Role of calcineurin (CN) in kidney glomerular podocyte: CN inhibitor ameliorated proteinuria by inhibiting the redistribution of CN at the slit diaphragm.
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ABSTRACT:
SUBMITTER: Wakamatsu A
PROVIDER: S-EPMC4814882 | biostudies-other | 2016 Mar
REPOSITORIES: biostudies-other
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Project description:The tight junction (TJ) has a key role in regulating paracellular permeability to water and solutes in the kidney. However, the functional role of the TJ in the glomerular podocyte is unclear. In diabetic nephropathy, the gene expression of claudins, in particular claudin-1, is markedly upregulated in the podocyte, accompanied by a tighter filtration slit and the appearance of TJ-like structures between the foot processes. However, there is no definitive evidence to show slit diaphragm (SD) to TJ transition in vivo Here, we report the generation of a claudin-1 transgenic mouse model with doxycycline-inducible transgene expression specifically in the glomerular podocyte. We found that induction of claudin-1 gene expression in mature podocytes caused profound proteinuria, and with deep-etching freeze-fracture electron microscopy, we resolved the ultrastructural change in the claudin-1-induced SD-TJ transition. Notably, immunolabeling of kidney proteins revealed that claudin-1 induction destabilized the SD protein complex in podocytes, with significantly reduced expression and altered localization of nephrin and podocin proteins. Mechanistically, claudin-1 interacted with both nephrin and podocin through cis- and trans-associations in cultured cells. Furthermore, the rat puromycin aminonucleoside nephrosis model, previously suspected of undergoing SD-TJ transition, exhibited upregulated expression levels of claudin-1 mRNA and protein in podocytes. Together, our data attest to the novel concept that claudins and the TJ have essential roles in podocyte pathophysiology and that claudin interactions with SD components may facilitate SD-TJ transition that appears to be common to many nephrotic conditions.
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Project description: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.
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Project description:We describe here the size and location of nephrin, the first protein to be identified at the glomerular podocyte slit diaphragm. In Western blots, nephrin antibodies generated against the two terminal extracellular Ig domains of recombinant human nephrin recognized a 180-kDa protein in lysates of human glomeruli and a 150-kDa protein in transfected COS-7 cell lysates. In immunofluorescence, antibodies to this transmembrane protein revealed reactivity in the glomerular basement membrane region, whereas the podocyte cell bodies remained negative. In immunogold-stained thin sections, nephrin label was found at the slit between podocyte foot processes. The congenital nephrotic syndrome of the Finnish type (NPHS1), a disease in which the nephrin gene is mutated, is characterized by massive proteinuria already in utero and lack of slit diaphragm and foot processes. These features, together with the now demonstrated localization of nephrin to the slit diaphragm area, suggests an essential role for this protein in the normal glomerular filtration barrier. A zipper-like model for nephrin assembly in the slit diaphragm is discussed, based on the present and previous data.
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Project description:The nephron is the basic structural and functional unit of the vertebrate kidney. It is composed of a glomerulus, the site of ultrafiltration, and a renal tubule, along which the filtrate is modified. Although widely regarded as a vertebrate adaptation, 'nephron-like' features can be found in the excretory systems of many invertebrates, raising the possibility that components of the vertebrate excretory system were inherited from their invertebrate ancestors. Here we show that the insect nephrocyte has remarkable anatomical, molecular and functional similarity to the glomerular podocyte, a cell in the vertebrate kidney that forms the main size-selective barrier as blood is ultrafiltered to make urine. In particular, both cell types possess a specialized filtration diaphragm, known as the slit diaphragm in podocytes or the nephrocyte diaphragm in nephrocytes. We find that fly (Drosophila melanogaster) orthologues of the major constituents of the slit diaphragm, including nephrin, NEPH1 (also known as KIRREL), CD2AP, ZO-1 (TJP1) and podocin, are expressed in the nephrocyte and form a complex of interacting proteins that closely mirrors the vertebrate slit diaphragm complex. Furthermore, we find that the nephrocyte diaphragm is completely lost in flies lacking the orthologues of nephrin or NEPH1-a phenotype resembling loss of the slit diaphragm in the absence of either nephrin (as in human congenital nephrotic syndrome of the Finnish type, NPHS1) or NEPH1. These changes markedly impair filtration function in the nephrocyte. The similarities we describe between invertebrate nephrocytes and vertebrate podocytes provide evidence suggesting that the two cell types are evolutionarily related, and establish the nephrocyte as a simple model in which to study podocyte biology and podocyte-associated diseases.
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Project description:LMX1B encodes a LIM-homeodomain transcription factor. Mutations in LMX1B cause nail-patella syndrome (NPS), an autosomal dominant disease with skeletal abnormalities, nail hypoplasia, and nephropathy. Expression of glomerular basement membrane (GBM) collagens is reduced in Lmx1b(-/-) mice, suggesting one basis for NPS nephropathy. Here, we show that Lmx1b(-/-) podocytes have reduced numbers of foot processes, are dysplastic, and lack typical slit diaphragms, indicating an arrest in development. Using antibodies to podocyte proteins important for podocyte function, we found that Lmx1b(-/-) podocytes express near-normal levels of nephrin, synaptopodin, ZO-1, alpha3 integrin, and GBM laminins. However, mRNA and protein levels for CD2AP and podocin were greatly reduced, suggesting a cooperative role for these molecules in foot process and slit diaphragm formation. We identified several LMX1B binding sites in the putative regulatory regions of both CD2AP and NPHS2 (podocin) and demonstrated that LMX1B binds to these sequences in vitro and can activate transcription through them in cotransfection assays. Thus, LMX1B regulates the expression of multiple podocyte genes critical for podocyte differentiation and function. Our results indicate that reduced levels of proteins associated with foot processes and the glomerular slit diaphragm likely contribute, along with reduced levels of GBM collagens, to the nephropathy associated with NPS.
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Project description:Both mTOR signaling and autophagy are important modulators of podocyte homeostasis, regeneration, and aging and have been implicated in glomerular diseases. However, the mechanistic role of these pathways for the glomerular filtration barrier remains poorly understood. We used Drosophila nephrocytes as an established podocyte model and found that inhibition of mTOR signaling resulted in increased spacing between slit diaphragms. Gain-of-function of mTOR signaling did not affect spacing, suggesting that additional cues limit the maximal slit diaphragm density. Interestingly, both activation and inhibition of mTOR signaling led to decreased nephrocyte function, indicating that a fine balance of signaling activity is needed for proper function. Furthermore, mTOR positively controlled cell size, survival, and the extent of the subcortical actin network. We also showed that basal autophagy in nephrocytes is required for survival and limits the expression of the sns (nephrin) but does not directly affect slit diaphragm formation or endocytic activity. However, using a genetic rescue approach, we demonstrated that excessive, mTOR-dependent autophagy is primarily responsible for slit diaphragm misspacing. In conclusion, we established this invertebrate podocyte model for mechanistic studies on the role of mTOR signaling and autophagy, and we discovered a direct mTOR/autophagy-dependent regulation of the slit diaphragm architecture.
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Project description:Background B-type ephrins are membrane-bound proteins that maintain tissue function in several organs. We previously reported that ephrin-B1 is localized at the slit diaphragm of glomerular podocytes. However, the function of ephrin-B1 at this location is unclear.Methods We analyzed the phenotype of podocyte-specific ephrin-B1 knockout mice and assessed the molecular association of ephrin-B1 and nephrin, a key molecule of the slit diaphragm, in HEK293 cells and rats with anti-nephrin antibody-induced nephropathy.Results Compared with controls, ephrin-B1 conditional knockout mice displayed altered podocyte morphology, disarrangement of the slit diaphragm molecules, and proteinuria. Ephrin-B1 expressed in HEK293 cells immunoprecipitated with nephrin, which required the basal regions of the extracellular domains of both proteins. Treatment of cells with an anti-nephrin antibody promoted the phosphorylation of nephrin and ephrin-B1. However, phosphorylation of ephrin-B1 did not occur in cells expressing a mutant nephrin lacking the ephrin-B1 binding site or in cells treated with an Src kinase inhibitor. The phosphorylation of ephrin-B1 enhanced the phosphorylation of nephrin and promoted the phosphorylation of c-Jun N-terminal kinase (JNK), which was required for ephrin-B1-promoted cell motility in wound-healing assays. Notably, phosphorylated JNK was detected in the glomeruli of control mice but not ephrin-B1 conditional knockout mice. In rats, the phosphorylation of ephrin-B1, JNK, and nephrin occurred in the early phase (24 hours) of anti-nephrin antibody-induced nephropathy.Conclusions Through interactions with nephrin, ephrin-B1 maintains the structure and barrier function of the slit diaphragm. Moreover, phosphorylation of ephrin-B1 and, consequently, JNK are involved in the development of podocyte injury.
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Project description:Phosphorylation (activation) and dephosphorylation (deactivation) of the slit diaphragm proteins NEPHRIN and NEPH1 are critical for maintaining the kidney epithelial podocyte actin cytoskeleton and, therefore, proper glomerular filtration. However, the mechanisms underlying these events remain largely unknown. Here we show that NEPHRIN and NEPH1 are novel receptor proteins for hepatocyte growth factor (HGF) and can be phosphorylated independently of the mesenchymal epithelial transition receptor in a ligand-dependent fashion through engagement of their extracellular domains by HGF. Furthermore, we demonstrate SH2 domain-containing protein tyrosine phosphatase-2-dependent dephosphorylation of these proteins. To establish HGF as a ligand, purified baculovirus-expressed NEPHRIN and NEPH1 recombinant proteins were used in surface plasma resonance binding experiments. We report high-affinity interactions of NEPHRIN and NEPH1 with HGF, although NEPHRIN binding was 20-fold higher than that of NEPH1. In addition, using molecular modeling we constructed peptides that were used to map specific HGF-binding regions in the extracellular domains of NEPHRIN and NEPH1. Finally, using an in vitro model of cultured podocytes and an ex vivo model of Drosophila nephrocytes, as well as chemically induced injury models, we demonstrated that HGF-induced phosphorylation of NEPHRIN and NEPH1 is centrally involved in podocyte repair. Taken together, this is the first study demonstrating a receptor-based function for NEPHRIN and NEPH1. This has important biological and clinical implications for the repair of injured podocytes and the maintenance of podocyte integrity.
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Project description:Neph1-deficient mice develop nephrotic syndrome at birth, indicating the importance of this protein in the development of a normal glomerular filtration barrier. While the precise subcellular localization of Neph1 remains unknown, its relationship with other components of the glomerular filtration barrier is of great interest in this field. In this paper, we localize the expression of Neph1 to the glomerular slit diaphragm by immunogold electron microscopy in rodents and describe its direct interaction with two other components of the slit diaphragm, nephrin and ZO-1. Both native and recombinant Neph1 associate with each other as dimers and multimers and interact with nephrin via their extracellular segments. Disruption of the Neph1-nephrin interaction in vivo by injecting combinations of individual subnephritogenic doses of anti-Neph1 and anti-nephrin results in complement- and leukocyte-independent proteinuria with preserved foot processes. This disruption modestly reduces Neph1 and nephrin protein expression in podocytes and dramatically reduces ZO-1 protein expression via the interaction of ZO-1 PDZ domains with the cytoplasmic tail of Neph1, independent of changes in mRNA expression of all three genes. The interaction between nephrin and Neph1 is specific and not shared by either protein with P-cadherin, another integral slit diaphragm protein. The interaction between nephrin and Neph1 therefore appears to be an important determinant of glomerular permeability.
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