Ghrelin enhances tubular magnesium absorption in the kidney.

Osteoporosis after bariatric surgery is an increasing health concern as the rate of bariatric surgery has risen. In animal studies mimicking bariatric procedures, bone disease, together with decreased serum levels of Ca2+, Mg2+ and the gastric hormone Ghrelin were described. Ghrelin regulates metabolism by binding to and activating the growth hormone secretagogue receptor (GHSR) which is also expressed in the kidney. As calcium and magnesium are key components of bone, we tested the hypothesis that Ghrelin-deficiency contributes to osteoporosis via reduced upregulation of the renal calcium channel TRPV5 and the heteromeric magnesium channel TRPM6/7. We expressed GHSR with TRPV5 or TRPM6/7 channel in HEK293 cells and treated them with purified Ghrelin. Whole-cell current density was analyzed by patch-clamp recording. Nephron-specific gene expression was performed by tubular microdissection followed by qPCR in wild-type (WT) mice, and immunofluorescent imaging of GHSR-eGFP mice. Tubular magnesium homeostasis was analyzed in GHSR-null and WT mice at baseline and after caloric restriction. After Ghrelin exposure, whole-cell current density did not change for TRPV5 but increased for TRPM6/7 in a dose-dependent fashion. Applying the Ghrelin-mimetic (D-Trp7, Ala8,D-Phe10)-α-MSH (6-11) amide without and with the GHSR antagonist (D-Lys3)-GHRP6, we confirmed the stimulatory role of Ghrelin towards TRPM6/7. As GHSR initiates downstream signaling via protein kinase A (PKA), we found that the PKA inhibitor H89 abrogated TRPM6/7 stimulation by Ghrelin. Similarly, transfected Gαs, but not the Gαs mutant Q227L, nor Gαi2, Gαq, or Gα13 upregulated TRPM6/7 current density. In microdissected TALs and DCTs similar levels of GHSR mRNA were detected. In contrast, TRPM6 mRNA was expressed in the DCT and also detected in the TAL at 25% expression compared to DCT. Immunofluorescent studies using reporter GHSR-eGFP mice showed a strong eGFP signal in the TAL but surprisingly displayed no eGFP signal in the DCT. In 3-, 6-, and 9-month-old GHSR-null and WT mice, baseline serum magnesium was not significantly different, but 24-h urinary magnesium excretion was elevated in 9-month-old GHSR-null mice. In calorically restricted GHSR-null mice, we detected excess urinary magnesium excretion and reduced serum magnesium levels compared to WT mice. The kidneys from calorically restricted WT mice showed upregulated gene expression of magnesiotropic genes Hnf1b, Cldn-16, Cldn-19, Fxyd-2b, and Parvalbumin compared to GHSR-null mice. Our in vitro studies show that Ghrelin stimulates TRPM6/7 via GHSR and Gαs-PKA signaling. The murine studies are consistent with Ghrelin-GHSR signaling inducing reduced urinary magnesium excretion, particularly in calorically restricted mice when Ghrelin levels are elevated. This effect may be mediated by Ghrelin-upregulation of TRPM6 in the TAL and/or upregulation of other magnesiotropic genes. We postulate that rising Ghrelin levels with hunger contribute to increased renal Mg2+ reabsorption to compensate for lack of enteral Mg2+ uptake.

Endoglycan (PODXL2) is proteolytically processed by ADAM10 (a disintegrin and metalloprotease 10) and controls neurite branching in primary neurons.

Cell adhesion is tightly controlled in multicellular organisms, for example, through proteolytic ectodomain shedding of the adhesion-mediating cell surface transmembrane proteins. In the brain, shedding of cell adhesion proteins is required for nervous system development and function, but the shedding of only a few adhesion proteins has been studied in detail in the mammalian brain. One such adhesion protein is the transmembrane protein endoglycan (PODXL2), which belongs to the CD34-family of highly glycosylated sialomucins. Here, we demonstrate that endoglycan is broadly expressed in the developing mouse brains and is proteolytically shed in vitro in mouse neurons and in vivo in mouse brains. Endoglycan shedding in primary neurons was mediated by the transmembrane protease a disintegrin and metalloprotease 10 (ADAM10), but not by its homolog ADAM17. Functionally, endoglycan deficiency reduced the branching of neurites extending from primary neurons in vitro, whereas deletion of ADAM10 had the opposite effect and increased neurite branching. Taken together, our study discovers a function for endoglycan in neurite branching, establishes endoglycan as an ADAM10 substrate and suggests that ADAM10 cleavage of endoglycan may contribute to neurite branching.

Novel truncating mutations in CTNND1 cause a dominant craniofacial and cardiac syndrome.

CTNND1 encodes the p120-catenin (p120) protein, which has a wide range of functions, including the maintenance of cell-cell junctions, regulation of the epithelial-mesenchymal transition and transcriptional signalling. Due to advances in next-generation sequencing, CTNND1 has been implicated in human diseases including cleft palate and blepharocheilodontic (BCD) syndrome albeit only recently. In this study, we identify eight novel protein-truncating variants, six de novo, in 13 participants from nine families presenting with craniofacial dysmorphisms including cleft palate and hypodontia, as well as congenital cardiac anomalies, limb dysmorphologies and neurodevelopmental disorders. Using conditional deletions in mice as well as CRISPR/Cas9 approaches to target CTNND1 in Xenopus, we identified a subset of phenotypes that can be linked to p120-catenin in epithelial integrity and turnover, and additional phenotypes that suggest mesenchymal roles of CTNND1. We propose that CTNND1 variants have a wider developmental role than previously described and that variations in this gene underlie not only cleft palate and BCD but may be expanded to a broader velocardiofacial-like syndrome.

Disruption of the beclin 1-BCL2 autophagy regulatory complex promotes longevity in mice.

Autophagy increases the lifespan of model organisms; however, its role in promoting mammalian longevity is less well-established1,2. Here we report lifespan and healthspan extension in a mouse model with increased basal autophagy. To determine the effects of constitutively increased autophagy on mammalian health, we generated targeted mutant mice with a Phe121Ala mutation in beclin 1 (Becn1F121A/F121A) that decreases its interaction with the negative regulator BCL2. We demonstrate that the interaction between beclin 1 and BCL2 is disrupted in several tissues in Becn1 F121A/F121A knock-in mice in association with higher levels of basal autophagic flux. Compared to wild-type littermates, the lifespan of both male and female knock-in mice is significantly increased. The healthspan of the knock-in mice also improves, as phenotypes such as age-related renal and cardiac pathological changes and spontaneous tumorigenesis are diminished. Moreover, mice deficient in the anti-ageing protein klotho 3 have increased beclin 1 and BCL2 interaction and decreased autophagy. These phenotypes, along with premature lethality and infertility, are rescued by the beclin 1(F121A) mutation. Together, our data demonstrate that disruption of the beclin 1-BCL2 complex is an effective mechanism to increase autophagy, prevent premature ageing, improve healthspan and promote longevity in mammals.

Nephronectin Regulates Mesangial Cell Adhesion and Behavior in Glomeruli.

A critical aspect of kidney function occurs at the glomerulus, the capillary network that filters the blood. The glomerular basement membrane (GBM) is a key component of filtration, yet our understanding of GBM interactions with mesangial cells, specialized pericytes that provide structural stability to glomeruli, is limited. We investigated the role of nephronectin (Npnt), a GBM component and known ligand of α8ß1 integrin. Immunolocalization and in situ hybridization studies in kidneys of adult mice revealed that nephronectin is produced by podocytes and deposited into the GBM. Conditional deletion of Npnt from nephron progenitors caused a pronounced increase in mesangial cell number and mesangial sclerosis. Nephronectin colocalized with α8ß1 integrin to novel, specialized adhesion structures that occurred at sites of mesangial cell protrusion at the base of the capillary loops. Absence of nephronectin disrupted these adhesion structures, leading to mislocalization of α8ß1. Podocyte-specific deletion of Npnt also led to mesangial sclerosis in mice. These results demonstrate a novel role for nephronectin and α8ß1 integrin in a newly described adhesion complex and begin to uncover the molecular interactions between the GBM and mesangial cells, which govern mesangial cell behavior and may have a role in pathologic states.

Afadin orients cell division to position the tubule lumen in developing renal tubules.

In many types of tubules, continuity of the lumen is paramount to tubular function, yet how tubules generate lumen continuity in vivo is not known. We recently found that the F-actin-binding protein afadin is required for lumen continuity in developing renal tubules, though its mechanism of action remains unknown. Here, we demonstrate that afadin is required for lumen continuity by orienting the mitotic spindle during cell division. Using an in vitro 3D cyst model, we find that afadin localizes to the cell cortex adjacent to the spindle poles and orients the mitotic spindle. In tubules, cell division may be oriented relative to two axes: longitudinal and apical-basal. Unexpectedly, in vivo examination of early-stage developing nephron tubules reveals that cell division is not oriented in the longitudinal (or planar-polarized) axis. However, cell division is oriented perpendicular to the apical-basal axis. Absence of afadin in vivo leads to misorientation of apical-basal cell division in nephron tubules. Together, these results support a model whereby afadin determines lumen placement by directing apical-basal spindle orientation, resulting in a continuous lumen and normal tubule morphogenesis.

Role of CD34 family members in lumen formation in the developing kidney.

Previous studies have shown CD34 family member Podocalyxin is required for epithelial lumen formation in vitro. We demonstrate that Endoglycan, a CD34 family member with homology to Podocalyxin, is produced prior to lumen formation in developing nephrons. Endoglycan localizes to Rab11-containing vesicles in nephron progenitors, and then relocalizes to the apical surface as progenitors epithelialize. Once an apical/luminal surface is formed, Endoglycan (and the actin-binding protein Ezrin) localize to large, intraluminal structures that may be vesicles/exosomes. We generated mice lacking Endoglycan and found mutants had timely initiation of lumen formation and continuous lumens, similar to controls. Mice with conditional deletion of both Endoglycan and Podocalyxin in developing nephrons also had normal tubular lumens. Despite this, Endoglycan/Podocalyxin is required for apical recruitment of the adaptor protein NHERF1, but not Ezrin, in podocyte precursors, a subset of the epithelia. In summary, while CD34 family members appear dispensable for lumen formation, our data identify Endoglycan as a novel pre-luminal marker and suggest lumen formation occurs via vesicular trafficking of apical cargo that includes Endoglycan.

Microfold Cells Actively Translocate Mycobacterium tuberculosis to Initiate Infection.

The prevailing paradigm is that tuberculosis infection is initiated when patrolling alveolar macrophages and dendritic cells within the terminal alveolus ingest inhaled Mycobacterium tuberculosis (Mtb). However, definitive data for this model are lacking. Among the epithelial cells of the upper airway, a specialized epithelial cell known as a microfold cell (M cell) overlies various components of mucosa-associated lymphatic tissue. Here, using multiple mouse models, we show that Mtb invades via M cells to initiate infection. Intranasal Mtb infection in mice lacking M cells either genetically or by antibody depletion resulted in reduced invasion and dissemination to draining lymph nodes. M cell-depleted mice infected via aerosol also had delayed dissemination to lymph nodes and reduced mortality. Translocation of Mtb across two M cell transwell models was rapid and transcellular. Thus, M cell translocation is a vital entry mechanism that contributes to the pathogenesis of Mtb.

Heme Oxygenase-1 Regulates Inflammation and Mycobacterial Survival in Human Macrophages during Mycobacterium tuberculosis Infection.

Mycobacterium tuberculosis, the causative agent of tuberculosis, is responsible for 1.5 million deaths annually. We previously showed that M. tuberculosis infection in mice induces expression of the CO-producing enzyme heme oxygenase (HO1) and that CO is sensed by M. tuberculosis to initiate a dormancy program. Further, mice deficient in HO1 succumb to M. tuberculosis infection more readily than do wild-type mice. Although mouse macrophages control intracellular M. tuberculosis infection through several mechanisms, such as NO synthase, the respiratory burst, acidification, and autophagy, how human macrophages control M. tuberculosis infection remains less well understood. In this article, we show that M. tuberculosis induces and colocalizes with HO1 in both mouse and human tuberculosis lesions in vivo, and that M. tuberculosis induces and colocalizes with HO1 during primary human macrophage infection in vitro. Surprisingly, we find that chemical inhibition of HO1 both reduces inflammatory cytokine production by human macrophages and restricts intracellular growth of mycobacteria. Thus, induction of HO1 by M. tuberculosis infection may be a mycobacterial virulence mechanism to enhance inflammation and bacterial growth.

Loss of diacylglycerol kinase epsilon in mice causes endothelial distress and impairs glomerular Cox-2 and PGE2 production.

Thrombotic microangiopathy (TMA) is a disorder characterized by microvascular occlusion that can lead to thrombocytopenia, hemolytic anemia, and glomerular damage. Complement activation is the central event in most cases of TMA. Primary forms of TMA are caused by mutations in genes encoding components of the complement or regulators of the complement cascade. Recently, we and others have described a genetic form of TMA caused by mutations in the gene diacylglycerol kinase-ε (DGKE) that encodes the lipid kinase DGKε (Lemaire M, Fremeaux-Bacchi V, Schaefer F, Choi MR, Tang WH, Le Quintrec M, Fakhouri F, Taque S, Nobili F, Martinez F, Ji WZ, Overton JD, Mane SM, Nurnberg G, Altmuller J, Thiele H, Morin D, Deschenes G, Baudouin V, Llanas B, Collard L, Majid MA, Simkova E, Nurnberg P, Rioux-Leclerc N, Moeckel GW, Gubler MC, Hwa J, Loirat C, Lifton RP. Nat Genet 45: 531-536, 2013; Ozaltin F, Li BH, Rauhauser A, An SW, Soylemezoglu O, Gonul II, Taskiran EZ, Ibsirlioglu T, Korkmaz E, Bilginer Y, Duzova A, Ozen S, Topaloglu R, Besbas N, Ashraf S, Du Y, Liang CY, Chen P, Lu DM, Vadnagara K, Arbuckle S, Lewis D, Wakeland B, Quigg RJ, Ransom RF, Wakeland EK, Topham MK, Bazan NG, Mohan C, Hildebrandt F, Bakkaloglu A, Huang CL, Attanasio M. J Am Soc Nephrol 24: 377-384, 2013). DGKε is unrelated to the complement pathway, which suggests that unidentified pathogenic mechanisms independent of complement dysregulation may result in TMA. Studying Dgke knockout mice may help to understand the pathogenesis of this disease, but no glomerular phenotype has been described in these animals so far. Here we report that Dgke null mice present subclinical microscopic anomalies of the glomerular endothelium and basal membrane that worsen with age and develop glomerular capillary occlusion when exposed to nephrotoxic serum. We found that induction of cyclooxygenase-2 and of the proangiogenic prostaglandin E2 are impaired in Dgke null kidneys and are associated with reduced expression of the antithrombotic cell adhesion molecule platelet endothelial cell adhesion molecule-1/CD31 in the glomerular endothelium. Notably, prostaglandin E2 supplementation was able to rescue motility defects of Dgke knockdown cells in vitro and to restore angiogenesis in a test in vivo. Our results unveil an unexpected role of Dgke in the induction of cyclooxygenase-2 and in the regulation of glomerular prostanoids synthesis under stress.

Cdc42 regulates epithelial cell polarity and cytoskeletal function during kidney tubule development.

The Rho GTPase Cdc42 regulates key signaling pathways required for multiple cell functions, including maintenance of shape, polarity, proliferation, migration, differentiation and morphogenesis. Although previous studies have shown that Cdc42 is required for proper epithelial development and maintenance, its exact molecular function in kidney development is not well understood. In this study, we define the specific role of Cdc42 during murine kidney epithelial tubulogenesis by deleting it selectively at the initiation of ureteric bud or metanephric mesenchyme development. Deletion in either lineage results in abnormal tubulogenesis, with profound defects in polarity, lumen formation and the actin cytoskeleton. Ultimately, these defects lead to renal failure. Additionally, in vitro analysis of Cdc42-null collecting duct cells shows that Cdc42 controls these processes by regulating the polarity Par complex (Par3-Par6-aPKC-Cdc42) and the cytoskeletal proteins N-Wasp and ezrin. Thus, we conclude that the principal role of Cdc42 in ureteric bud and metanephric mesenchyme development is to regulate epithelial cell polarity and the actin cytoskeleton.

De novo lumen formation and elongation in the developing nephron: a central role for afadin in apical polarity.

A fundamental process in biology is the de novo formation and morphogenesis of polarized tubules. Although these processes are essential for the formation of multiple metazoan organ systems, little is known about the molecular mechanisms that regulate them. In this study, we have characterized several steps in tubule formation and morphogenesis using the mouse kidney as a model system. We report that kidney mesenchymal cells contain discrete Par3-expressing membrane microdomains that become restricted to an apical domain, coinciding with lumen formation. Once lumen formation has been initiated, elongation occurs by simultaneous extension and additional de novo lumen generation. We demonstrate that lumen formation and elongation require afadin, a nectin adaptor protein implicated in adherens junction formation. Mice that lack afadin in nephron precursors show evidence of Par3-expressing membrane microdomains, but fail to develop normal apical-basal polarity and generate a continuous lumen. Absence of afadin led to delayed and diminished integration of nectin complexes and failure to recruit R-cadherin. Furthermore, we demonstrate that afadin is required for Par complex formation. Together, these results suggest that afadin acts upstream of the Par complex to regulate the integration and/or coalescence of membrane microdomains, thereby establishing apical-basal polarity and lumen formation/elongation during kidney tubulogenesis.

p120 catenin is required for normal renal tubulogenesis and glomerulogenesis.

Defects in the development or maintenance of tubule diameter correlate with polycystic kidney disease. Here, we report that absence of the cadherin regulator p120 catenin (p120ctn) from the renal mesenchyme prior to tubule formation leads to decreased cadherin levels with abnormal morphologies of early tubule structures and developing glomeruli. In addition, mutant mice develop cystic kidney disease, with markedly increased tubule diameter and cellular proliferation, and detached luminal cells only in proximal tubules. The p120ctn homolog Arvcf is specifically absent from embryonic proximal tubules, consistent with the specificity of the proximal tubular phenotype. p120ctn knockdown in renal epithelial cells in 3D culture results in a similar cystic phenotype with reduced levels of E-cadherin and active RhoA. We find that E-cadherin knockdown, but not RhoA inhibition, phenocopies p120ctn knockdown. Taken together, our data show that p120ctn is required for early tubule and glomerular morphogenesis, as well as control of luminal diameter, probably through regulation of cadherins.