Ischemic Injury to Kidney Induces Glomerular Podocyte Effacement and Dissociation of Slit Diaphragm Proteins Neph1 and ZO-1

Abstract

Glomerular injury is often characterized by the effacement of podocytes, loss of slit diaphragms, and proteinuria. Renal ischemia or the loss of blood flow to the kidneys has been widely associated with tubular and endothelial injury but rarely has been shown to induce podocyte damage and disruption of the slit diaphragm. In this study, we have used an in vivo rat ischemic model to demonstrate that renal ischemia induces podocyte effacement with loss of slit diaphragm and proteinuria. Biochemical analysis of the ischemic glomerulus shows that ischemia induces rapid loss of interaction between slit diaphragm junctional proteins Neph1 and ZO-1. To further understand the effect of ischemia on molecular interactions between slit diaphragm proteins, a cell culture model was employed to study the binding between Neph1 and ZO-1. Under physiologic conditions, Neph1 co-localized with ZO-1 at cell-cell contacts in cultured human podocytes. Induction of injury by ATP depletion resulted in rapid loss of Neph1 and ZO-1 binding and redistribution of Neph1 and ZO-1 proteins from cell membrane to the cytoplasm. Recovery resulted in increased Neph1 tyrosine phosphorylation, restoring Neph1 and ZO-1 binding and their localization at the cell membrane. We further demonstrate that tyrosine phosphorylation of Neph1 mediated by Fyn results in significantly increased Neph1 and ZO-1 binding, suggesting a critical role for Neph1 tyrosine phosphorylation in reorganizing the Neph1-ZO-1 complex. This study documents that renal ischemia induces dynamic changes in the molecular interactions between slit diaphragm proteins, leading to podocyte damage and proteinuria. Glomerular injury is often characterized by the effacement of podocytes, loss of slit diaphragms, and proteinuria. Renal ischemia or the loss of blood flow to the kidneys has been widely associated with tubular and endothelial injury but rarely has been shown to induce podocyte damage and disruption of the slit diaphragm. In this study, we have used an in vivo rat ischemic model to demonstrate that renal ischemia induces podocyte effacement with loss of slit diaphragm and proteinuria. Biochemical analysis of the ischemic glomerulus shows that ischemia induces rapid loss of interaction between slit diaphragm junctional proteins Neph1 and ZO-1. To further understand the effect of ischemia on molecular interactions between slit diaphragm proteins, a cell culture model was employed to study the binding between Neph1 and ZO-1. Under physiologic conditions, Neph1 co-localized with ZO-1 at cell-cell contacts in cultured human podocytes. Induction of injury by ATP depletion resulted in rapid loss of Neph1 and ZO-1 binding and redistribution of Neph1 and ZO-1 proteins from cell membrane to the cytoplasm. Recovery resulted in increased Neph1 tyrosine phosphorylation, restoring Neph1 and ZO-1 binding and their localization at the cell membrane. We further demonstrate that tyrosine phosphorylation of Neph1 mediated by Fyn results in significantly increased Neph1 and ZO-1 binding, suggesting a critical role for Neph1 tyrosine phosphorylation in reorganizing the Neph1-ZO-1 complex. This study documents that renal ischemia induces dynamic changes in the molecular interactions between slit diaphragm proteins, leading to podocyte damage and proteinuria. Glomerular diseases of the kidney are the major cause of end stage renal disease worldwide. Diseases, including diabetes, hypertension, and ischemia, are associated with glomerular dysfunction that leads to proteinuria and loss of kidney function. Renal ischemia-reperfusion injury is the major cause of acute renal failure as observed in the native kidneys and after renal transplantation with a more than 50! mortality rate (1Molitoris B.A. Melnikov V.Y. Okusa M.D. Himmelfarb J. Nat. Clin. Pract. Nephrol. 2008; 4: 154-165Crossref PubMed Scopus (59) Google Scholar, 2Molitoris B.A. Sutton T.A. Kidney Int. 2004; 66: 496-499Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar). There is no specific treatment for such a devastating disease, primarily due to lack of understanding of the pathophysiology and the mechanism of progression of this disease. Identification of major cellular pathways affected by ischemia-reperfusion is an active area of research and has revealed many molecular targets for therapeutic intervention (3Kelly K.J. Molitoris B.A. Semin Nephrol. 2000; 20: 4-19PubMed Google Scholar, 4Molitoris B.A. J. Am. Soc. Nephrol. 2003; 14: 265-267Crossref PubMed Scopus (99) Google Scholar, 5Parikh C.R. Edelstein C.L. Devarajan P. Cantley L. J. Investig. Med. 2007; 55: 333-340Crossref PubMed Scopus (22) Google Scholar). Glomerular podocytes contribute to the kidney filtration barrier and have a footlike appearance of numerous interdigitating processes that arise from their cell bodies and completely cover the glomerular basement membrane. The interdigitation of foot processes is linked via 40 nm wide porous slits commonly known as slit diaphragms (6Tryggvason K. Pikkarainen T. Patrakka J. Cell. 2006; 125: 221-224Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). These are specialized cell junctions that constitute an important component of the glomerular filtration barrier, restricting the passage of proteins and solutes into urine. Increasing evidence now suggests that injuries to the glomerulus leads to podocyte effacement and loss of slit diaphragm structure/function, resulting in proteinuria. Although a significant amount of work has been done to study the effects of renal ischemia on kidney tubules and endothelium (2Molitoris B.A. Sutton T.A. Kidney Int. 2004; 66: 496-499Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar), relatively less is known about the effects of ischemia on glomerular podocytes. Moreover, ischemia-induced changes in the molecular organization of slit diaphragm proteins have never been studied. Recent efforts to understand the mechanisms underlying the loss of slit diaphragm have led to the identification of a number of proteins that are specifically localized at the foot process in podocytes. It is believed that the slit diaphragm is formed by homophilic and heterophilic interactions between the extracellular domains of various podocyte-specific proteins, including Nephrin, Neph1, Fat1, and Fat2 (6Tryggvason K. Pikkarainen T. Patrakka J. Cell. 2006; 125: 221-224Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Proteins such as and are to the slit diaphragm membrane proteins to the P. J. Pikkarainen T. K. K. J. PubMed Scopus Google Scholar, K. J. Clin. 2006; PubMed Scopus Google Scholar). In to their to to the slit that proteins in the important that the physiologic glomerular filtration K. J. Clin. 2006; PubMed Scopus Google Scholar, Nat. Clin. Pract. Nephrol. 2006; PubMed Scopus Google Scholar, K. Patrakka J. J. J. Med. 2006; PubMed Scopus Google Scholar). The study evidence that the ischemic injury induces dynamic changes in the at the slit diaphragm with a loss of and proteinuria. Neph1, a of the is to PubMed Scopus Google and of extracellular by a and a of L. P. J. 2003; PubMed Scopus Google Scholar). In the and Neph1 are in podocytes and are co-localized at the of the slit diaphragm J. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, P. L. T. J. Am. Soc. Nephrol. 2003; 14: PubMed Scopus Google Scholar). Nephrin, is primarily localized in the Neph1 is in including the and PubMed Scopus Google Scholar). analysis of the suggests that to a of proteins and that to the of Although of Neph1 has been of Neph1 results in a podocyte effacement with proteinuria and to PubMed Scopus Google Scholar). It is widely believed that the extracellular domains of Neph1 and a that is at the foot process and a for the slit diaphragm. of Neph1 and at the with various proteins, including and suggests their in that induce (6Tryggvason K. Pikkarainen T. Patrakka J. Cell. 2006; 125: 221-224Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, P. Med. 2006; PubMed Scopus Google Scholar, J. K. Med. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). of the extracellular domains of or Neph1 results in tyrosine phosphorylation of proteins on specific tyrosine and of various proteins, including and K. J. Clin. 2006; PubMed Scopus Google Scholar, P. 2007; PubMed Scopus Google Scholar, P. J. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). to was for to Neph1 was for K. J. Clin. 2006; PubMed Scopus Google Scholar, P. 2007; PubMed Scopus Google Scholar). was that and Neph1 to induce in a cell culture model P. 2007; PubMed Scopus Google Scholar). from interaction with a binding the of Neph1 with the of ZO-1 Nat. Clin. Pract. Nephrol. 2006; PubMed Scopus Google Scholar, P. L. T. J. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). ZO-1 is a of the J. Full Text Full Text PDF PubMed Scopus Google Scholar). ZO-1 a role by junctions and the of the and to the J. Full Text Full Text PDF PubMed Scopus Google Scholar, Am. J. PubMed Google Scholar, 2008; PubMed Scopus Google Scholar). ZO-1 is localized at the of junctions and with proteins such as and and P. L. T. J. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, Am. J. PubMed Google Scholar). ZO-1 in and with Neph1 at the slit diaphragm. from various cell that in ZO-1 and phosphorylation are associated with increased PubMed Scopus Google Scholar, Am. J. Google Scholar, K. 2006; 55: PubMed Scopus Google Scholar). changes in ZO-1 and localization in a was associated with proteinuria and loss of glomerular K. 2006; 55: PubMed Scopus Google Scholar). now that the foot processes of podocytes to glomerular injury in a dynamic by their associated resulting in of the (6Tryggvason K. Pikkarainen T. Patrakka J. Cell. 2006; 125: 221-224Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, P. Med. 2006; PubMed Scopus Google Scholar, T. J. Am. Soc. Nephrol. 2004; PubMed Scopus Google Scholar, Kidney Int. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, L. T.A. K. J. Am. Soc. Nephrol. PubMed Scopus Google Scholar). is that glomerular injury the interactions of and of resulting in loss of slit diaphragm interaction of ZO-1 with Neph1 Neph1 to the we to the that glomerular injury leads to the of this complex. In this study, we demonstrate that renal ischemia induces significant podocyte effacement with a loss of interaction between slit diaphragm proteins Neph1 and ZO-1. The from injury induces Neph1 tyrosine phosphorylation and this results further a critical role for Neph1 tyrosine phosphorylation mediated by Fyn in the of Neph1 and ZO-1 renal and to Neph1 was J. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). including ZO-1 number and Fyn number The cell was from including and from and active was from J. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, P. 2007; PubMed Scopus Google and various P. 2007; PubMed Scopus Google Scholar, 2007; Google Scholar). has been P. 2007; PubMed Scopus Google Scholar). The was a from of used proteins and from as P. 2007; PubMed Scopus Google Scholar). was in and from as P. 2007; PubMed Scopus Google Scholar). Fyn and Neph1 and in and used to cultured in with and and The to the The human podocyte cell was by and was cultured in as L. K. P. Am. J. Full Text Full Text PDF PubMed Scopus Google Scholar). The to at to L. K. P. Am. J. Full Text Full Text PDF PubMed Scopus Google Scholar). ATP depletion was as Molitoris B.A. J. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, J. PubMed Google Scholar). with or was to and was to the The to the for the ATP depletion The in and at in with and of was to by with a and by a The was at for in a and the was and membrane by for at at in a The was and the was with in and by and Renal from in with of The for the and of and by the and with an of on a to a of was and the renal ischemia was by the renal for with of the was observed of was into the to of the to on the to the was from to The was from the blood from and ischemic the The was from of and ischemic in by a for and the for Glomerular by as P. 2007; PubMed Scopus Google Scholar). The of the rat glomerular was at a of of was and from to induce tyrosine proteins with ZO-1 on with and with was by to with the and and the P. 2007; PubMed Scopus Google Scholar, 2007; Google Scholar). of Neph1 and from kidneys of by as P. 2007; PubMed Scopus Google Scholar), and by in was by for on and in by with in in and for at by and for at the a on a The is with an by a of for ZO-1 and the and of the and between the and was to analysis was the ZO-1 was and used to a for analysis of the of to junctional and The of the of was on the after from a of are and analysis was on at for of for was by P. 2007; PubMed Scopus Google Scholar). was and kidneys by with a at for The kidneys and in and by and from kidney to Kidney and of effect of ischemia on tubular cell damage has been ischemia-induced loss of glomerular filtration barrier slit diaphragm has rarely been To the changes by ischemia, we used an in vivo rat model that of the renal P. Molitoris B.A. Am. J. 2004; PubMed Scopus Google Scholar). The kidney for changes in the glomerulus and podocytes by and and podocytes including effacement in to ischemia of ischemia, podocytes with filtration slits of loss of the of podocytes and their processes with the loss of slit diaphragms was observed In an in was the analysis of from ischemic a significant in to in of Neph1 with ZO-1 and to further ischemia induces changes in the molecular interactions between slit diaphragm proteins that are critical for the of of the slit diaphragm (6Tryggvason K. Pikkarainen T. Patrakka J. Cell. 2006; 125: 221-224Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, P. 2007; PubMed Scopus Google Scholar, J. J. Clin. 2003; PubMed Scopus Google Scholar). of to of ischemia for Neph1 interaction with ZO-1 and by shown in interaction of Neph1 with ZO-1 and was significantly after of of this interaction and was associated with Neph1 tyrosine phosphorylation results evidence for a dynamic interaction between slit diaphragm proteins Neph1, and that is significantly by glomerular injury has the to It is to that interaction between slit diaphragm proteins was of glomerular and podocyte was observed This suggests that the of for molecular interaction is than the at the and This to further the effect of ischemia on the molecular interactions of slit diaphragm proteins and that ischemia-induced changes in the binding of slit diaphragm proteins used as for the glomerular injury or as targets for renal Neph1 and ZO-1 at the in suggests that ischemia Neph1 including binding with ZO-1 and Although Neph1 tyrosine phosphorylation has been shown to a critical role in Neph1 and interaction P. 2007; PubMed Scopus Google Scholar), is Neph1 binding with ZO-1 is ZO-1 is a that with Neph1 and has been shown in to we to the of this interaction as a model to study the effect of ischemia on the molecular interactions of slit diaphragm a human podocyte cell was employed proteins are L. K. P. Am. J. Full Text Full Text PDF PubMed Scopus Google Scholar). from this cell that interaction between Neph1 and ZO-1 in To further the localization of proteins in this cell the to and with Neph1 and ZO-1 by The results in the that Neph1 and ZO-1 are primarily localized at the cell-cell The of proteins is shown in have the of ZO-1 as a J. Full Text Full Text PDF PubMed Scopus Google Scholar, Am. J. PubMed Google Scholar, Am. J. 2006; PubMed Scopus Google Scholar). The that Neph1 is localized in this and with ZO-1 evidence that this interaction a role in the Neph1 at the slit diaphragm junctions in podocytes. of with ATP in of Neph1 and ZO-1 further the effect of ischemia on Neph1 and ZO-1 interaction in podocyte the effect of ATP depletion was in the cultured human podocyte cell shown in organization was significantly affected the of It is to that the ATP this more than this effect was the to by in and To the effect of ATP depletion on the binding of Neph1 and a was from Neph1 was and the for the of ZO-1 and Neph1 tyrosine phosphorylation and after the of with ischemia resulted in a significant in Neph1 and ZO-1 binding the interaction between Neph1 and ZO-1 was to from suggesting that effects are It has been that the Neph1 tyrosine phosphorylation is P. 2007; PubMed Scopus Google Scholar). In we that the from ischemia results in Neph1 tyrosine phosphorylation suggesting that of junctions in vivo In disruption of organization in cell is an important of ischemia-induced acute renal injury and in J. PubMed Google Scholar, P. Molitoris B.A. Am. J. 2004; PubMed Scopus Google Scholar, Google Scholar, C.L. Molitoris B.A. Am. J. Renal PubMed Scopus Google Scholar, J. PubMed Google Scholar). evidence that glomerular injuries that organization the interaction between Neph1 and ZO-1. to in of Neph1 and ZO-1 from the further the dynamic interaction between Neph1 and we analysis of cultured podocytes and In and and of ZO-1 and Neph1 from and of ATP depletion and of ATP depletion by for or are of for for and for that was no a of ZO-1 and Neph1 at the cell in was with significant ATP depletion that results in an of proteins at the cell Recovery ATP was with and Neph1 significant To further of Neph1 and ZO-1 was in to In with the results of ischemia and shows that Neph1 and ZO-1 are primarily localized in the membrane and ischemia, in the and leads to their in the membrane To the changes and the interaction between Neph1 and we the between Neph1 and ZO-1 and the for the was as the at the cell ZO-1. In was for the and the was with the an with a of by an Although was no significant at the a significant in the ATP this after of ATP the of and proteins that with ATP the lack of at the To this we the in the for Neph1 and ZO-1 and the as the of for In this the and significant in this for ZO-1 and Neph1, from the area and the rapid with ATP analysis of the changes by and cell of and changes in localization of ZO-1 and Neph1 podocyte and to ischemia, by The to and membrane and the of Neph1 and ZO-1 in by was to the interaction between ZO-1 and Neph1 and the in in the ATP of ZO-1 and Neph1 in the and used to the The of for is to a of with the analysis of more than cell between Neph1 and ZO-1 on the of was that the binding of Neph1 with and is by tyrosine phosphorylation of Neph1 P. 2007; PubMed Scopus Google Scholar). from ischemia tyrosine phosphorylation of Neph1, we to the effect of tyrosine phosphorylation of Neph1 on the interaction between Neph1 and ZO-1. interaction between Neph1 and ZO-1 was by Neph1 and ZO-1 proteins in the of Fyn or Fyn of Neph1 or ZO-1 from cell a significant in the binding between Neph1 and ZO-1 in the of Fyn this in the interaction between Neph1 and ZO-1 was in the of Fyn or the with To further this the interaction between Neph1 and ZO-1 was by and in the of of Fyn in increased of Fyn resulted in a significant in Neph1 with ZO-1. This was further by the Neph1 and ZO-1 interaction in Fyn was by Neph1 was from the glomerular from and Fyn and the for the of ZO-1. shown in the amount of ZO-1 with Neph1 was significantly in with Fyn results that the of Fyn significantly the between Neph1 and ZO-1. of Neph1 by Fyn for between Neph1 and further a effect of Neph1 tyrosine phosphorylation on the binding between Neph1 and the binding of tyrosine of Neph1 with ZO-1 was a was in or was with binding of Neph1 was with the Neph1 This further that the tyrosine phosphorylation of Neph1 for ZO-1. In we tyrosine phosphorylation in To further this a was various Neph1 The results from this analysis that the or on phosphorylation and in Neph1 resulted in a loss of increased interaction between Neph1 and ZO-1 on Fyn phosphorylation and in Neph1 significantly Neph1 and ZO-1 binding results that the Neph1 tyrosine phosphorylation by Fyn the interaction between Neph1 and ZO-1. on we that the of Neph1 and ZO-1 from ischemia is by Neph1 tyrosine injury is a observed in a number of glomerular including disease, and leading to end stage renal disease Nat. Clin. Pract. Nephrol. 2006; PubMed Scopus Google Scholar, Kidney Int. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, Am. J. 2006; PubMed Scopus Google Scholar, J. J. Med. 2006; Google Scholar, L. J. Clin. 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In this study, of ischemia a loss of renal and podocyte Although of Neph1 and ZO-1 interaction the of podocyte was It is that a at the from various molecular interactions and pathways that are affected by have the effects of renal ischemia on that is mediated by (2Molitoris B.A. Sutton T.A. Kidney Int. 2004; 66: 496-499Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar). Moreover, Neph1 with proteins, including Nephrin, and proteins and P. J. T. J. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar), is that ischemia Neph1 interactions that in the the from ischemia is on a effect of interactions and pathways a of and damage in glomerular and diseases has been the of many the identification of various proteins localized at the slit has been that the interactions by proteins are critical for the slit diaphragm to as a filtration Biochemical analysis of the slit diaphragm suggests that of and to the is by the of was the slit diaphragm J. 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Nephrol. 2000; PubMed Google Scholar). is that the proteins localized at the slit diaphragm in a to proteins to the of The that the Neph1 and ZO-1 is localized at cell-cell junctions in podocytes and to the changes in evidence for this on we further that Neph1 and ZO-1 interaction is an important that the of slit diaphragm Induction including glomerular injury by ischemia, to the of Neph1 and ZO-1 and loss of slit diaphragm of loss of Neph1 and ZO-1 binding induces loss of slit diaphragm further Recovery from injury results in Neph1 tyrosine of the Neph1 of junctions This is in by the a dynamic interaction between Neph1 and is on Neph1 tyrosine phosphorylation by the that glomerular injury to podocyte effacement and proteinuria with a loss of interaction between Neph1 and ZO-1 a critical role for this interaction in podocyte have now ZO-1 as a that as a and the J. Full Text Full Text PDF PubMed Scopus Google Scholar, 2008; PubMed Scopus Google Scholar, Am. 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These results further evidence that in to ischemia, Neph1 a ZO-1. in to Neph1 the Neph1 and ZO-1 the Neph1 to the and is by Neph1 tyrosine It is that the Neph1 binding to ZO-1 and are of the that in podocytes in to interactions of Neph1 in the of Neph1 and ZO-1 interaction is by the tyrosine phosphorylation and that Neph1 and interaction P. 2007; PubMed Scopus Google Scholar, T. T. T. J. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). This is have been shown to induce loss of in Neph1 by induce P. 2007; PubMed Scopus Google Scholar). on we that phosphorylation of Neph1 on and induces changes in Neph1 that to increased for ZO-1. a analysis of the and Neph1 more into the phosphorylation associated changes in It is widely that ischemia induces loss of the barrier of the with from ischemia of the Am. J. PubMed Google Scholar, J. PubMed Google Scholar, P. J. PubMed Scopus Google Scholar). In ischemic injury the of membrane proteins and results in significant changes in the Am. J. PubMed Google Scholar, T.A. Molitoris B.A. Nephrol. Google Scholar). the cell culture and we to study changes in the molecular interactions of Neph1 injury and of podocytes with ATP depletion redistribution of Neph1 and ZO-1 to the redistribution of ZO-1 from podocyte membrane has been various of including the cell culture model Am. J. PubMed Scopus Google Scholar), the ischemic cell culture model J. T. Am. J. PubMed Google Scholar), and the in vivo model of K. 2006; 55: PubMed Scopus Google Scholar). The of ZO-1 from membrane to the is associated with the and disruption of junctions K. 2006; 55: PubMed Scopus Google Scholar, L. 2000; PubMed Scopus Google Scholar, K.J. J. PubMed Scopus Google Scholar). In this study, increased of Neph1 and ZO-1 injury was associated with a in Neph1 and ZO-1 binding and This suggests that Neph1 and ZO-1 to on we that injury induces disruption and redistribution of the Neph1-ZO-1 from the cell junctions that to an in in was associated with increased Neph1 phosphorylation and of Neph1 and ZO-1 interaction and their redistribution at the cell membrane and tyrosine of proteins, including and has been in an ATP depletion model kidney T. Am. J. Google Scholar). Neph1 with is Neph1 phosphorylation with ZO-1. on the of we that of cell increased localization of ZO-1 at the cell is an interaction between Neph1 and ZO-1. We further that increased binding between Neph1 and ZO-1 this at the membrane and in cell and have been shown to their interaction with Neph1 P. 2007; PubMed Scopus Google Scholar), is that a of Neph1 interaction with and results P. 2007; PubMed Scopus Google and the results in further this In a study by J. J. Clin. 2003; PubMed Scopus Google suggests that interaction of Neph1 with the binding between Neph1 and further the role of Neph1 interactions in the Neph1 and ZO-1 complex. the of this a study was that interaction of Neph1 with proteins and P. J. T. J. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). This further suggests that from pathways critical in the of slit diaphragm. Neph1 interactions in the in to cellular injury a of this study into the molecular mechanisms that are affected by renal The results from this study that in to changes in podocyte and ischemia induces loss of slit diaphragm and of slit diaphragm proteins Neph1 and ZO-1. on we that Neph1 tyrosine phosphorylation is the from renal injury and leads to the of the that is for It to interactions are critical for the from at of and their effect on podocyte injury further into their in renal with