Quantitative phenotyping of Nphs1 knockout mice as a prerequisite for gene replacement studies.

Steroid-resistant nephrotic syndrome (SRNS) is the second most frequent cause of chronic kidney disease before the age of 25 yr. Nephrin, encoded by NPHS1, localizes to the slit diaphragm of glomerular podocytes and is the predominant structural component of the glomerular filtration barrier. Biallelic variants in NPHS1 can cause congenital nephrotic syndrome of the Finnish type, for which, to date, no causative therapy is available. Recently, adeno-associated virus (AAV) vectors targeting the glomerular podocyte have been assessed as a means for gene replacement therapy. Here, we established quantitative and reproducible phenotyping of a published, conditional Nphs1 knockout mouse model (Nphs1tm1.1Pgarg/J and Nphs2-Cre+) in preparation for a gene replacement study using AAV vectors. Nphs1 knockout mice (Nphs1fl/fl Nphs2-Cre+) exhibited 1) a median survival rate of 18 days (range: from 9 to 43 days; males: 16.5 days and females: 20 days); 2) an average foot process (FP) density of 1.0 FP/µm compared with 2.0 FP/µm in controls and a mean filtration slit density of 2.64 µm/µm2 compared with 4.36 µm/µm2 in controls; 3) a high number of proximal tubular microcysts; 4) the development of proteinuria within the first week of life as evidenced by urine albumin-to-creatinine ratios; and 5) significantly reduced levels of serum albumin and elevated blood urea nitrogen and creatinine levels. For none of these phenotypes, significant differences between sexes in Nphs1 knockout mice were observed. We quantitatively characterized five different phenotypic features of congenital nephrotic syndrome in Nphs1fl/fl Nphs2-Cre+ mice. Our results will facilitate future gene replacement therapy projects by allowing for sensitive detection of even subtle molecular effects.NEW & NOTEWORTHY To evaluate potential, even subtle molecular, therapeutic effects of gene replacement therapy (GRT) in a mouse model, prior rigorous quantifiable and reproducible disease phenotyping is necessary. Here, we, therefore, describe such a phenotyping effort in nephrin (Nphs1) knockout mice to establish the basis for GRT for congenital nephrotic syndrome. We believe that our findings set an important basis for upcoming/ongoing gene therapy approaches in the field of nephrology, especially for monogenic nephrotic syndrome.

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.

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.

CNNM proteins selectively bind to the TRPM7 channel to stimulate divalent cation entry into cells.

Magnesium is essential for cellular life, but how it is homeostatically controlled still remains poorly understood. Here, we report that members of CNNM family, which have been controversially implicated in both cellular Mg2+ influx and efflux, selectively bind to the TRPM7 channel to stimulate divalent cation entry into cells. Coexpression of CNNMs with the channel markedly increased uptake of divalent cations, which is prevented by an inactivating mutation to the channel's pore. Knockout (KO) of TRPM7 in cells or application of the TRPM7 channel inhibitor NS8593 also interfered with CNNM-stimulated divalent cation uptake. Conversely, KO of CNNM3 and CNNM4 in HEK-293 cells significantly reduced TRPM7-mediated divalent cation entry, without affecting TRPM7 protein expression or its cell surface levels. Furthermore, we found that cellular overexpression of phosphatases of regenerating liver (PRLs), known CNNMs binding partners, stimulated TRPM7-dependent divalent cation entry and that CNNMs were required for this activity. Whole-cell electrophysiological recordings demonstrated that deletion of CNNM3 and CNNM4 from HEK-293 cells interfered with heterologously expressed and native TRPM7 channel function. We conclude that CNNMs employ the TRPM7 channel to mediate divalent cation influx and that CNNMs also possess separate TRPM7-independent Mg2+ efflux activities that contribute to CNNMs' control of cellular Mg2+ homeostasis.

Diagnostic Dilemma in an Adolescent Girl with an Eating Disorder, Intellectual Disability, and Hypomagnesemia.

Neurological disorders, including seizures, migraine, depression, and intellectual disability, are frequently associated with hypomagnesemia. Specifically, magnesium (Mg2+) channel transient receptor potential melastatin (TRPM) 6 and TRPM7 are essential for brain function and development. Both channels are also localized in renal and intestinal epithelia and are crucial for Mg2+(re)absorption. Cyclin M2 (CNNM2) is located on the basolateral side of the distal convoluted tubule. In addition, it plays a role in the maintenance of plasma Mg2+ levels along with TRPM6, which is present at the apical level. The CNNM2 gene is crucial for renal magnesium handling, brain development, and neurological functioning. Here, we identified a novel mutation in the CNNM2 gene causing a cognitive delay in a girl with hypomagnesemia. We suggest testing for CNNM2 mutation in patients with neurological impairment and hypomagnesemia.

ARL15 modulates magnesium homeostasis through N-glycosylation of CNNMs.

Cyclin M (CNNM1-4) proteins maintain cellular and body magnesium (Mg2+) homeostasis. Using various biochemical approaches, we have identified members of the CNNM family as direct interacting partners of ADP-ribosylation factor-like GTPase 15 (ARL15), a small GTP-binding protein. ARL15 interacts with CNNMs at their carboxyl-terminal conserved cystathionine-ß-synthase (CBS) domains. In silico modeling of the interaction between CNNM2 and ARL15 supports that the small GTPase specifically binds the CBS1 and CNBH domains. Immunocytochemical experiments demonstrate that CNNM2 and ARL15 co-localize in the kidney, with both proteins showing subcellular localization in the endoplasmic reticulum, Golgi apparatus and the plasma membrane. Most importantly, we found that ARL15 is required for forming complex N-glycosylation of CNNMs. Overexpression of ARL15 promotes complex N-glycosylation of CNNM3. Mg2+ uptake experiments with a stable isotope demonstrate that there is a significant increase of 25Mg2+ uptake upon knockdown of ARL15 in multiple kidney cancer cell lines. Altogether, our results establish ARL15 as a novel negative regulator of Mg2+ transport by promoting the complex N-glycosylation of CNNMs.

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.

The phenotypic and genetic spectrum of patients with heterozygous mutations in cyclin M2 (CNNM2).

Hypomagnesemia, seizures, and intellectual disability (HSMR) syndrome is a rare disorder caused by mutations in the cyclin M2 (CNNM2) gene. Due to the limited number of cases, extensive phenotype analyses of these patients have not been performed, hindering early recognition of patients. In this study, we established the largest cohort of HSMR to date, aiming to improve recognition and diagnosis of this complex disorder. Eleven novel variants in CNNM2 were identified in nine single sporadic cases and in two families with suspected HSMR syndrome. 25 Mg2+ uptake assays demonstrated loss-of-function in seven out of nine variants in CNNM2. Interestingly, the pathogenic mutations resulted in decreased plasma membrane expression. The phenotype of those affected by pathogenic CNNM2 mutations was compared with five previously reported cases of HSMR. All patients suffered from hypomagnesemia (0.44-0.72 mmol/L), which could not be fully corrected by Mg2+ supplementation. The majority of patients (77%) experienced generalized seizures and exhibited mild to moderate intellectual disability and speech delay. Moreover, severe obesity was present in most patients (89%). Our data establish hypomagnesemia, seizures, intellectual disability, and obesity as hallmarks of HSMR syndrome. The assessment of these major features offers a straightforward tool for the clinical diagnosis of HSMR.

Mechanisms coupling sodium and magnesium reabsorption in the distal convoluted tubule of the kidney.

Hypomagnesaemia is a common feature of renal Na+ wasting disorders such as Gitelman and EAST/SeSAME syndrome. These genetic defects specifically affect Na+ reabsorption in the distal convoluted tubule, where Mg2+ reabsorption is tightly regulated. Apical uptake via TRPM6 Mg2+ channels and basolateral Mg2+ extrusion via a putative Na+ -Mg2+ exchanger determines Mg2+ reabsorption in the distal convoluted tubule. However, the mechanisms that explain the high incidence of hypomagnesaemia in patients with Na+ wasting disorders of the distal convoluted tubule are largely unknown. In this review, we describe three potential mechanisms by which Mg2+ reabsorption in the distal convoluted tubule is linked to Na+ reabsorption. First, decreased activity of the thiazide-sensitive Na+ /Cl- cotransporter (NCC) results in shortening of the segment, reducing the Mg2+ reabsorption capacity. Second, the activity of TRPM6 and NCC are determined by common regulatory pathways. Secondary effects of NCC dysregulation such as hormonal imbalance, therefore, might disturb TRPM6 expression. Third, the basolateral membrane potential, maintained by the K+ permeability and Na+ -K+ -ATPase activity, provides the driving force for Na+ and Mg2+ extrusion. Depolarisation of the basolateral membrane potential in Na+ wasting disorders of the distal convoluted tubule may therefore lead to reduced activity of the putative Na+ -Mg2+ exchanger SLC41A1. Elucidating the interconnections between Mg2+ and Na+ transport in the distal convoluted tubule is hampered by the currently available models. Our analysis indicates that the coupling of Na+ and Mg2+ reabsorption may be multifactorial and that advanced experimental models are required to study the molecular mechanisms.