Glomerular Endothelial Cell Receptor ADGRF5 and the Integrity of the Glomerular Filtration Barrier.

BACKGROUND: Glomerular endothelial cells are recognized to be important for maintaining the glomerular filtration barrier. ADGRF5, an adhesion G protein-coupled receptor, has been suggested to be involved in endothelial cell function. However, the role of ADGRF5 in the glomerular filtration barrier integrity remains elusive. METHODS: Cellular expression of ADGRF5 in mouse glomerulus was determined by histological analyses. The impact of ADGRF5 deletion on the glomerular morphology, kidney function, and glomerular endothelial gene/protein expression was then analyzed using ADGRF5 knockout (Adgrf5-/-) mice and human primary glomerular endothelial cells. RESULTS: ADGRF5 was specifically expressed in the capillary endothelial cells within the glomerulus. Adgrf5-/- mice developed albuminuria and impaired kidney function with morphological defects in the glomeruli, namely glomerular hypertrophy, glomerular basement membrane splitting and thickening, diaphragmed fenestration and detachment of the glomerular endothelial cells, and mesangial interposition. These defects were accompanied by the altered expression of genes responsible for glomerular basement membrane organization (type IV collagens and laminins) and Krüppel-like factor 2 (Klf2) in glomerular endothelial cells. Moreover, ADGRF5 knockdown decreased COL4A3 and COL4A4 expression and increased KLF2 expression in human primary glomerular endothelial cells. CONCLUSIONS: The loss of ADGRF5 resulted in altered gene expression in glomerular endothelial cells, and perturbed the structure and permselectivity of the glomerular filtration barrier.

Distinguishing two distinct types of salivary extracellular vesicles: a potential tool for understanding their pathophysiological roles.

Extracellular vesicles (EVs), which are found in almost all cells and human body fluids, are currently being studied as a source of pathophysiological information. Previously, we demonstrated that at least two types of EVs can be isolated from human whole saliva (WS) using enzymatic activity of dipeptidyl peptidase IV (DPP IV) as a marker for differentiating the EV subsets. In the present study, EV fractions, termed EV-I 20 k-ppt and EV-II 100 k-ppt, were prepared by a combination of size-exclusion chromatography of improved condition and sequential centrifugation. The EV-I 20 k-ppt fraction contained medium/large EVs with a diameter of 100-1,000 nm, including aminopeptidase N (APN), mucin 1, ezrin, and Annexin A1. EV-II 100 k-ppt contained small EVs with a diameter of 20-70 nm, with DPP IV and CD9, programmed cell death 6-interacting protein, and tumor susceptibility gene 101 as characteristic proteins. Proteomic analyses also revealed distinctive repertoires of constituent proteins. Immunoprecipitation of several membrane proteins of the EVs with respective antibodies suggested their differential local membrane environment between the two types of salivary vesicles. Thus, we identified two distinctive types of EVs, one is APN/MUC1- rich EVs (EV-I, large/medium EVs) and the other is DPP IV/CD9-rich EVs (EV-II, small EVs). Furthermore, analysis of the binding of the EVs to coronavirus spike proteins showed that EV-II 100 k-ppt, but not EV-I 20 k-ppt, significantly bound to the spike protein of Middle East respiratory syndrome coronavirus (MERS-CoV). Finally, we developed a simple method to prepare two distinctive EVs from only 1 mL of human WS using sequential immunoprecipitation. Elucidating the features and functions of these two types of salivary EVs may help us understand their pathophysiological roles in the oral cavity and gastrointestinal tract.

Endothelial tissue remodeling induced by intraluminal pressure enhances paracellular solute transport.

The endothelial layers of the microvasculature regulate the transport of solutes to the surrounding tissues. It remains unclear how this barrier function is affected by blood flow-induced intraluminal pressure. Using a 3D microvessel model, we compare the transport of macromolecules through endothelial tissues at mechanical rest or with intraluminal pressure, and correlate these data with electron microscopy of endothelial junctions. On application of an intraluminal pressure of 100 Pa, we demonstrate that the flow through the tissue increases by 2.35 times. This increase is associated with a 25% expansion of microvessel diameter, which leads to tissue remodeling and thinning of the paracellular junctions. We recapitulate these data with the deformable monopore model, in which the increase in paracellular transport is explained by the augmentation of the diffusion rate across thinned junctions under mechanical stress. We therefore suggest that the deformation of microvasculatures contributes to regulate their barrier function.

5-Aminosalicylic acid alters the gut microbiota and altered microbiota transmitted vertically to offspring have protective effects against colitis.

Although many therapeutic options are available for inflammatory bowel disease (IBD), 5-aminosalicylic acid (5-ASA) is still the key medication, particularly for ulcerative colitis (UC). However, the mechanism of action of 5-ASA remains unclear. The intestinal microbiota plays an important role in the pathophysiology of IBD, and we hypothesized that 5-ASA alters the intestinal microbiota, which promotes the anti-inflammatory effect of 5-ASA. Because intestinal inflammation affects the gut microbiota and 5-ASA can change the severity of inflammation, assessing the impact of inflammation and 5-ASA on the gut microbiota is not feasible in a clinical study of patients with UC. Therefore, we undertook a translational study to demonstrate a causal link between 5-ASA administration and alterations of the intestinal microbiota. Furthermore, by rigorously controlling environmental confounders and excluding the effect of 5-ASA itself with a vertical transmission model, we observed that the gut microbiota altered by 5-ASA affected host mucosal immunity and decreased susceptibility to dextran sulfate sodium-induce colitis. Although the potential intergenerational transmission of epigenetic changes needs to be considered in this study, these findings suggested that alterations in the intestinal microbiota induced by 5-ASA directed the host immune system towards an anti-inflammatory state, which underlies the mechanism of 5-ASA efficacy.

Exploring the pathophysiological mechanism of interstitial edema focusing on the role of macrophages and their interaction with the glycocalyx.

OBJECTIVES: Glycocalyx lines the vascular intraluminal space that regulates fluid movement between the intra- and extra-vascular compartments. The depletion of glycocalyx (GCX) is associated with leukocyte accumulation, possibly causing the endothelial cells to become hyperpermeable in various organs, including oral tissues. Whether neutrophils or macrophages are responsible for developing interstitial edema remains controversial. We explored the pathophysiological mechanism of interstitial edema by examining the role of reactive neutrophils and macrophages and their interactions with GCX. METHODS: An anti-MHC class I antibody was administered intravenously to male BALB/c mice to induce pulmonary edema. Pulmonary edema was evaluated by measuring the lung wet-to-dry weight ratio. Changes in the GCX were evaluated by electron microscopy and measurements of the serum level of soluble syndecan-1. Heparin sulfate was administered to examine its protective effect on the GCX. The macrophages were depleted using clodronate to examine their role in developing edema. RESULTS: The GCX degradation induced by the anti-MHC class I antibody was accompanied by increased serum syndecan-1 and heparan sulfate levels. Macrophage depletion inhibited the development of pulmonary edema, and the administration of supplemental heparin suppressed the edema. CONCLUSIONS: We demonstrated that the degradation of the GCX induced by the anti-MHC class I antibody was suppressed by macrophage depletion. These results suggest that macrophages may play a key role in interstitial edema. Heparin inhibited both the degradation of the GCX and interstitial edema. This study's results may be extrapolated to develop an interventional strategy for inhibiting interstitial edema in various organs.

A possible function of Nik-related kinase in the labyrinth layer of delayed delivery mouse placentas.

In mice and humans, Nik-related protein kinase (Nrk) is an X-linked gene that encodes a serine/threonine kinase belonging to GCK group 4. Nrk knockout (Nrk KO) mice exhibit delayed delivery, possibly due to defective communication between the Nrk KO conceptus and its mother. However, the mechanism of delayed labor remains largely unknown. Here, we found that in pregnant mothers with the Nrk KO conceptus, the serum progesterone (P4) and placental lactogen (PL-2) concentrations in late pregnancy were higher than those in the wild type. Moreover, we demonstrated that Nrk is expressed in trophoblast giant cells (TGCs) and syncytiotrophoblast-2 (SynT-2) in the labyrinth layer of the mouse placenta. In the human placenta, NRK is also expressed in Syn-T in villi. Both human Syn-T and mouse TGCs of the labyrinth layer are present within fetal tissues that are in direct contact with the maternal blood. The labyrinth layer of the Nrk KO conceptus was gigantic, with enlarged cytoplasm and Golgi bodies in the TGCs. To investigate the function of Nrk in the labyrinth layer, a differentially expressed gene (DEG) analysis was performed. The DEG analysis revealed that labor-promoting factors, such as prostaglandins, were decreased, and pregnancy-maintaining factors, such as the prolactin family and P4 receptor, were increased. These findings suggest that the Nrk KO mice exhibit delayed delivery owing to high P4 concentrations caused by the hypersecretion of pregnancy-maintaining factors, such as PL-2, from the placenta.

The effect of tetrastarch on the endothelial glycocalyx layer in early hemorrhagic shock using fluorescence intravital microscopy: a mouse model.

PURPOSE: To investigate vascular endothelial dysfunction based on glycocalyx impairment in massive hemorrhage and to evaluate fluid therapy. METHODS: In this randomized controlled animal study, we withdrew 1.5 mL blood and administered 1.5 mL resuscitation fluid. Mice were divided into six groups according to the infusion type and administration timing: NS-NS (normal saline), NS-HES ([hydroxyethyl starch]130), HES-NS, NS-ALB (albumin), ALB-NS, and C (control) groups. RESULTS: The glycocalyx index (GCXI) of a 40-µm artery was significantly larger in group C than in other groups (P < 0.01). Similarly, the GCXI for a 60-µm artery was significantly higher in group C than in NS-NS (P ≤ 0.05), NS-HES (P ≤ 0.01), and NS-ALB groups (P ≤ 0.05). The plasma syndecan-1 concentration, at 7.70 ± 5.71 ng/mL, was significantly lower in group C than in group NS-NS (P ≤ 0.01). The tetramethylrhodamine-labeled dextran (TMR-DEX40) fluorescence intensity in ALB-NS and HES-NS groups and the fluorescein isothiocyanate-labeled hydroxyethyl starch (FITC-HES130) fluorescence intensity in NS-HES and HES-NS groups were not significantly different from those of group C at any time point. FITC-HES130 was localized on the inner vessel wall in groups without HES130 infusion but uniformly distributed in HES130-treated groups in intravital microscopy. FITC-FITC-HES130 was localized remarkably in the inner vessel walls in group HES-NS in electron microscopy. CONCLUSIONS: In an acute massive hemorrhage mouse model, initial fluid resuscitation therapy with saline administration impaired glycocalyx and increased vascular permeability. Prior colloid-fluid administration prevented the progression of glycocalyx damage and improve prognosis. Prior HES130 administration may protect endothelial cell function.

A new beta cell-specific mitophagy reporter mouse shows that metabolic stress leads to accumulation of dysfunctional mitochondria despite increased mitophagy.

AIMS/HYPOTHESIS: Mitophagy, the selective autophagy of mitochondria, is essential for maintenance of mitochondrial function. Recent studies suggested that defective mitophagy in beta cells caused diabetes. However, because of technical difficulties, the development of a convenient and reliable method to evaluate mitophagy in beta cells in vivo is needed. The aim of this study was to establish beta cell-specific mitophagy reporter mice and elucidate the role of mitophagy in beta cell function under metabolically stressed conditions induced by a high-fat diet (HFD). METHODS: Mitophagy was assessed using newly generated conditional mitochondrial matrix targeting mitophagy reporter (CMMR) mice, in which mitophagy can be visualised specifically in beta cells in vivo using a fluorescent probe sensitive to lysosomal pH and degradation. Metabolic stress was induced in mice by exposure to the HFD for 20 weeks. The accumulation of dysfunctional mitochondria was examined by staining for functional/total mitochondria and reactive oxygen species (ROS) using specific fluorescent dyes and antibodies. To investigate the molecular mechanism underlying mitophagy in beta cells, overexpression and knockdown experiments were performed. HFD-fed mice were examined to determine whether chronic insulin treatment for 6 weeks could ameliorate mitophagy, mitochondrial function and impaired insulin secretion. RESULTS: Exposure to the HFD increased the number of enlarged (HFD-G) islets with markedly elevated mitophagy. Mechanistically, HFD feeding induced severe hypoxia in HFD-G islets, which upregulated mitophagy through the hypoxia-inducible factor 1-ɑ (Hif-1ɑ)/BCL2 interacting protein 3 (BNIP3) axis in beta cells. However, HFD-G islets unexpectedly showed the accumulation of dysfunctional mitochondria due to excessive ROS production, suggesting an insufficient capacity of mitophagy for the degradation of dysfunctional mitochondria. Chronic administration of insulin ameliorated hypoxia and reduced ROS production and dysfunctional mitochondria, leading to decreased mitophagy and restored insulin secretion. CONCLUSIONS/INTERPRETATION: We demonstrated that CMMR mice enabled the evaluation of mitophagy in beta cells. Our results suggested that metabolic stress induced by the HFD caused the aberrant accumulation of dysfunctional mitochondria, which overwhelmed the mitophagic capacity and was associated with defective maintenance of mitochondrial function and impaired insulin secretion.

Successful pregnancy and childbirth without metabolic abnormality in a patient with holocarboxylase synthetase deficiency.

Holocarboxylase synthetase deficiency (HSD), an autosomal recessive biotin cycle disorder, is caused by holocarboxylase synthetase (HLCS) genetic variants, resulting in multiple carboxylase deficiency. Catabolic stress can induce metabolic crises in patients with HSD. Although pharmacological doses of biotin have improved HLCS enzyme activity and HSD prognosis, the prolonged life expectancy has gradually highlighted novel issues in adult patients with HSD. To the best of our knowledge, there is only one report on a case of HSD during pregnancy and childbirth, and the metabolic profile was not well defined. In this report, we present the history and metabolic profile of a woman with HSD who had an uncomplicated pregnancy and childbirth. A high pharmacological dose of biotin, 100 mg/day, had no effect on the fetus. Even during the emergency cesarean section, the detailed metabolic assessments revealed no significant laboratory findings, such as ketolactic acidosis, hyperammonemia, and remarkable acylcarnitine change. This report suggests that a woman with HSD who regularly takes biotin can conceive and give birth safely, and biotin doses of 100 mg/day may not influence the growth and development of the fetus. Further research and case studies on pregnant women with HSD are required to determine an acceptable maximum dosage of biotin for human fetuses.

Prevention of Shiga toxin 1-caused colon injury by plant-derived recombinant IgA.

Immunoglobulin A (IgA) is a candidate antibody for oral passive immunization against mucosal pathogens like Shiga toxin-producing Escherichia coli (STEC). We previously established a mouse IgG monoclonal antibody (mAb) neutralizing Shiga toxin 1 (Stx1), a bacterial toxin secreted by STEC. We designed cDNA encoding an anti-Stx1 antibody, in which variable regions were from the IgG mAb and all domains of the heavy chain constant region from a mouse IgA mAb. Considering oral administration, we expressed the cDNA in a plant expression system aiming at the production of enough IgA at low cost. The recombinant-IgA expressed in Arabidopsis thaliana formed the dimeric IgA, bound to the B subunit of Stx1, and neutralized Stx1 toxicity to Vero cells. Colon injury was examined by exposing BALB/c mice to Stx1 via the intrarectal route. Epithelial cell death, loss of crypt and goblet cells from the distal colon were observed by electron microscopy. A loss of secretory granules containing MUC2 mucin and activation of caspase-3 were observed by immunohistochemical methods. Pretreatment of Stx1 with the plant-based recombinant IgA completely suppressed caspase-3 activation and loss of secretory granules. The results indicate that a plant-based recombinant IgA prevented colon damage caused by Stx1 in vivo.

A Novel Mouse Model of Idiopathic Nephrotic Syndrome Induced by Immunization with the Podocyte Protein Crb2.

BACKGROUND: The cause of podocyte injury in idiopathic nephrotic syndrome (INS) remains unknown. Although recent evidence points to the role of B cells and autoimmunity, the lack of animal models mediated by autoimmunity limits further research. We aimed to establish a mouse model mimicking human INS by immunizing mice with Crb2, a transmembrane protein expressed at the podocyte foot process. METHODS: C3H/HeN mice were immunized with the recombinant extracellular domain of mouse Crb2. Serum anti-Crb2 antibody, urine protein-to-creatinine ratio, and kidney histology were studied. For signaling studies, a Crb2-expressing mouse podocyte line was incubated with anti-Crb2 antibody. RESULTS: Serum anti-Crb2 autoantibodies and significant proteinuria were detected 4 weeks after the first immunization. The proteinuria reached nephrotic range at 9-13 weeks and persisted up to 29 weeks. Initial kidney histology resembled minimal change disease in humans, and immunofluorescence staining showed delicate punctate IgG staining in the glomerulus, which colocalized with Crb2 at the podocyte foot process. A subset of mice developed features resembling FSGS after 18 weeks. In glomeruli of immunized mice and in Crb2-expressing podocytes incubated with anti-Crb2 antibody, phosphorylation of ezrin, which connects Crb2 to the cytoskeleton, increased, accompanied by altered Crb2 localization and actin distribution. CONCLUSION: The results highlight the causative role of anti-Crb2 autoantibody in podocyte injury in mice. Crb2 immunization could be a useful model to study the immunologic pathogenesis of human INS, and may support the role of autoimmunity against podocyte proteins in INS.

USP40 deubiquitinates HINT1 and stabilizes p53 in podocyte damage.

Podocyte damage is a major pathological lesion leading to focal segmental glomerulosclerosis (FSGS). Podocytes damaged by cellular stress undergo hypertrophy to compensate for podocytopenia. It is known that cyclin-dependent kinase inhibitors induced by p53 ensure podocytes hypertrophy; however, its precise mechanism remains to be further investigated. In this study, we found that ubiquitin specific protease 40 (USP40) is a novel regulator of p53. Although USP40 knockout mice established in the present study revealed no abnormal kidney phenotype, intermediate filament Nestin was upregulated in the glomeruli, and was bound to and colocalized with USP40. We also found that USP40 deubiquitinated histidine triad nucleotide-binding protein 1 (HINT1), an inducer of p53. Gene knockdown experiments of USP40 in cultured podocytes revealed the reduction of HINT1 and p53 protein expression. Finally, in glomerular podocytes of mouse FSGS, upregulation of HINT1 occurred in advance of the proteinuria, which was followed by upregulation of USP40, p53 and Nestin. In conclusion, USP40 bound to Nestin deubiquitinates HINT1, and in consequence upregulates p53. These results provide additional insight into the pathological mechanism of podocyte hypertrophy in FSGS.

Piezo2 expression and its alteration by mechanical forces in mouse mesangial cells and renin-producing cells.

The kidney plays a central role in body fluid homeostasis. Cells in the glomeruli and juxtaglomerular apparatus sense mechanical forces and modulate glomerular filtration and renin release. However, details of mechanosensory systems in these cells are unclear. Piezo2 is a recently identified mechanically activated ion channel found in various tissues, especially sensory neurons. Herein, we examined Piezo2 expression and regulation in mouse kidneys. RNAscope in situ hybridization revealed that Piezo2 expression was highly localized in mesangial cells and juxtaglomerular renin-producing cells. Immunofluorescence assays detected GFP signals in mesangial cells and juxtaglomerular renin-producing cells of Piezo2GFP reporter mice. Piezo2 transcripts were observed in the Foxd1-positive stromal progenitor cells of the metanephric mesenchyme in the developing mouse kidney, which are precursors of mesangial cells and renin-producing cells. In a mouse model of dehydration, Piezo2 expression was downregulated in mesangial cells and upregulated in juxtaglomerular renin-producing cells, along with the overproduction of renin and enlargement of the area of renin-producing cells. Furthermore, the expression of the renin coding gene Ren1 was reduced by Piezo2 knockdown in cultured juxtaglomerular As4.1 cells under static and stretched conditions. These data suggest pivotal roles for Piezo2 in the regulation of glomerular filtration and body fluid balance.

MAB21L1 modulates gene expression and DNA metabolic processes in the lens placode.

Mutations in human MAB21L1 cause aberrations in lens ectoderm morphogenesis and lead to congenital cerebellar, ocular, craniofacial and genital (COFG) syndrome. Murine Mab21l1-null mutations cause severe cell-autonomous defects in lens formation, leading to microphthalmia; therefore, Mab21l1-null mice are used as a mouse model for COFG syndrome. In this study, we investigated the early-onset single-cell-level phenotypes of murine Mab21l1-null lens ectoderms using electron microscopy and single-cell RNA sequencing (scRNA-seq). Electron microscopy and immunohistochemical analyses indicated endoplasmic reticulum stress at the 24- to 26-somite stage in Mab21l1-null lens placodes. scRNA-seq analysis revealed that 131 genes were downregulated and 148 were upregulated in Mab21l1-null lens ectoderms relative to the wild type. We successfully identified 21 lens-specific genes that were downregulated in Mab21l1-null cells, including three key genes involved in lens formation: Pitx3, Maf and Sfrp2. Moreover, gene ontology analysis of the 279 differentially expressed genes indicated enrichment in housekeeping genes associated with DNA/nucleotide metabolism prior to cell death. These findings suggest that MAB21L1 acts as a nuclear factor that modulates not only lens-specific gene expression but also DNA/nucleotide metabolic processes during lens placode formation.

Stability of human salivary extracellular vesicles containing dipeptidyl peptidase IV under simulated gastrointestinal tract conditions.

BACKGROUND: Extracellular vesicles (EVs) have been isolated from various sources, including primary and cultured cell lines and body fluids. Previous studies, including those conducted in our laboratory, have reported the stability of EVs under various storage conditions. METHODS: EVs from human whole saliva were separated via size-exclusion chromatography. To simulate the effects of gastric or intestinal fluids on the stability of EVs, pepsin or pancreatin was added to the samples. Additionally, to determine the effect of bile acids, sodium cholate was added. The samples were then subjected to western blotting, dynamic light scattering, and transmission electron microscopy analyses. In addition, the activity of dipeptidyl peptidase (DPP) IV retained in the samples was examined to monitor the stability of EVs. RESULTS: Under acidic conditions, with pepsin mimicking the milieu of the stomach, the EVs remained stable. However, they partially lost their membrane integrity in the presence of pancreatin and sodium cholate, indicating that they may be destabilized after passing through the duodenum. Although several associated proteins, such as mucin 5B and CD9 were degraded, DPP IV was stable, and its activity was retained under the simulated gastrointestinal conditions. CONCLUSION: Our data indicate that although EVs can pass through the stomach without undergoing significant damage, they may be disrupted in the intestine to release their contents. The consistent delivery of active components such as DPP IV from EVs into the intestine might play a role in the efficient modulation of homeostasis of the signal transduction pathways occurring in the gastrointestinal tract.

Cdc42 has important roles in postnatal angiogenesis and vasculature formation.

Cdc42, a Rho family low molecular weight G protein, has important roles in various cell functions, including cytoskeletal rearrangement, cell adhesion and cell proliferation and differentiation. To investigate the involvement of Cdc42 in the activities of vascular endothelial cells, we generated Cdc42 conditional knockout mice in which Cdc42 was time -specifically deficient in vascular endothelial cells (Cdc42 â€‹fl/fl; VE-Cad CreERT: Cdc42 cKO). When the Cdc42 gene was deleted after birth, Cdc42 cKO mice were smaller than the control mice, and died between postnatal day 8 (P8) and P10. Necropsy findings confirmed that these mice had various pathological aberrances in the vessels of most organs, such as blood flow congestion and blood cell invasion. Electron microscopic observations also revealed that capillary endothelial cells were detached from the basement membrane as well as phagocytosis of dead endothelial cells induced by macrophages. Moreover, vascular sprouting from aortic rings induced by VEGF-A was diminished in samples from the Cdc42 cKO mice because of an endothelial cell proliferation defect. These results suggest that Cdc42 in vascular endothelial cells has important roles in blood vessel formation after birth.

A novel podocyte protein, R3h domain containing-like, inhibits TGF-ß-induced p38 MAPK and regulates the structure of podocytes and glomerular basement membrane.

Not only in kidney glomerular physiological function but also glomerular pathology especially in diabetic condition, glomerular podocytes play pivotal roles. Therefore, it is important to increase our knowledge about the genes and proteins expressed in podocytes. Recently, we have identified a novel podocyte-expressed gene, R3h domain containing-like (R3hdml) and analyzed its function in vivo as well as in vitro. Transforming growth factor-ß (TGF-ß) signaling regulated the expression of R3hdml. And R3hdml inhibited p38 mitogen-activated protein kinase phosphorylation, which was induced by TGF-ß, leading to the amelioration of podocyte apoptosis. Furthermore, a lack of R3hdml in mice significantly worsened glomerular function in streptozotocin (STZ)-induced diabetes, while overexpression of R3hdml ameliorated albuminuria in STZ-induced diabetes. Our results surmise that the functional analyses of R3hdml may lead to the development of novel therapeutic strategies for diabetic nephropathy in the future. KEY MESSAGES: • A novel podocyte expressed protein R3h domain containing-like was identified. • R3HDML inhibits podocyte apoptosis by inhibiting TGF-ß-mediated p38 MAPK signaling. • Overexpression of R3HDML ameliorates albuminuria in STZ-induced diabetes mice. • R3HDML may prove to be a novel therapeutic strategy for diabetic nephropathy.

Mucin-type O-glycosylation controls pluripotency in mouse embryonic stem cells via Wnt receptor endocytosis.

Mouse embryonic stem cells (ESCs) can differentiate into a range of cell types during development, and this pluripotency is regulated by various extrinsic and intrinsic factors. Mucin-type O-glycosylation has been suggested to be a potential factor in the control of ESC pluripotency, and is characterized by the addition of N-acetylgalactosamine (GalNAc) to serine or threonine residues of membrane-anchored proteins and secreted proteins. To date, the relationship between mucin-type O-glycosylation and signaling in ESCs remains undefined. Here, we identify the elongation pathway via C1GalT1 that synthesizes T antigen (Galß1-3GalNAc) as the most prominent among mucin-type O-glycosylation modifications in ESCs. Moreover, we show that mucin-type O-glycosylation on the Wnt signaling receptor frizzled-5 (Fzd5) regulates its endocytosis via galectin-3 binding to T antigen, and that reduction of T antigen results in the exit of the ESCs from pluripotency via canonical Wnt signaling activation. Our findings reveal a novel regulatory mechanism that modulates Wnt signaling and, consequently, ESC pluripotency.This article has an associated First Person interview with the first author of the paper.