Localization of the succinate receptor in the distal nephron and its signaling in polarized MDCK cells

When the succinate receptor (SUCNR1) is activated in the afferent arterioles of the glomerulus it increases renin release and induces hypertension. To study its location in other nephron segments and its role in kidney function, we performed immunohistochemical analysis and found that SUCNR1 is located in the luminal membrane of macula densa cells of the juxtaglomerular apparatus in close proximity to renin-producing granular cells, the cortical thick ascending limb, and cortical and inner medullary collecting duct cells. In order to study its signaling, SUCNR1 was stably expressed in Madin-Darby Canine Kidney (MDCK) cells, where it localized to the apical membrane. Activation of the cells by succinate caused Gq and Gi-mediated intracellular calcium mobilization, transient phosphorylation of extracellular regulated kinase (ERK)1/2 and the release of arachidonic acid along with prostaglandins E2 and I2. Signaling was desensitized without receptor internalization but rapidly resensitized upon succinate removal. Immunohistochemical evidence of phosphorylated ERK1/2 was found in cortical collecting duct cells of wild type but not SUCNR1 knockout streptozotocin-induced diabetic mice, indicating in vivo relevance. Since urinary succinate concentrations in health and disease are in the activation range of the SUCNR1, this receptor can sense succinate in the luminal fluid. Our study suggests that changes in the luminal succinate concentration may regulate several aspects of renal function

A human polymorphism affects NEDD4L subcellular targeting by leading to two isoforms that contain or lack a C2 domain

<p>Abstract</p> <p>Background</p> <p>Ubiquitination serves multiple cellular functions, including proteasomal degradation and the control of stability, function, and intracellular localization of a wide variety of proteins. NEDD4L is a member of the HECT class of E3 ubiquitin ligases. A defining feature of NEDD4L protein isoforms is the presence or absence of an amino-terminal C2 domain, a class of subcellular, calcium-dependent targeting domains. We previously identified a common variant in human <it>NEDD4L </it>that generates isoforms that contain or lack a C2 domain.</p> <p>Results</p> <p>To address the potential functional significance of the <it>NEDD4L </it>common variant on NEDD4L subcellular localization, NEDD4L isoforms that either contained or lacked a C2 domain were tagged with enhanced green fluorescent protein, transfected into <it>Xenopus laevis </it>kidney epithelial cells, and imaged by performing confocal microscopy on live cells. We report that the presence or absence of this C2 domain exerts differential effects on the subcellular distribution of NEDD4L, the ability of C2 containing and lacking NEDD4L isoforms to mobilize in response to a calcium stimulus, and the intracellular transport of subunits of the NEDD4L substrate, ENaC. Furthermore, the ability of the C2-containing isoform to influence β-ENaC mobilization from intracellular pools involves the NEDD4L active site for ubiquitination. We propose a model to account for the potential impact of this common genetic variant on protein function at the cellular level.</p> <p>Conclusion</p> <p>NEDD4L isoforms that contain or lack a C2 domain target different intracellular locations. Additionally, whereas the C2-containing NEDD4L isoform is capable of shuttling between the plasma membrane and intracellular compartments in response to calcium stimulus the C2-lacking isoform can not. The C2-containing isoform differentially affects the mobilization of ENaC subunits from intracellular pools and this trafficking step requires NEDD4L ubiquitin ligase activity. This observation suggests a new mechanism for the requirement for the PY motif in cAMP-mediated exocytosis of ENaC. We have elucidated how a common genetic variant can underlie significant functional diversity in NEDD4L at the cellular level. We propose a model that describes how that functional variation may influence blood pressure. Moreover, our observations regarding differential function of the NEDD4L isoforms may impact other aspects of physiology that involve this ubiquitin ligase.</p

A conditionally immortalized cell line from murine proximal tubule

A conditionally immortalized cell line from murine proximal tubule. We have developed a conditionally immortalized murine cell line with proximal tubule characteristics (tsMPT) and a background suitable for genetic manipulations. tsMPT was derived from the F1 progeny of crosses between: [1] a transgenic mouse harboring a γ-interferon (IFN-γ)-inducible, temperature sensitive SV40 large T antigen gene (tsA58) and [2] mice of the 129/SvEv strain, the background from which most embryonic stem (ES) cells are derived. Under permissive conditions (33°C and in the presence of IFN-γ), tsMPT cells grow rapidly as monolayers with a doubling time of 23 hours; the large T antigen can be detected by immunocytochemistry and by Western blotting. When transferred to non-permissive conditions (39°C, without IFN-γ), the cells undergo differentiation coinciding with the disappearance of the large T antigen. By electron microscopy, tsMPT cells are polarized and show microvilli at their apical surface. tsMPT cells express brush border enzymes γ-glutamyl transpeptidase and carbonic anhydrase IV. They possess Na+-dependent transport systems for Pi, D-glucose and L-proline as well as an amiloride-insensitive Na+-H+ exchanger. Intracellular cAMP generation is stimulated by parathyroid hormone but not by arginine vasopressin. Angiotensinogen mRNA and protein are present in tsMPT with markedly higher levels at non-permissive conditions. tsMPT cells should be a useful model for investigation of the functional features of the proximal tubule epithelium in relation to cellular differentiation

Phenotypic dissection of the mouse Ren-1(d) knockout by complementation with human renin

Normal renin synthesis and secretion is important for the maintenance of juxtaglomerular apparatus architecture. Mice lacking a functional Ren-1d gene are devoid of renal juxtaglomerular cell granules and exhibit an altered macula densa morphology. Due to the species-specificity of renin activity, transgenic mice are ideal models for experimentally investigating and manipulating expression patterns of the human renin gene in a native cellular environment without confounding Renin-angiotensin-system interactions. A 55 kb transgene encompassing the human renin locus was crossed onto the mouse Ren-1d-null background, restoring granulation in juxtaglomerular cells.  Correct processing of human renin in dense core granules was confirmed by immunogold labelling. After stimulation of the renin-angiotensin system, juxtaglomerular cells contained rhomboid protogranules with paracrystalline contents, dilated rough endoplasmic reticulum and electron-lucent granular structures. However, complementation of Ren-1d-/- mice with human renin was unable to rescue the abnormality seen in macula densa structure. The juxtaglomerular apparatus was still able to respond to tubuloglomerular feedback in isolated perfused juxtaglomerular apparatus preparations, although minor differences in glomerular tuft contractility and macula densa cell calcium handling were observed. This study reveals that the human renin protein is able to complement the mouse Ren-1d-/- non-granulated defect and suggests that granulopoiesis requires a structural motif that is conserved between the mouse Ren-1d and human renin proteins. It also suggests that the altered macula densa phenotype is related to the activity of the renin-1d enzyme in a local juxtaglomerular renin-angiotensin system

Simulation of Vehicle-Pedestrian Interaction

The literature on vehicle crash reconstruction provides a number of empirical or classical theoretical models for the distance pedestrians are thrown in impacts with various types of vehicles and impact speeds. The aim of this research was to compare the predictions offered by computer simulation to those obtained using the empirical and classical theoretical models traditionally utilised in vehicle-pedestrian accident reconstruction. Particular attention was paid to the pedestrian throw distance versus vehicle impact speed relationship and the determination of pedestrian injury patterns and associated severity. It was discovered that computer simulation offered improved pedestrian kinematic prediction in comparison to traditional vehicle-pedestrian accident reconstruction techniques. The superior kinematic prediction was found to result in a more reliable pedestrian throw distance versus vehicle impact speed relationship, particularly in regard to varying vehicle and pedestrian parameters such as shape, size and orientation. The pedestrian injury prediction capability of computer simulation was found to be very good for head and lower extremity injury determination. Such injury prediction capabilities were noted to be useful in providing additional correlation of vehicle impact speed predictions, whether these predictions were made using computer simulation, traditional vehicle-pedestrian accident reconstruction methods or a combination of both. A generalised approach to the use of computer simulation for the reconstruction of vehicle-pedestrian accidents was also offered. It is hoped that this approach is developed and improved by other researchers so that over time guidelines for a standardised approach to the simulation of vehicle-pedestrian accidents might evolve. Thoracic injury prediction, particularly for frontal impacts, was found to be less than ideal. It is suspected that the relatively poor thoracic biofidelity stems from the development of pedestrian mathematical models from occupant mathematical models, which were in turn developed from cadaver and dummy tests. It is hoped that future research will result in improved thoracic biofidelity in human mathematical models

Scalable Newborn Screening Solutions: Bioinformatics and Next-Generation Sequencing

Expansion of the newborn disorder panel requires the incorporation of new testing modalities. This is especially true for disorders lacking robust biomarkers for detection in primary screening methods and for disorders requiring genotyping or sequencing as a second-tier and/or diagnostic test. In this commentary, we discuss how next-generation sequencing (NGS) methods can be used as a secondary testing method in NBS. Additionally, we elaborate on the importance of genomic variant repositories for the annotation and interpretation of variants. Barriers to the incorporation of NGS and bioinformatics within NBS are discussed, and ideas for a regional bioinformatics model and shared variant repository are presented as potential solutions

Papier, Schrift und Feuer. Zu einer produktiven Konstellation

Körte M. Papier, Schrift und Feuer. Zu einer produktiven Konstellation . In: Lethen H, Pelz A, Rohrwasser M, Universität Wien, eds. Konstellationen - Versuchsanordnungen des Schreibens. Schriften der Wiener Germanistik. Vol 1. Wien: Vienna Univ. Press; 2013: 243