Robin Kunkel

In the present paper, we characterize an antibody, mAb BV13, directed to mouse vascular endothelial (VE)-cadherin, a major adhesive protein of interendothelial adherens junctions. When added to cultured endothelial cells, BV13 induces a redistribution of VE-cadherin from intercellular junctions. VE-cadherin redistribution did not change the localization of platelet endothelial cell adhesion molecule or tight junction markers such as zonula occludens 1, cingulin, and junctional adhesion molecule. Intravenous administration of mAb BV13 induced a concentration- and time-dependent increase in vascular permeability in heart and lungs. By electron microscopy, interstitial edema and accumulation of mixed types of inflammatory cells in heart and lungs were observed. Injection of (rhodamine-labeled) Ricinus communis I lectin showed focal spots of exposed basement membrane in the alveolar capillaries and in some larger pulmonary vessels. These data indicate that VE-cadherin is required for vascular integrity and normal organ functions.

Intravascular activation of the complement system with cobra venom factor results in acute lung injury, which has been quantitated by increases in lung vascular permeability. Cobra venom factor preparations devoid of phospholipase A2 activity retain full lung-damaging capacity. The lung injury is associated with the preceding appearance of chemotactic activity in the serum coincident with the development of a profound neutropenia. The chemotactic activity is immunochemically related to human C5a. Morphologic studies have revealed discontinuities in the endothelial cell lining of lung alveolar capillaries, damage and/or destruction of endothelial cells in these areas, plugging of pulmonary capillaries with neutrophils that are in direct contact with vascular basement membrane, the presence of fibrin in alveolar spaces and in areas adjacent to damaged endothelial cells, and intraalveolar hemorrhage. Lung injury is dramatically attenuated in animals that have been previously neutrophil depleted. Teh intravenous injection of superoxide dismutase or catalase also provides significant protection from the pulmonary damage. Very little protection from the pulmonary damage. Very little protection is afforded by pretreatment of rats with antihistamine. These studies suggest that intravascular activation of the complement system leads to neutrophil aggregation and activation, intrapulmonary capillary sequestration of neutrophils, and vascular injury, which may be related to production of toxic oxygen metabolites by complement-activated neutrophils.

Using our recently described model of acute lung injury in rats after systemic activation of complement by cobra venom factor (CVF), we demonstrated that pretreatment of animals with human milk apolactoferrin (in its native or derivatized form), but not iron-saturated lactoferrin, provides significant protection against complement- and neutrophil-mediated lung injury. The synthetic iron chelator deferoxamine mesylate also affords protection from lung injury. The protective effects of apolactoferrin are not related to a blocking of CVF-induced complement activation. We also demonstrated that infusion of ionic iron, especially Fe3+, greatly potentiates lung vascular injury after systemic complement activation. Finally, protection from lung injury occurs in animals pretreated with the potent scavenger of hydroxyl radicals (OH.), dimethyl sulfoxide. Based on transmission electron microscopy, CVF-treated rats show leukoaggregates and endothelial cell destruction in interstitial pulmonary capillaries, along with intraalveolar hemorrhage and fibrin deposition. In animals protected with apolactoferrin, deferoxamine mesylate, or dimethyl sulfoxide, the morphological studies reveal leukoaggregates but no endothelial cell damage, hemorrhage, or fibrin deposition. These data support the concept that tissue injury that is complement and neutrophil dependent may be related to generation of OH. derived from H2O2 after leukocytic activation.

Recent investigations have focused on characterizing the molecular components of the podocyte intercellular junction, because several of these components, including Nephrin, are functionally necessary for development of normal podocyte structure and filter integrity. Accumulating evidence suggests that the Nephrin-associated protein complex is a signaling nexus. As such, Nephrin-dependent signaling might be mediated in part through Nephrin phosphorylation. Described are biochemical and mouse genetics experiments demonstrating that membrane-associated Nephrin is tyrosine-phosphorylated by the Src family kinase Fyn. Nephrin fractionated in detergent-resistant glomerular membrane fractions with Fyn and Yes. Fyn directly bound Nephrin via its SH3 domain, and Fyn directly phosphorylated Nephrin. Glomeruli in which Fyn, Yes, or Fyn and Yes were genetically deleted in mice were characterized to explore the relationship between these kinases and Nephrin. Fyn deletion resulted in coarsening of podocyte foot processes and marked attenuation of Nephrin phosphorylation in isolated glomerular detergent-resistant membrane fractions. Yes deletion had no identifiable effect on podocyte morphology but dramatically increased Nephrin phosphorylating activity. Similar to Fyn deletion, simultaneous deletion of Fyn and Yes reduced Nephrin phosphorylating activity. These results demonstrate that endogenous Fyn catalyzes Nephrin phosphorylation in podocyte detergent-resistant membrane fractions. Although Yes appears to effect the regulation of Nephrin phosphorylation, the mechanism by which this occurs requires investigation. Recent investigations have focused on characterizing the molecular components of the podocyte intercellular junction, because several of these components, including Nephrin, are functionally necessary for development of normal podocyte structure and filter integrity. Accumulating evidence suggests that the Nephrin-associated protein complex is a signaling nexus. As such, Nephrin-dependent signaling might be mediated in part through Nephrin phosphorylation. Described are biochemical and mouse genetics experiments demonstrating that membrane-associated Nephrin is tyrosine-phosphorylated by the Src family kinase Fyn. Nephrin fractionated in detergent-resistant glomerular membrane fractions with Fyn and Yes. Fyn directly bound Nephrin via its SH3 domain, and Fyn directly phosphorylated Nephrin. Glomeruli in which Fyn, Yes, or Fyn and Yes were genetically deleted in mice were characterized to explore the relationship between these kinases and Nephrin. Fyn deletion resulted in coarsening of podocyte foot processes and marked attenuation of Nephrin phosphorylation in isolated glomerular detergent-resistant membrane fractions. Yes deletion had no identifiable effect on podocyte morphology but dramatically increased Nephrin phosphorylating activity. Similar to Fyn deletion, simultaneous deletion of Fyn and Yes reduced Nephrin phosphorylating activity. These results demonstrate that endogenous Fyn catalyzes Nephrin phosphorylation in podocyte detergent-resistant membrane fractions. Although Yes appears to effect the regulation of Nephrin phosphorylation, the mechanism by which this occurs requires investigation. Fyn binds to and phosphorylates the kidney slit diaphragm component Nephrin. Vol. 278 (2003) 20716-20723Journal of Biological ChemistryVol. 280Issue 28PreviewPages 20719 and 20720 (Fig. 5): Fig. 5 in this paper was inadvertently mislabeled. A revision of this figure is shown below. In Fig. 5, A, and B, the labels GST-FynSH2 and GST-FynSH3 were reversed. As a result, the conclusion reached in the text on page 20719 (second full paragraph) is incorrect. It should properly read: “In these experiments, Fyn SH2 domain but not its SH3 domain interacted with Nephrin obtained from COS7 cells pretreated with pervanadate (Fig. 5A).... In similar pull-down experiments, GST-Fyn SH2 but not Fyn SH3 interacted with Nephrin obtained from lysates of isolated glomeruli (Fig. Full-Text PDF Open Access Diseases of the renal glomerulus that result in the nephrotic syndrome are important causes of morbidity and mortality affecting both adults and children. Unfortunately, the molecular mechanisms governing development of the nephrotic syndrome remain poorly understood (1Somlo S. Mundel P. Nat. Genet. 2000; 24: 333-335Crossref PubMed Scopus (235) Google Scholar, 2Smoyer W.E. Mundel P. J. Mol. Med. 1998; 76: 172-183Crossref PubMed Scopus (141) Google Scholar). Glomerular visceral epithelial cells appear to play a central role in maintaining the selective filtration barrier of the renal glomerulus. These cells are also termed podocytes to describe the foot-like appearance of numerous interdigitating processes that arise from their cell bodies and cover glomerular capillary walls. Glomerular filtrate passes across the specialized intercellular junction-also termed the “slit diaphragm” formed at the interface of these interdigitated foot processes (3Reiser J. Kriz W. Kretzler M. Mundel P. J. Am. Soc. Nephrol. 2000; 11: 1-8Crossref PubMed Google Scholar). In response to glomerular injury, podocytes undergo a dramatic change in morphology termed “foot process effacement” resulting in retraction and spreading of foot processes and alteration in their intercellular junctions (4Seiler, M. W., Rennke, H. G., Venkatachalam, M. A., and Cotran, R. S. (1997) 36, 48–61Google Scholar). Foot process effacement is a fluid and reversible process that correlates closely with the development of proteinuria both in human disease and in experimental models (2Smoyer W.E. Mundel P. J. Mol. Med. 1998; 76: 172-183Crossref PubMed Scopus (141) Google Scholar, 5Kriz W. Gretz N. Lemley K.V. Kidney. Int. 1998; 54: 687-697Abstract Full Text Full Text PDF PubMed Scopus (513) Google Scholar). Clearly, foot process effacement requires precise interplay of multiple cellular processes, including alterations in the structure of the cytoskeleton, movement of foot process over the basement membrane, and disassembly or re-assembly of the intercellular junction that comprises the slit diaphragm. Each of these unique but interdependent cellular processes are in and of themselves complex. The cellular and molecular mechanisms governing podocyte morphology and filter integrity are incompletely defined. Recent investigations have focused on identifying and characterizing the interrelationships and functions of the molecular components of the foot process intercellular junction, because several of these components, including Nephrin (6Kestila M. Lenkkeri U. Mannikko M. Lamerdin J. McCready P. Putaala H. Ruotsalainen V. Morita T. Nissinen M. Herva R. Kashtan C.E. Peltonen L. Holmberg C. Olsen A. Tryggvason K. Mol. Cell. 1998; 1: 575-582Abstract Full Text Full Text PDF PubMed Scopus (1566) Google Scholar), Podocin (7Boute N. Gribouval O. Roselli S. Benessy F. Lee H. Fuchshuber A. Dahan K. Gubler M.C. Niaudet P. Antignac C. Nat. Genet. 2000; 24: 349-354Crossref PubMed Scopus (1199) Google Scholar), and CD2ap (8Shih N.Y. Li J. Karpitskii V. Nguyen A. Dustin M.L. Kanagawa O. Miner J.H. Shaw A.S. Science. 1999; 286: 312-315Crossref PubMed Scopus (698) Google Scholar), have been demonstrated to be necessary for development of normal podocyte structure and filter integrity. Nephrin is encoded by NPHS1, the gene mutated in congenital nephrotic syndrome of the Finnish type, a rare autosomal recessive developmental disorder manifest at birth by heavy proteinuria and diffuse podocyte foot process effacement (9Holmberg C. Antikainen M. Ronnholm K. Ala-Houhala M. Jalanko H. Pediatr. Nephrol. 1995; 9: 87-93Crossref PubMed Scopus (172) Google Scholar, 10Laine J. Jalanko H. Holthofer H. Krogerus L. Rapola J. von Willebrand E. Lautenschlager I. Salmela K. Holmberg C. Kidney. Int. 1993; 44: 867-874Abstract Full Text PDF PubMed Scopus (52) Google Scholar). Deletion of Nephrin by gene targeting in mice results in a similar phenotype (11Putaala H. Soininen R. Kilpelainen P. Wartiovaara J. Tryggvason K. Hum. Mol. Genet. 2001; 10: 1-8Crossref PubMed Scopus (423) Google Scholar). In the kidney, Nephrin expression is podocyte-specific, and the protein is targeted to the lateral aspect of foot process cell membranes in the region of the slit diaphragm (12Ruotsalainen V. Ljungberg P. Wartiovaara J. Lenkkeri U. Kestila M. Jalanko H. Holmberg C. Tryggvason K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7962-7967Crossref PubMed Scopus (610) Google Scholar, 13Holzman L.B. St John P.L. Kovari I.A. Verma R. Holthofer H. Abrahamson D.R. Kidney Int. 1999; 56: 1481-1491Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). Nephrin is a member of the immunoglobulin superfamily. For this reason, it has been proposed that Nephrin is a cell adhesion molecule (CAM) 1The abbreviations used are: CAM, cell adhesion molecule; GST, glutathione S-transferase; RIPA, radioimmune precipitation assay; PBS, phosphate-buffered saline; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; DRM, detergent-resistant membrane fraction; CD, cytoplasmic domain; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; PP3, 4-amino-7-phenylpyrazol[3,4-d]pyrimidine; SFK, Src family kinase. that participates in forming the glomerular filter via interactions involving its extracellular domain (12Ruotsalainen V. Ljungberg P. Wartiovaara J. Lenkkeri U. Kestila M. Jalanko H. Holmberg C. Tryggvason K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7962-7967Crossref PubMed Scopus (610) Google Scholar). More than simply mediating cellular interactions, various Ig superfamily CAMs behave as signal transducing molecules. Depending on the particular CAM involved, Ig superfamily CAMs can play a role in either “outside-in” signaling in which a signal is transduced into the cell following ligand binding or can participate in “inside-out” signaling in which signals originating within the cell result in changes in binding characteristics of the CAM. Like other transmembrane proteins, Nephrin participates in a protein complex at the cytoplasmic face of the plasma membrane. To date, Nephrin has been shown to associate with Podocin, the protein product of NPHS2 that is mutated in an inherited form of steroid-resistant focal and segmental glomerulosclerosis (7Boute N. Gribouval O. Roselli S. Benessy F. Lee H. Fuchshuber A. Dahan K. Gubler M.C. Niaudet P. Antignac C. Nat. Genet. 2000; 24: 349-354Crossref PubMed Scopus (1199) Google Scholar). Nephrin also binds CD2ap, an adapter protein whose genetic deletion in mice results in foot process effacement and proteinuria (8Shih N.Y. Li J. Karpitskii V. Nguyen A. Dustin M.L. Kanagawa O. Miner J.H. Shaw A.S. Science. 1999; 286: 312-315Crossref PubMed Scopus (698) Google Scholar, 14Shih N.Y. Li J. Cotran R. Mundel P. Miner J.H. Shaw A.S. Am. J. Pathol. 2001; 159: 2303-2308Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). These observations suggest that, in a fashion similar to other cell adhesion molecules, Nephrin might function in intracellular signal transduction. Nephrin-dependent signaling might be mediated in part through Nephrin phosphorylation. Indeed, as described herein, biochemical and genetic evidence demonstrates that, in the plasma membrane of podocytes, Nephrin is tyrosine-phosphorylated by the Src family protein kinase Fyn. Reagents—Rabbit polyclonal antibodies to the cytoplasmic domain of mouse Nephrin (13Holzman L.B. St John P.L. Kovari I.A. Verma R. Holthofer H. Abrahamson D.R. Kidney Int. 1999; 56: 1481-1491Abstract Full Text Full Text PDF PubMed Scopus (258) Google and to the of human or mouse Podocin A. L.B. J. Am. Soc. Nephrol. PubMed Scopus Google were described to a protein the and SH3 of mouse CD2ap was by at Biological and antibodies to Fyn or Yes and and mouse and immunoglobulin were obtained and GST-FynSH2 were described A. C. Cell. 1998; Full Text Full Text PDF Scopus Google Scholar). GST-Fyn and Nephrin cytoplasmic domain (13Holzman L.B. St John P.L. Kovari I.A. Verma R. Holthofer H. Abrahamson D.R. Kidney Int. 1999; 56: 1481-1491Abstract Full Text Full Text PDF PubMed Scopus (258) Google were and from lysates as described M. L.B. J. Full Text Full Text PDF PubMed Scopus Google Scholar, M. C. L.B. J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). expression Fyn, Yes, and were described A. C. Cell. 1998; Full Text Full Text PDF Scopus Google Scholar). expression mouse was in a similar to that described S. L.B. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). The expression a form of the cytoplasmic domain of mouse Nephrin was by a PP2, a of Src kinases and PP3, its were obtained from Fyn and were obtained were in COS7 The cells were in with and and were cells were in the of pervanadate for to For of cells were and with with for 5 at and and experiments were the antibodies as described S. L.B. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). Nephrin and Src family protein kinases were from plasma membranes in and was as described M. L.B. J. Full Text Full Text PDF PubMed Scopus Google Scholar). Nephrin was by and to a membrane, and the of the molecular was were by were by and was by a and and for mice and mice were obtained from Yes mouse and Fyn mouse were obtained by mice were obtained by mice with and with was from at a A was to mice as described on the For Fyn the following were from The of the product is from the For Yes the following were and A product was from the Yes A and a product were from the targeted mice were obtained from The of on and of and was in with the and in the of for the and of Glomerular were obtained from or Glomeruli were isolated by as described (13Holzman L.B. St John P.L. Kovari I.A. Verma R. Holthofer H. Abrahamson D.R. Kidney Int. 1999; 56: 1481-1491Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). The of the glomerular was for mice and for Glomeruli were in the at a of glomeruli of glomeruli from or mouse were at with of a in a 5 and To the was to a of and The was on for with to a of of this was to with of and of were obtained by with Tricine, and The was at in a for at The membrane membrane was at the interface between 5 and to fractions were and as from mice were for a and for by the In on Glomerular fractions were in kinase 5 5 and a of and by at at These were in kinase 5 of and at for were in the of or were and with kinase and were with and at and the obtained were or as in the were either with isolated glomerular or with obtained from COS7 cells with Nephrin. The were to with PBS, and of glutathione was to The were at for in the were and to the The bound protein was 5 reduced The were on and by as In of Nephrin cytoplasmic domain was with of Fyn or in kinase 5 of and at for were in the of or were with and for 5 were by to and by of for was by of kidney in were and with glomeruli were and by M. L.B. J. 2000; PubMed Scopus Google Scholar). For of glomerular from mice at and for of the genetic were glomeruli from kidney were were for was of and multiple was used for The Nephrin of the cytoplasmic domain of Nephrin that Nephrin might be To this Nephrin cytoplasmic domain was by in COS7 was as described in Fig. by and phosphorylation of the was following with the pervanadate (Fig. phosphorylation of was not in the of pervanadate of in COS7 cells that were with that Nephrin phosphorylation on and not on and (Fig. these the phosphorylation of endogenous Nephrin in to of renal were with or not Nephrin obtained by from the lysates of isolated glomeruli with pervanadate was tyrosine-phosphorylated (Fig. In experiments, phosphorylation was not on Nephrin obtained from kidney with in experiments, of to or the of a to the of Nephrin phosphorylation not Nephrin with Fyn and Yes in Glomerular Nephrin with a membrane obtained from isolated glomeruli M. K. Kriz W. A. J. Mundel P. Holthofer H. Am. J. Pathol. 2001; 159: Full Text Full Text PDF PubMed Scopus Google Scholar). In a similar Src family protein kinases associate with detergent-resistant membrane fractions in a of cell K. Nat. Mol. Cell. 2000; 1: PubMed Scopus Google Scholar, 2000; PubMed Scopus Google Scholar). this the that an kinase or kinases are at in for Nephrin phosphorylation. the of Nephrin and several Src family kinases in detergent-resistant glomerular membrane fractions was (Fig. glomeruli were at in and the was fractionated by Like Nephrin, Src family kinases Fyn and Yes but not were in the detergent-resistant membrane As described Podocin and CD2ap fractionated in the Nephrin as a for kinase was to of protein kinases that Nephrin as in the isolated glomerular the glomerular membrane was and at for in a kinase and and was with that had been to membrane-associated Nephrin. Nephrin was from this and were by and As shown in Fig. Nephrin, at the molecular of phosphorylated the of this this was in the of PP2, a of Src family kinase Nephrin was not in the of PP3, an of PP2, Nephrin in kinase fractions were also by and A of with a with that of Src family kinases were at phosphorylation of these was not of was in the of It was that Nephrin might be phosphorylated by a Src family protein kinase in a podocyte detergent-resistant membrane To the that Nephrin as a for Src family protein the of endogenous Nephrin to with endogenous Fyn or Yes was (Fig. obtained from isolated glomeruli was with These were by and by for the of Fyn or Yes. Fyn and Yes both with Nephrin. In experiments, Nephrin with Fyn. to Nephrin with not with The Fyn domain for with Nephrin was in pull-down experiments (Fig. or or were from to COS7 cells with Nephrin were or with pervanadate to Nephrin phosphorylation. In these experiments, Fyn SH3 domain but not its SH2 domain interacted with Nephrin obtained from COS7 cells pretreated with pervanadate (Fig. In Nephrin obtained from COS7 cells that were not pretreated with pervanadate not with either the Fyn SH3 or its SH2 In similar pull-down experiments, GST-Fyn SH3 but not Fyn SH2 interacted with Nephrin obtained from lysates of isolated glomeruli (Fig. The binding of Nephrin for Fyn or Yes was also in experiments (Fig. As Nephrin with Fyn and Yes. with results of the pull-down experiments described Fyn or Yes deleted of their SH3 had for Nephrin in these pervanadate of cells to resulted in attenuation of the of Fyn and Yes for Nephrin. these results demonstrate that Fyn Yes can with Nephrin via their SH3 As regulation of this is complex but appears to be Yes, and with Nephrin in COS7 COS7 cells were as with Fyn, Yes, or and with Nephrin as cells were with pervanadate for Fyn and were with was with are were by with antibodies were used to demonstrate expression of various Fyn deleted of its SH3 is of Nephrin can as a of Fyn was in an in protein kinase (Fig. Nephrin cytoplasmic domain in was with Fyn, and at for in a and and these Nephrin was this was in the of the PP2, Nephrin was not In similar experiments, was for used as a not Nephrin not Fyn can directly Nephrin in a Fyn and Yes in the of glomerular of mice deleted of Fyn or of Fyn and Yes by have been described and mice proteinuria and foot process effacement 2001; 11: Full Text Full Text PDF PubMed Scopus Google Scholar). mice were to foot process and marked with and of P.L. H. P. PubMed Scopus Google Scholar). glomerular phenotype was not described for In for Nephrin phosphorylation in these mouse and mouse were and In mice proteinuria to In mice increased by to mice (Fig. by renal of or mice were from that of In of both and mice and that was by not obtained from and mice were also a and mice glomerular from that of and In podocyte foot processes of and mice were The of foot processes were or but in slit intercellular junctions and and podocytes forming a epithelial with of slit intercellular glomeruli demonstrated with and increased Glomeruli of mice demonstrated a and phenotype similar to that and described and In glomerular capillary and were in with of the and focal of and of Fyn, Yes, and podocyte capillary are of in foot process in mouse models at B, that several of changes are in mice foot In and are in to foot process A kinase similar to that described was used to the role of Fyn or Yes in Nephrin phosphorylation in isolated glomerular detergent-resistant membrane fractions of Fyn, Yes, and mouse (Fig. and obtained from multiple experiments in Fig. In these experiments, plasma membrane fractions from isolated glomeruli obtained from of mice were to Deletion of Fyn was with attenuation of Nephrin phosphorylation in glomerular fractions. In deletion of Yes not result in attenuation of Nephrin phosphorylation the of this deletion of Yes resulted in increased Nephrin phosphorylation in this was isolated from no attenuation in Nephrin phosphorylation was that in glomerular isolated from mice (Fig. and with the observations these results genetic evidence that endogenous Fyn catalyzes Nephrin phosphorylation in podocyte detergent-resistant membrane fractions. Although Yes appears to effect the regulation of Nephrin phosphorylation, the mechanism by which this occurs requires investigation. evidence suggests that the molecular of the intercellular junction between podocyte foot processes is complex. is not epithelial cell intercellular the podocyte intercellular junction is a this is podocyte or the process of foot process effacement that occurs in response to podocyte the molecular these of multiple cellular processes via intracellular observations suggest that Nephrin, or the complex of with which it is at the podocyte intercellular junction, as a signaling nexus. Deletion of Nephrin either in human disease (6Kestila M. Lenkkeri U. Mannikko M. Lamerdin J. McCready P. Putaala H. Ruotsalainen V. Morita T. Nissinen M. Herva R. Kashtan C.E. Peltonen L. Holmberg C. Olsen A. Tryggvason K. Mol. Cell. 1998; 1: 575-582Abstract Full Text Full Text PDF PubMed Scopus (1566) Google or in genetically mice (9Holmberg C. Antikainen M. Ronnholm K. Ala-Houhala M. Jalanko H. Pediatr. Nephrol. 1995; 9: 87-93Crossref PubMed Scopus (172) Google results in on multiple cellular processes that are via signal Although it is that of the components of the Nephrin-associated protein complex have been it is that deletion of Podocin and CD2ap, the to date, result in a phenotype similar to that of Nephrin deletion (7Boute N. Gribouval O. Roselli S. Benessy F. Lee H. Fuchshuber A. Dahan K. Gubler M.C. Niaudet P. Antignac C. Nat. Genet. 2000; 24: 349-354Crossref PubMed Scopus (1199) Google Scholar, N.Y. Li J. Karpitskii V. Nguyen A. Dustin M.L. Kanagawa O. Miner J.H. Shaw A.S. Science. 1999; 286: 312-315Crossref PubMed Scopus (698) Google Scholar). Nephrin and its protein complex are in signaling has also been by M. T. J. 2001; Full Text Full Text PDF PubMed Scopus Google that of Podocin with Nephrin in cells the of Nephrin to via of a these of the and signaling function of the Nephrin complex The suggests that signaling mechanisms participate in Nephrin signal transduction. both biochemical and genetic this that the Src family kinase Fyn is a component of the Nephrin-associated protein complex. Fyn can directly to the Nephrin cytoplasmic domain via its SH3 domain and can directly Nephrin both in and in the of that the expression of Fyn is necessary for the development or of normal foot process morphology 2001; 11: Full Text Full Text PDF PubMed Scopus Google Scholar). In to the of 2001; 11: Full Text Full Text PDF PubMed Scopus Google Scholar), in Fyn mouse foot process morphology not result in increased the of these observations is but be to in genetic It is to that deletion of Fyn results in the podocyte alterations to in Nephrin phosphorylation. this to be because Fyn deletion effect podocyte structure at multiple within the The functions of Fyn and Yes in the glomerulus are the that Yes can be shown to associate with Nephrin in biochemical experiments, the results of genetic experiments with Yes not to that Yes to Nephrin in a signaling deletion of Yes results in increased phosphorylation of Nephrin in isolated membrane fractions. The used to the role of Yes in Nephrin phosphorylation were in isolated membrane fractions Nephrin. it is to but not to that Yes is in the Nephrin-associated protein complex. deletion of both Fyn and Yes also results in Nephrin phosphorylation in these fractions suggests that Fyn in an relationship between Yes and Nephrin. In this Yes be to Fyn either directly or The mechanism by which this occurs requires investigation. The mechanism by which Src family kinases are has been in PubMed Scopus Google Scholar). are are phosphorylated on a results in a change in which the SH2 domain of the binds to the phosphorylated domain of the molecule of and of the with its The is a protein the mechanism might the that of COS7 cells with the protein pervanadate results in of Nephrin for Fyn. similar the isolated Fyn SH3 domain have for Nephrin obtained with because endogenous Fyn, which might for binding of Fyn SH3 domain, should not be with Nephrin these The For the of Nephrin phosphorylation H. Am. J. 1995; Google a between increased phosphorylation of at the podocyte intercellular junction and the of podocyte effacement and of glomerular filter integrity in a Fyn and Nephrin phosphorylation might as a result of a podocyte that results in foot process The mechanism by which Fyn is in this is also via an via the Nephrin complex or via an signal of the complex. The of Nephrin complex phosphorylation requires investigation. Nephrin phosphorylation might result in protein complex or the of signaling that signaling or alterations in the of Nephrin for its extracellular or it might Nephrin and S. PubMed Scopus Google Scholar). of these of mechanisms are in but are described for the complex E. PubMed Scopus Google Scholar, J. PubMed Scopus Google or at the M. Science. PubMed Scopus Google Scholar). Each of these requires investigation.