg2+ absorption. We show that the butyrate concentration in the colon negatively correlates with serum Mg2+ levels in wildtype mice. Moreover, Na-butyrate significantly inhibited Mg2+ uptake in Caco-2 cells, while Ca2+ uptake was unaffected. Although Na-butyrate significantly lowered total ATP production rate, and resulted in increased phosphorylation of AMP-activated protein kinase (AMPK), inhibition of Mg2+ uptake by butyrate preceded these consequences. Importantly, electrophysiological examinations demonstrated that intracellular butyrate directly reduced the activity of the heteromeric Mg2+ channel complex, transient receptor potential melastatin (TRPM)6/7. Blocking cellular butyrate uptake prevented its inhibitory effect on Mg2+ uptake, demonstrating that butyrate acts intracellularly. Our work identified butyrate as novel regulator of intestinal Mg2+ uptake that works independently from metabolic regulation. This finding further highlights the role of microbial fermentation in the regulation of mineral absorption.