Structural Basis of Heterochromatin Formation by Human HP1.

Heterochromatin plays important roles in transcriptional silencing and genome maintenance by the formation of condensed chromatin structures, which determine the epigenetic status of eukaryotic cells. The trimethylation of histone H3 lysine 9 (H3K9me3), a target of heterochromatin protein 1 (HP1), is a hallmark of heterochromatin formation. However, the mechanism by which HP1 folds chromatin-containing H3K9me3 into a higher-order structure has not been elucidated. Here we report the three-dimensional structure of the H3K9me3-containing dinucleosomes complexed with human HP1α, HP1ß, and HP1γ, determined by cryogenic electron microscopy with a Volta phase plate. In the structures, two H3K9me3 nucleosomes are bridged by a symmetric HP1 dimer. Surprisingly, the linker DNA between the nucleosomes does not directly interact with HP1, thus allowing nucleosome remodeling by the ATP-utilizing chromatin assembly and remodeling factor (ACF). The structure depicts the fundamental architecture of heterochromatin.

Cryo-EM structure of the Ebola virus nucleoprotein-RNA complex at 3.6 Å resolution.

Ebola virus causes haemorrhagic fever with a high fatality rate in humans and non-human primates. It belongs to the family Filoviridae in the order Mononegavirales, which are viruses that contain linear, non-segmented, negative-sense, single-stranded genomic RNA1,2. The enveloped, filamentous virion contains the nucleocapsid, consisting of the helical nucleoprotein-RNA complex, VP24, VP30, VP35 and viral polymerase1,3. The nucleoprotein-RNA complex acts as a scaffold for nucleocapsid formation and as a template for RNA replication and transcription by condensing RNA into the virion4,5. RNA binding and nucleoprotein oligomerization are synergistic and do not readily occur independently6. Although recent cryo-electron tomography studies have revealed the overall architecture of the nucleocapsid core4,5, there has been no high-resolution reconstruction of the nucleocapsid. Here we report the structure of a recombinant Ebola virus nucleoprotein-RNA complex expressed in mammalian cells without chemical fixation, at near-atomic resolution using single-particle cryo-electron microscopy. Our structure reveals how the Ebola virus nucleocapsid core encapsidates its viral genome, its sequence-independent coordination with RNA by nucleoprotein, and the dynamic transition between the RNA-free and RNA-bound states. It provides direct structural evidence for the role of the N terminus of nucleoprotein in subunit oligomerization, and for the hydrophobic and electrostatic interactions that lead to the formation of the helical assembly. The structure is validated as representative of the native biological assembly of the nucleocapsid core by consistent dimensions and symmetry with the full virion5. The atomic model provides a detailed mechanistic basis for understanding nucleocapsid assembly and highlights key structural features that may serve as targets for anti-viral drug development.

Fuzzy RNA recognition by the Trypanosoma brucei editosome.

The assembly of high molecular mass ribonucleoprotein complexes typically relies on the binary interaction of defined RNA sequences or precisely folded RNA motifs with dedicated RNA-binding domains on the protein side. Here we describe a new molecular recognition principle of RNA molecules by a high molecular mass protein complex. By chemically probing the solvent accessibility of mitochondrial pre-mRNAs when bound to the Trypanosoma brucei editosome, we identified multiple similar but non-identical RNA motifs as editosome contact sites. However, by treating the different motifs as mathematical graph objects we demonstrate that they fit a consensus 2D-graph consisting of 4 vertices (V) and 3 edges (E) with a Laplacian eigenvalue of 0.5477 (λ2). We establish that synthetic 4V(3E)-RNAs are sufficient to compete for the editosomal pre-mRNA binding site and that they inhibit RNA editing in vitro. Furthermore, we demonstrate that only two topological indices are necessary to predict the binding of any RNA motif to the editosome with a high level of confidence. Our analysis corroborates that the editosome has adapted to the structural multiplicity of the mitochondrial mRNA folding space by recognizing a fuzzy continuum of RNA folds that fit a consensus graph descriptor.

First record of Trypanosoma (Ornithotrypanum) infecting Neotropical birds.

Parasites play a pivotal role in ecosystem health, influencing human and zoonotic diseases, as well as biodiversity preservation. The genus Trypanosoma comprises approximately 500 species mostly found in wildlife animals. This study focuses on identifying trypanosomes found in the white-necked thrush (Turdus albicollis) and the yellow-legged thrush (Turdus flavipes) in the Neotropics. First, we demonstrate the utility of an 18S rDNA sequence-structure phylogeny as an alternative method for trypanosome classification, especially when gGAPDH sequences are unavailable. Subsequently, the sequence-structure phylogeny is employed to classify new trypanosome sequences discovered in wild birds, placing them within the Ornithotrypanum subgenus. This marks the first identification of Ornithotrypanum in Neotropical birds, contributing to the understanding of the distribution and ecological adaptation of avian trypanosomes. Beyond taxonomy, this study broadens our comprehension of the ecological implications of avian trypanosomes in the Neotropics, emphasizing the need for continued research in this field. These findings underscore the importance of alternative classification methods, which are essential to unravel the complex interactions between parasites, wildlife hosts, and their ecosystems.

Paramecium: RNA sequence-structure phylogenetics.

Organisms classified as members of the genus Paramecium belong to the best-known group of single-celled eukaryotes. Nevertheless, the phylogeny within the genus Paramecium has been discussed and revisited in recent decades and remains partly unresolved. By applying an RNA sequence-structure approach, we attempt to increase accuracy and robustness of phylogenetic trees. For each individual 18S and internal transcribed spacer 2 (ITS2) sequence, a putative secondary structure was predicted through homology modelling. While searching for a structural template, we found, in contrast to the available literature, that the ITS2 molecule consists of three helices in members of the genus Paramecium and four helices in members of the genus Tetrahymena. Two sequencestructure neighbor-joining overall trees were reconstructed with (1) more than 400 taxa (ITS2) and (2) more than 200 taxa (18S). For smaller subsets, neighbor-joining, maximum-parsimony, and maximum-likelihood analyses were executed using sequence-structure information simultaneously. Based on a combined data set (ITS2+18S rDNA) a well-supported tree was reconstructed with bootstrap values over 50 in at least one of the applied analyses. Our results are in general agreement with those published in the available literature based on multi-gene analyses. Our study supports the simultaneous use of sequence-structure data to reconstruct accurate and robust phylogenetic trees.

18S rDNA sequence-structure phylogeny of the Euglenophyceae (Euglenozoa, Euglenida).

The phylogeny of Euglenophyceae (Euglenozoa, Euglenida) has been discussed for decades with new genera being described in the last few years. In this study, we reconstruct a phylogeny using 18S rDNA sequence and structural data simultaneously. Using homology modeling, individual secondary structures were predicted. Sequence-structure data are encoded and automatically aligned. Here, we present a sequence-structure neighbor-joining tree of more than 300 taxa classified as Euglenophyceae. Profile neighbor-joining was used to resolve the basal branching pattern. Neighbor-joining, maximum parsimony, and maximum likelihood analyses were performed using sequence-structure information for manually chosen subsets. All analyses supported the monophyly of Eutreptiella, Discoplastis, Lepocinclis, Strombomonas, Cryptoglena, Monomorphina, Euglenaria, and Colacium. Well-supported topologies were generally consistent with previous studies using a combined dataset of genetic markers. Our study supports the simultaneous use of sequence and structural data to reconstruct more accurate and robust trees. The average bootstrap value is significantly higher than the average bootstrap value obtained from sequence-only analyses, which is promising for resolving relationships between more closely related taxa.

Nephronophthisis: a pathological and genetic perspective.

Nephronophthisis (NPHP) is an autosomal recessive cystic kidney disease and is one of the most frequent genetic causes for kidney failure (KF) in children and adolescents. Over 20 genes cause NPHP and over 90 genes contribute to renal ciliopathies often involving multiple organs. About 15-20% of NPHP patients have additional extrarenal symptoms affecting other organs than the kidneys. The involvement of additional organ systems in syndromic forms of NPHP is explained by shared expression of most NPHP gene products in centrosomes and primary cilia, a sensory organelle present in most mammalian cells. This finding resulted in the classification of NPHP as a ciliopathy. If extrarenal symptoms are present in addition to NPHP, these disorders are defined as NPHP-related ciliopathies (NPHP-RC) and can involve the retina (e.g., with Senior-Løken syndrome), CNS (central nervous system) (e.g., with Joubert syndrome), liver (e.g., Boichis and Arima syndromes), or bone (e.g., Mainzer-Saldino and Sensenbrenner syndromes). This review focuses on the pathological findings and the recent genetic advances in NPHP and NPHP-RC. Different mechanisms and signaling pathways are involved in NPHP ranging from planar cell polarity, sonic hedgehog signaling (Shh), DNA damage response pathway, Hippo, mTOR, and cAMP signaling. A number of therapeutic interventions appear to be promising, ranging from vasopressin receptor 2 antagonists such as tolvaptan, cyclin-dependent kinase inhibitors such as roscovitine, Hh agonists such as purmorphamine, and mTOR inhibitors such as rapamycin.

Gentamicin Inhibits Ca2+ Channel TRPV5 and Induces Calciuresis Independent of the Calcium-Sensing Receptor-Claudin-14 Pathway.

BACKGROUND: Treatment with the aminoglycoside antibiotic gentamicin can be associated with severe adverse effects, including renal Ca2+ wasting. The underlying mechanism is unknown but it has been proposed to involve activation of the Ca2+-sensing receptor (CaSR) in the thick ascending limb, which would increase expression of claudin-14 (CLDN14) and limit Ca2+ reabsorption. However, no direct evidence for this hypothesis has been presented. METHODS: We studied the effect of gentamicin in vivo using mouse models with impaired Ca2+ reabsorption in the proximal tubule and the thick ascending limb. We used a Cldn14 promoter luciferase reporter assay to study CaSR activation and investigated the effect of gentamicin on activity of the distal nephron Ca2+ channel transient receptor potential vanilloid 5 (TRPV5), as determined by patch clamp in HEK293 cells. RESULTS: Gentamicin increased urinary Ca2+ excretion in wild-type mice after acute and chronic administration. This calciuretic effect was unaltered in mice with genetic CaSR overactivation and was present in furosemide-treated animals, whereas the calciuretic effect in Cldn14-/- mice and mice with impaired proximal tubular Ca2+ reabsorption (claudin-2 [CLDN2]-deficient Cldn2-/- mice) was equivalent to that of wild-type mice. In vitro, gentamicin failed to activate the CaSR. In contrast, patch clamp analysis revealed that gentamicin strongly inhibited rabbit and human TRPV5 activity and chronic gentamicin administration downregulated distal nephron Ca2+ transporters. CONCLUSIONS: Gentamicin does not cause hypercalciuria via activation of the CaSR-CLDN14 pathway or by interfering with proximal tubular CLDN2-dependent Ca2+ reabsorption. Instead, gentamicin blocks distal Ca2+ reabsorption by direct inhibition of the Ca2+ channel TRPV5. These findings offer new insights into Ca2+ wasting in patients treated with gentamicin.

Core-Shell DNA-Cholesterol Nanoparticles Exert Lysosomolytic Activity in African Trypanosomes.

Trypanosoma brucei is the causal infectious agent of African trypanosomiasis in humans and Nagana in livestock. Both diseases are currently treated with a small number of chemotherapeutics, which are hampered by a variety of limitations reaching from efficacy and toxicity complications to drug-resistance problems. Here, we explore the forward design of a new class of synthetic trypanocides based on nanostructured, core-shell DNA-lipid particles. In aqueous solution, the particles self-assemble into micelle-type structures consisting of a solvent-exposed, hydrophilic DNA shell and a hydrophobic lipid core. DNA-lipid nanoparticles have membrane-adhesive qualities and can permeabilize lipid membranes. We report the synthesis of DNA-cholesterol nanoparticles, which specifically subvert the membrane integrity of the T. brucei lysosome, killing the parasite with nanomolar potencies. Furthermore, we provide an example of the programmability of the nanoparticles. By functionalizing the DNA shell with a spliced leader (SL)-RNA-specific DNAzyme, we target a second trypanosome-specific pathway (dual-target approach). The DNAzyme provides a backup to counteract the recovery of compromised parasites, which reduces the risk of developing drug resistance.

The roles of homocysteinemia and methylmalonic acidemia in kidney injury in atypical hemolytic uremic syndrome caused by cobalamin C deficiency.

BACKGROUND: Cobalamin C (cblC), a vitamin B12 processing protein, plays a crucial role in metabolism for the conversion of homocysteine to methionine and methylmalonyl-CoA to succinyl-CoA. CblC deficiency, an inborn error of cobalamin processing, is a rare cause of atypical hemolytic-uremic syndrome (aHUS) and results in hyperhomocysteinemia and methylmalonic aciduria. Both substances are thought to contribute to thrombotic microangiopathy (TMA) in cblC deficiency patients. However, the roles of homocysteine and methylmalonic acid (MMA) in these patients remain unclear. We want to shed more light on the contributions of homocysteine and MMA levels as contributing factors for thrombotic microangiopathy (TMA)/aHUS by a follow-up of a cblC deficiency patient over 6 years. CASE DIAGNOSIS: A 27-day-old Hispanic female presented with abnormal C3-carnitine on her newborn screen, poor feeding, decreased activity, and oligouria. She was diagnosed with cblC deficiency after laboratory results revealed elevated serum homocysteine, and serum MMA along with genetic testing showing a homozygous pathogenic frameshift variant in MMACHC. The patient developed aHUS and acute kidney injury (AKI), which resolved after appropriate therapy. Over 6 years, she continued to have normal kidney function with no thrombocytopenia despite persistently elevated homocysteine and MMA levels. CONCLUSION: We question the roles of homocysteine and MMA as causative of aHUS/TMA in cblC deficiency as they remained elevated during follow-up but did not result in aHUS/TMA or AKI. Hyperhomocysteinemia and/or MMA caused by other metabolic diseases do not result in aHUS/TMA or AKI. This suggests that other nephrotoxic factors may trigger aHUS/TMA in cblC patients.

Autosomal dominant tubulointerstitial kidney disease: more than just HNF1ß.

Autosomal dominant tubulointerstitial kidney disease (ADTKD) refers to a group of disorders with a bland urinary sediment, slowly progressive chronic kidney disease (CKD), and autosomal dominant inheritance. Due to advances in genetic diagnosis, ADTKD is becoming increasingly recognized as a cause of CKD in both children and adults. ADTKD-REN presents in childhood with mild hypotension, CKD, hyperkalemia, acidosis, and anemia. ADTKD-UMOD is associated with gout and CKD that may present in adolescence and slowly progresses to kidney failure. HNF1ß mutations often present in childhood with anatomic abnormalities such as multicystic or dysplastic kidneys, as well as CKD and a number of other extra-kidney manifestations. ADTKD-MUC1 is less common in childhood, and progressive CKD is its sole clinical manifestation, usually beginning in the late teenage years. This review describes the pathophysiology, genetics, clinical characteristics, diagnosis, and treatment of the different forms of ADTKD, with an emphasis on diagnosis. We also present data on kidney function in children with ADTKD from the Wake Forest Rare Inherited Kidney Disease Registry.

Analyzing editosome function in high-throughput.

Mitochondrial gene expression in African trypanosomes and other trypanosomatid pathogens requires a U-nucleotide specific insertion/deletion-type RNA-editing reaction. The process is catalyzed by a macromolecular protein complex known as the editosome. Editosomes are restricted to the trypanosomatid clade and since editing is essential for the parasites, the protein complex represents a near perfect target for drug intervention strategies. Here, we report the development of an improved in vitro assay to monitor editosome function. The test system utilizes fluorophore-labeled substrate RNAs to analyze the processing reaction by automated, high-throughput capillary electrophoresis (CE) in combination with a laser-induced fluorescence (LIF) readout. We optimized the assay for high-throughput screening (HTS)-experiments and devised a multiplex fluorophore-labeling regime to scrutinize the U-insertion/U-deletion reaction simultaneously. The assay is robust, it requires only nanogram amounts of materials and it meets all performance criteria for HTS-methods. As such the test system should be helpful in the search for trypanosome-specific pharmaceuticals.

Phosphorylated claudin-16 interacts with Trpv5 and regulates transcellular calcium transport in the kidney.

Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) was previously considered to be a paracellular channelopathy caused by mutations in the claudin-16 and claudin-19 genes. Here, we provide evidence that a missense FHHNC mutation c.908C>G (p.T303R) in the claudin-16 gene interferes with the phosphorylation in the claudin-16 protein. The claudin-16 protein carrying phosphorylation at residue T303 is localized in the distal convoluted tubule (DCT) but not in the thick ascending limb (TAL) of the mouse kidney. The phosphomimetic claudin-16 protein carrying the T303E mutation but not the wildtype claudin-16 or the T303R mutant protein increases the Trpv5 channel conductance and membrane abundance in human kidney cells. Phosphorylated claudin-16 and Trpv5 are colocalized in the luminal membrane of the mouse DCT tubule; phosphomimetic claudin-16 and Trpv5 interact in the yeast and mammalian cell membranes. Knockdown of claudin-16 gene expression in transgenic mouse kidney delocalizes Trpv5 from the luminal membrane in the DCT. Unlike wildtype claudin-16, phosphomimetic claudin-16 is delocalized from the tight junction but relocated to the apical membrane in renal epithelial cells because of diminished binding affinity to ZO-1. High-Ca2+ diet reduces the phosphorylation of claudin-16 protein at T303 in the DCT of mouse kidney via the PTH signaling cascade. Knockout of the PTH receptor, PTH1R, from the mouse kidney abrogates the claudin-16 phosphorylation at T303. Together, these results suggest a pathogenic mechanism for FHHNC involving transcellular Ca2+ pathway in the DCT and identify a molecular component in renal Ca2+ homeostasis under direct regulation of PTH.

High-resolution archaellum structure reveals a conserved metal-binding site.

Many archaea swim by means of archaella. While the archaellum is similar in function to its bacterial counterpart, its structure, composition, and evolution are fundamentally different. Archaella are related to archaeal and bacterial type IV pili. Despite recent advances, our understanding of molecular processes governing archaellum assembly and stability is still incomplete. Here, we determine the structures of Methanococcus archaella by X-ray crystallography and cryo-EM The crystal structure of Methanocaldococcus jannaschii FlaB1 is the first and only crystal structure of any archaellin to date at a resolution of 1.5 Å, which is put into biological context by a cryo-EM reconstruction from Methanococcus maripaludis archaella at 4 Å resolution created with helical single-particle analysis. Our results indicate that the archaellum is predominantly composed of FlaB1. We identify N-linked glycosylation by cryo-EM and mass spectrometry. The crystal structure reveals a highly conserved metal-binding site, which is validated by mass spectrometry and electron energy-loss spectroscopy. We show in vitro that the metal-binding site, which appears to be a widespread property of archaellin, is required for filament integrity.

Structural basis for anthrax toxin receptor 1 recognition by Seneca Valley Virus.

Recently, the use of oncolytic viruses in cancer therapy has become a realistic therapeutic option. Seneca Valley Virus (SVV) is a newly discovered picornavirus, which has earned a significant reputation as a potent oncolytic agent. Anthrax toxin receptor 1 (ANTXR1), one of the cellular receptors for the protective antigen secreted by Bacillus anthracis, has been identified as the high-affinity cellular receptor for SVV. Here, we report the structure of the SVV-ANTXR1 complex determined by single-particle cryo-electron microscopy analysis at near-atomic resolution. This is an example of a shared receptor structure between a mammalian virus and a bacterial toxin. Our structure shows that ANTXR1 decorates the outer surface of the SVV capsid and interacts with the surface-exposed BC loop and loop II of VP1, "the puff" of VP2 and "the knob" of VP3. Comparison of the receptor-bound capsid structure with the native capsid structure reveals that receptor binding induces minor conformational changes in SVV capsid structure, suggesting the role of ANTXR1 as an attachment receptor. Furthermore, our results demonstrate that the capsid footprint on the receptor is not conserved in anthrax toxin receptor 2 (ANTXR2), thereby providing a molecular mechanism for explaining the exquisite selectivity of SVV for ANTXR1.

An international cohort study of autosomal dominant tubulointerstitial kidney disease due to REN mutations identifies distinct clinical subtypes.

There have been few clinical or scientific reports of autosomal dominant tubulointerstitial kidney disease due to REN mutations (ADTKD-REN), limiting characterization. To further study this, we formed an international cohort characterizing 111 individuals from 30 families with both clinical and laboratory findings. Sixty-nine individuals had a REN mutation in the signal peptide region (signal group), 27 in the prosegment (prosegment group), and 15 in the mature renin peptide (mature group). Signal group patients were most severely affected, presenting at a mean age of 19.7 years, with the prosegment group presenting at 22.4 years, and the mature group at 37 years. Anemia was present in childhood in 91% in the signal group, 69% prosegment, and none of the mature group. REN signal peptide mutations reduced hydrophobicity of the signal peptide, which is necessary for recognition and translocation across the endoplasmic reticulum, leading to aberrant delivery of preprorenin into the cytoplasm. REN mutations in the prosegment led to deposition of prorenin and renin in the endoplasmic reticulum-Golgi intermediate compartment and decreased prorenin secretion. Mutations in mature renin led to deposition of the mutant prorenin in the endoplasmic reticulum, similar to patients with ADTKD-UMOD, with a rate of progression to end stage kidney disease (63.6 years) that was significantly slower vs. the signal (53.1 years) and prosegment groups (50.8 years) (significant hazard ratio 0.367). Thus, clinical and laboratory studies revealed subtypes of ADTKD-REN that are pathophysiologically, diagnostically, and clinically distinct.

Re-Evaluation of a Fulvene-Based Self-Replicating Diels-Alder Reaction System.

We re-evaluate our claim of a high diastereoselectivity in the self-relicating Diels-Alder reaction between maleimide 1 and fulvene 3. It was shown that the system has a diastereoselectivity of 1.8:1 for NN-4:NX-4, which is contrary to the 16:1 ratio claimed by Dieckmann et al. The analysis of 1 H NMR monitoring of the reaction revealed that both replicators show sigmoidal growth which is typical for auto-catalytic systems.

Greater activity, better range of motion and higher quality of life following unicompartmental knee arthroplasty: a comparative case-control study.

PURPOSE: The purpose of this study was to provide a matched cohort comparison of clinical and functional outcome scores, range of motion and quality of life following unicompartmental knee arthroplasty (UKA) or total knee arthroplasty (TKA). The hypothesis was that patients receiving UKA report better results than comparable patients who receive conventional TKA. METHODS: Clinical and functional results of 35 patients with medial end-stage osteoarthritis who had received a fixed-bearing UKA were compared with the results of 35 matched patients who had received a TKA from the same manufacturer by the same surgeon. Outcome scores were measured before surgery and at final follow-up using Tegner Activity Scale (TAS), range of motion (ROM) and Short Form 36 Health Survey (SF-36). The Knee Society Score (KSS) was assessed at final follow-up. The mean observation period was 2.3 years in both groups. RESULTS: The preoperative knee scores had no statistically significant differences between the two groups. Postoperatively, however, UKAs performed significantly better regarding TAS and ROM (4 vs. 3 and 118.4 vs. 103.7, respectively). The results of the SF-36 showed significantly better results for the UKA group in the mental component summary score and in the subscale of social function. CONCLUSIONS: The present study suggests that UKA is associated with higher activity level, higher quality of life, and greater ROM when compared with TKA on comparable patients. Prolonged clinical follow-up in a larger patient cohort with a randomised-controlled study design would be beneficial to confirm these findings. LEVEL OF EVIDENCE: III.

Uromodulin regulates renal magnesium homeostasis through the ion channel transient receptor potential melastatin 6 (TRPM6).

Up to 15% of the population have mild to moderate chronic hypomagnesemia, which is associated with type 2 diabetes mellitus, hypertension, metabolic syndrome, and chronic kidney disease. The kidney is the key organ for magnesium homeostasis, but our understanding of renal magnesium regulation is very limited. Uromodulin (UMOD) is the most abundant urinary protein in humans, and here we report that UMOD has a role in renal magnesium homeostasis. Umod-knockout (Umod-/-) mice excreted more urinary magnesium than WT mice and displayed up-regulation of genes promoting magnesium absorption. The majority of magnesium is absorbed in the thick ascending limb. However, both mouse strains responded similarly to the diuretic agent furosemide, indicating appropriate function of the thick ascending limb in the Umod-/- mice. Magnesium absorption is fine-tuned in the distal convoluted tubule (DCT) via the apical magnesium channel transient receptor potential melastatin 6 (TRPM6). We observed decreased apical Trpm6 staining in the DCT of Umod-/- mice. Applying biotinylation assays and whole-cell patch-clamp recordings, we found that UMOD enhances TRPM6 cell-surface abundance and current density from the extracellular space. UMOD physically interacted with TRPM6 and thereby impaired dynamin-dependent TRPM6 endocytosis. WT mice fed a low-magnesium diet had an increased urinary UMOD secretion compared with the same mice on a regular diet. Our results suggest that increased urinary UMOD secretion in low-magnesium states reduces TRPM6 endocytosis and thereby up-regulates TRPM6 cell-surface abundance to defend against further urinary magnesium losses.