Podocyte dysfunction, represented by foot process effacement and proteinuria, is often the starting point for progressive kidney disease. Therapies aimed at the cellular level of the disease are currently not available. Here we show that induction of urokinase receptor (uPAR) signaling in podocytes leads to foot process effacement and urinary protein loss via a mechanism that includes lipid-dependent activation of alphavbeta3 integrin. Mice lacking uPAR (Plaur-/-) are protected from lipopolysaccharide (LPS)-mediated proteinuria but develop disease after expression of a constitutively active beta3 integrin. Gene transfer studies reveal a prerequisite for uPAR expression in podocytes, but not in endothelial cells, for the development of LPS-mediated proteinuria. Mechanistically, uPAR is required to activate alphavbeta3 integrin in podocytes, promoting cell motility and activation of the small GTPases Cdc42 and Rac1. Blockade of alphavbeta3 integrin reduces podocyte motility in vitro and lowers proteinuria in mice. Our findings show a physiological role for uPAR signaling in the regulation of kidney permeability.
Kidney podocytes and their slit diaphragms form the final barrier to urinary protein loss. This explains why podocyte injury is typically associated with nephrotic syndrome. The present study uncovered an unanticipated novel role for costimulatory molecule B7-1 in podocytes as an inducible modifier of glomerular permselectivity. B7-1 in podocytes was found in genetic, drug-induced, immune-mediated, and bacterial toxin-induced experimental kidney diseases with nephrotic syndrome. The clinical significance of our results is underscored by the observation that podocyte expression of B7-1 correlated with the severity of human lupus nephritis. In vivo, exposure to low-dose LPS rapidly upregulates B7-1 in podocytes of WT and SCID mice, leading to nephrotic-range proteinuria. Mice lacking B7-1 are protected from LPS-induced nephrotic syndrome, suggesting a link between podocyte B7-1 expression and proteinuria. LPS signaling through toll-like receptor-4 reorganized the podocyte actin cytoskeleton in vitro, and activation of B7-1 in cultured podocytes led to reorganization of vital slit diaphragm proteins. In summary, upregulation of B7-1 in podocytes may contribute to the pathogenesis of proteinuria by disrupting the glomerular filter and provides a novel molecular target to tackle proteinuric kidney diseases. Our findings suggest a novel function for B7-1 in danger signaling by nonimmune cells. 1390The Nonstandard abbreviations used: foot process (FP); glomerular basement membrane (GBM); puromycin aminonucleoside (PAN); slit diaphragm (SD); toll-like receptor (TLR).
The spine apparatus is a cellular organelle that is present in many dendritic spines of excitatory neurons in the mammalian forebrain. Despite its discovery >40 years ago, the function of the spine apparatus is still unknown although calcium buffering functions as well as roles in synaptic plasticity have been proposed. We have recently shown that the 100-kDa protein synaptopodin is associated with the spine apparatus. Here, we now report that mice homozygous for a targeted deletion of the synaptopodin gene completely lack spine apparatuses. Interestingly, this absence of the spine apparatus is accompanied by a reduction in hippocampal long-term potentiation (LTP) in the CA1 region of the hippocampus and by an impairment of spatial learning in the radial arm maze test. This genetic analysis points to a role of the spine apparatus in synaptic plasticity.
We report the cloning and functional characterization of myopodin, the second member of the synaptopodin gene family. Myopodin shows no significant homology to any known protein except synaptopodin. Northern blot analysis resulted in a 3.6-kb transcript for mouse skeletal and heart muscle. Western blots showed an 80-kD signal for skeletal and a 95-kD signal for heart muscle. Myopodin contains one PPXY motif and multiple PXXP motifs. Myopodin colocalizes with α-actinin and is found at the Z-disc as shown by immunogold electron microscopy. In myoblasts, myopodin shows preferential nuclear localization. During myotube differentiation, myopodin binds to stress fibers in a punctuated pattern before incorporation into the Z-disc. Myopodin can directly bind to actin and contains a novel actin binding site in the center of the protein. Myopodin has actin-bundling activity as shown by formation of latrunculin-A–sensitive cytosolic actin bundles and nuclear actin loops in transfected cells expressing green fluorescent protein–myopodin. Under stress conditions, myopodin accumulates in the nucleus and is depleted from the cytoplasm. Nuclear export of myopodin is sensitive to leptomycin B, despite the absence of a classical nuclear export sequence. We propose a dual role for myopodin as a structural protein also participating in signaling pathways between the Z-disc and the nucleus.
Synaptic ribbons are large, dynamic structures in the active zone complex of ribbon synapses and important for the physiological properties of these tonically active synapses. RIBEYE is a unique and major protein component of synaptic ribbons. The aim of the present study was to understand how the synaptic ribbon is built and how the construction of the ribbon could contribute to its ultrastructural plasticity. In the present study, we demonstrate that RIBEYE self-associates using different independent approaches (yeast two-hybrid analyses, protein pull downs, synaptic ribbon-RIBEYE interaction assays, coaggregation experiments, transmission electron microscopy and immunogold electron microscopy). The A -domain [RIBEYE(A)] and B-domain [RIBEYE(B)] of RIBEYE contain five distinct sites for RIBEYE-RIBEYE interactions. Three interaction sites are present in the A-domain of RIBEYE and mediate RIBEYE(A)-RIBEYE(A) homodimerization and heterodimerization with the B-domain. The docking site for RIBEYE(A) on RIBEYE(B) is topographically and functionally different from the RIBEYE(B) homodimerization interface and is negatively regulated by nicotinamide adenine dinucleotide. The identified multiple RIBEYE-RIBEYE interactions have the potential to build the synaptic ribbon:heterologously expressed RIBEYE forms large electron-dense aggregates that are in part physically associated with surrounding vesicles and membrane compartments. These structures resemble spherical synaptic ribbons. These ribbon-like structures coassemble with the active zone protein bassoon, an interaction partner of RIBEYE at the active zone of ribbon synapses, emphasizing the physiological relevance of these RIBEYE-containing aggregates. Based on the identified multiple RIBEYE-RIBEYE interactions, we provide a molecular mechanism for the dynamic assembly of synaptic ribbons from individual RIBEYE subunits.
NPHS1 has recently been identified as the gene whose mutations cause congenital nephrotic syndrome of the Finnish type. The respective gene product nephrin is a transmembrane protein expressed in glomerular podocytes and primarily localized to the glomerular slit diaphragm. This interpodocyte junction functions in the glomerular filtration by restricting the passage of plasma proteins into the urinary space in a size-selective manner. The functional role of nephrin in this filtration process is so far not very well understood. In this study, we show that nephrin associates in an oligomerized form with signaling microdomains, also known as lipid rafts, and that these localize to the slit diaphragm. We also show that the nephrin-containing rafts can be immunoisolated with the 27A antibody recognizing a podocyte-specific 9-Oacetylated GD3 ganglioside. In a previous study it has been shown that the in vivo injection of this antibody leads to morphological changes of the filtration slits resembling foot process effacement. Here, we report that, in this model of foot process effacement, nephrin dislocates to the apical pole of the narrowed filtration slits and also that it is tyrosine phosphorylated. We suggest that lipid rafts are important in the spatial organization of the glomerular slit diaphragm under physiological and pathological conditions. One of the main functions of the kidney is formation of the primary urine in the glomerulus by plasma ultrafiltration. The structural correlate of ultrafiltration is the glomerular capillary wall with its distinctly layered filtration barrier. It consists of the fenestrated vascular endothelium, the glomerular basement membrane, and a layer of morphologically highly specialized epithelial cells, podocytes.Podocytes form a tight network of multiple interdigitating cellular extensions, called foot processes, which are bridged by so-called "slit diaphragms." 1 The filtration barrier is freely permeable for water, small solutes, and ions but does not allow passage for proteins larger than albumin and other large molecules. The charge selectivity in this process has been attributed to the glomerular basement membrane whereas the slit diaphragm supposedly plays a major role in the size selectivity. 2,3 The importance of the filtration barrier is reflected by a number of human diseases in which the filtration barrier function is disrupted resulting in the loss of plasma proteins into the urine. Patients with persistent proteinuria ultimately develop a nephrotic syndrome with a variety of symptoms including edema, hypoalbuminemia, and hyperlipidemia. 4
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