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.

Formation of tight junctions between neighboring podocytes is an early ultrastructural feature in experimental crescentic glomerulonephritis

“…The loss of ZO-1 expression at the slit diaphragm in a profibrotic ischemic model of renal injury, where ZO-1 was reduced to sub-basal levels in rats' post-ATP depletion leading to podocyte damage and proteinuria, is consistent with the reduction in ZO-1 expression after day 7 in NSN. 37 Rincon-Choles et al 23 also demonstrated a decrease in ZO-1 expression in glomeruli of rats with type 1 and type 2 diabetes, suggesting that alterations in the localization of ZO-1 may be relevant to the pathogenesis of proteinuria and fibrosis in diabetes as well. Hence, reduction in ZO-1 expression appears to associate with worsening of renal injury and proteinuria.…”

Does prolonged pneumoperitoneum affect the kidney? Oxidative stress, stereological and electron microscopy study in a rat model

“…Podocyte effacement is associated with loss of slit diaphragm, leading to proteinuria and loss of kidney function. Podocyte effacement is a common event observed in a number of glomerular diseases, including renal ischemia (26).…”

Structural Analysis of the Myo1c and Neph1 Complex Provides Insight into the Intracellular Movement of Neph1

“…Mammalian expression plasmids encoding GFP-tagged Myo1c-FL (full length), the Myo1c tail without the IQ domain, and the Myo1c tail with the 3IQ domain were obtained as described previously (14,15). The Myo1c head construct was generated by PCR-based cloning at EcoRI and SalI sites in the pEGFP-N1 vector (Clontech).…”

“…COS7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS; Invitrogen) and 200 U/ml of penicillin and streptomycin (Invitrogen). RPMI 1640-based medium supplemented with 10% FBS (Invitrogen), 2 g/liter of sodium bicarbonate (NaHCO 3 ), insulin-transferrin-selenium (38) supplement (Sigma-Aldrich), and 200 U/ml of penicillin and streptomycin (Invitrogen) was used to culture the human podocytes (14,15). Lipofectamine 2000 (Invitrogen) was used to perform transfection according to the manufacturer's protocol.…”

“…Proteins, including Neph1 and nephrin, have been shown to contribute toward the backbone of this structure, thus maintaining the integrity of the slit diaphragm (2,9,10). Localization of these proteins at the podocyte cell membrane is significantly altered in many glomerular disorders, which is commonly associated with the loss of glomerular filtration function (11)(12)(13)(14). Recent evidence suggests that the trafficking of slit diaphragm proteins, including nephrin and Neph1, might be a critical determinant in the reorganization of podocyte cell membrane that restores the glomerular filtration function during recovery from various glomerular diseases (12)(13)(14)(15).…”