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Podocytes overexpressing Neph1 are resistant to PAN-induced injury. A, schematic representation of the construction of Cherry-Neph1 construct (where Cherry was introduced after the transmembrane domain and before any potential tyrosine phosphorylation sites in Neph1) is shown. B, expression pattern of Cherry-Neph1 was similar to endogenous Neph1 in podocytes, where both proteins localized at the cell-cell junctions (arrows). C, podocytes without and with stable expression of Cherry-Neph1 were created and subjected to PAN treatment for 48 h. Unlike the control cells transfected with empty vector, the Cherry-Neph1-transfected cells were resistant to PAN-induced cytoskeletal damage, and their cell-cell junctions were well maintained. D, BSA permeability assay was performed with vector-transfected (control) and Cherry-Neph1-overexpressing podocytes that were cultured as a monolayer on Transwell filters and treated with PAN. The passage of albumin across the podocytes monolayer was assessed by a paracellular albumin flux assay using Texas red-labeled albumin. The Cherry-Neph1-overexpressing cells showed increased resistance to PAN-induced albumin leakage as compared with control podocytes . ns, nonsignificant. Scale bar, 20 m (B); 10 m (C).
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Podocytes are specialized epithelial cells that are critical components of the glomerular filtration barrier and their dysfunction leads to proteinuria and renal failure. Therefore, preserving podocyte function is therapeutically significant. In this study, we identified Neph1 signaling as a therapeutic target which upon inhibition prevented podocy...
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Defects in the glomerular filtration barrier (GFB) play a major role in the onset of human renal diseases. Highly ramified glomerular cells named podocytes are a critical component of the GFB. Injury to podocytes results in abnormal excretion of plasma proteins, which can lead to chronic kidney disease. The conserved paired nephron of larval zebraf...
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... We previously showed that the slit-diaphragm protein NEPH1 mis-localizes and is lost from the podocyte cell membrane in response to injury, but re-localizes during recovery from injury [15,21,22]. To further evaluate the ability of β 2 -AR knockout mice to recover from injury, kidney sections from wild-type and β 2 -AR knockout mice injured with NTS and treated with formoterol were immunostained with NEPH1 and SYNAPTOPODIN antibodies. ...
Podocytes have a remarkable ability to recover from injury; however, little is known about the recovery mechanisms involved in this process. We recently showed that formoterol, a long-acting β2-adrenergic receptor (β2-AR) agonist, induced mitochondrial biogenesis (MB) in podocytes and led to renoprotection in mice. However, it is not clear whether this effect was mediated by formoterol acting through the β2-AR or if it occurred through “off-target” effects.
We genetically deleted the β2-AR specifically in murine podocytes and used these mice to determine whether formoterol acting through the podocyte β2-AR alone is sufficient for recovery of renal filtration function following injury. The podocyte-specific β2-AR knockout mice (β2-ARfl/fl/PodCre) were generated by crossing β2-AR floxed mice with podocin Cre (B6.Cg-Tg(NPHS2-cre)295Lbh/J) mice. These mice were then subjected to both acute and chronic glomerular injury using nephrotoxic serum (NTS) and adriamycin (ADR), respectively. The extent of injury was evaluated by measuring albuminuria and histological and immunostaining analysis of the murine kidney sections.
A similar level of injury was observed in β2-AR knockout and control mice; however, the β2-ARfl/fl/PodCre mice failed to recover in response to formoterol. Functional evaluation of the β2-ARfl/fl/PodCre mice following injury plus formoterol showed similar albuminuria and glomerular injury to control mice that were not treated with formoterol.
These results indicate that the podocyte β2-AR is a critical component of the recovery mechanism and may serve as a novel therapeutic target for treating podocytopathies.
... Lipid raft microdomains are critical for normal podocyte morphology and glomerular barrier function [19,36]. In this study, we identified and described the functional role of raft protein Flot2 in recruiting SD proteins podocin and nephrin into rafts and mediating podocyte injury and proteinuric glomerular disease. ...
Podocyte injury is a common hallmark of chronic kidney disease (CKD). The podocin-nephrin complex localized in lipid rafts of podocyte is vital to reduce podocyte injury and proteinuria, however, the mechanism underlying its localization remains unclear. This study uncovers an important role of Flot2 in stabilizing the podocin-nephrin complex localized in lipid rafts. We first confirmed that Flot2 was expressed in podocyte and demenstrated that podocyte-specific Flot2 deletion worsen albuminuria, podocyte injury and glomerular pathology in LPS/ADR-induced nephropathy mouse models. Meanwhile, podocyte injury, albuminuria and pathologic aberrance were prevented in podocyte-specific Flot2 overexpression transgenic mice when challenged with LPS or ADR. Further found that Flot2 was vital to recruit podocin and nephrin into rafts and ameliorated podocyte injury. Flot2 and podocin directly interacted with each other via their SPFH domain. Meanwhile, we also showed that Flot-2 is a direct target of Krüppel-like factor (KLF15). Importanly, we observed that Flot2 was downregulated in renal biopsies from patients with podocytopathies and its expression negatively correlated with proteinuria and positively correlated with eGFR, indicating that Flot2 may be a novel therapeutic target for proteinuric kidney disease.
... The Drosophila homolog of KIRREL is roughest, which is required for controlling cell numbers in the Drosophila retinal epithelium (21). The zebrafish KIRREL homolog Enph1 is a podocyte protein that is critical for maintaining renal function (22). However, the role of KIR-REL in tumorigenesis is poorly understood. ...
Cell surface proteins play essential roles in various biological processes and are highly related to cancer development. They also serve as important markers for cell identity and targets for pharmacological intervention. Despite their great potentials in biomedical research, comprehensive functional analysis of cell surface proteins remains scarce. Here, with a de novo designed library targeting cell surface proteins, we performed in vivo CRISPR screens to evaluate the effects of cell surface proteins on tumor survival and proliferation. We found that Kirrel1 loss markedly promoted tumor growth in vivo. Moreover, KIRREL was significantly enriched in a separate CRISPR screen based on a specific Hippo pathway reporter. Further studies revealed that KIRREL binds directly to SAV1 to activate the Hippo tumor suppressor pathway. Together, our integrated screens reveal a cell surface tumor suppressor involved in the Hippo pathway and highlight the potential of these approaches in biomedical research.
... The reduced phosphorylation level of nephrin tyrosine residues is detected in MCD and MN, and phosphorylation stabilizes and restores podocyte foot process architecture [70][71][72]. On the contrary, phosphorylation of NEPH1 tyrosine residues is found in several podocyte injury models, and this can be assumed as a therapeutic target [73][74][75]. Another molecule, ephrin-B1, at the slit-diaphragm also involves the signaling network. ...
Treatment for glomerular diseases has been extrapolated from the experience of other autoimmune disorders while the underlying pathogenic mechanisms were still not well understood. As the classification of glomerular diseases was based on patterns of juries instead of mechanisms, treatments were typically the art of try and error. With the advancement of molecular biology, the role of the immune agent in glomerular diseases is becoming more evident. The four-hit theory based on the discovery of gd-IgA1 gives a more transparent outline of the pathogenesis of IgA nephropathy (IgAN), and dysregulation of Treg plays a crucial role in the pathogenesis of minimal change disease (MCD). An epoch-making breakthrough is the discovery of PLA2R antibodies in the primary membranous nephropathy (pMN). This is the first biomarker applied for precision medicine in kidney disease. Understanding the immune system’s role in glomerular diseases allows the use of various immunosuppressants or other novel treatments, such as complement inhibitors, to treat glomerular diseases more reasonable. In this era of advocating personalized medicine, it is inevitable to develop precision medicine with mechanism-based novel biomarkers and novel therapies in kidney disease.
... We next sought to investigate the mechanism by which KIRREL1-SAV1 interaction modulates the Hippo pathway. Previous studies suggest that KIRREL1 undergoes trans-dimerization and mediates cell-cell interaction of kidney podocytes [23][24][25][26] . Moreover, recent findings suggest that membrane SAV1 recruits MST kinases to the plasma membrane to phosphorylate membrane-localized LATS kinases 15 . ...
The Hippo/YAP pathway controls cell proliferation through sensing physical and spatial organization of cells. How cell-cell contact is sensed by Hippo signaling is poorly understood. Here, we identified the cell adhesion molecule KIRREL1 as an upstream positive regulator of the mammalian Hippo pathway. KIRREL1 physically interacts with SAV1 and recruits SAV1 to cell-cell contact sites. Consistent with the hypothesis that KIRREL1-mediated cell adhesion suppresses YAP activity, knockout of KIRREL1 increases YAP activity in neighboring cells. Analyzing pan-cancer CRISPR proliferation screen data reveals KIRREL1 as the top plasma membrane protein showing strong correlation with known Hippo regulators, highlighting a critical role of KIRREL1 in regulating Hippo signaling and cell proliferation. During liver regeneration in mice, KIRREL1 is upregulated, and its genetic ablation enhances hepatic YAP activity, hepatocyte reprogramming and biliary epithelial cell proliferation. Our data suggest that KIRREL1 functions as a feedback regulator of the mammalian Hippo pathway through sensing cell-cell interaction and recruiting SAV1 to cell-cell contact sites. How cell-cell contact is sensed by Hippo pathway is poorly understood. Here, the authors show that KIRREL1 functions as a feedback regulator of the mammalian Hippo pathway by sensing cell-cell interaction and recruiting SAV1 to cell-cell contacts.
... Podocytes play a vital part in maintaining glomerular structure and function, and the progression of chronic kidney diseases always has podocytes involvement [7]. Although accumulating evidence has indicated that signaling pathways play a pivotal role in the pathogenesis of podocyte injury [8,9], the underlying mechanisms of podocyte-associated damage remain to be elucidated. Activated T nuclear factor (NFAT) is the most widely studied calcineurin substrate (can), which belongs to the Ca2 + dependent transcription factor family. ...
Podocyte injury is sufficient to cause glomerulosclerosis and proteinuria, eventually leading to kidney failure. Previous studies found that podocytes and neurons had similar biological characteristics. Growth-associated protein-43 (GAP-43) is a growth cone protein in neurons, and a marker of axonal and synaptic growth. However, it is not known whether GAP-43 is expressed in podocytes. Compared with normal glomerular podocytes, GAP-43 was significantly reduced in patients with glomerular diseases. GAP-43 also significantly reduced in lipopolysaccharide (LPS)-treated podocytes. We found that the decreased expression of nephrin, the cell marker of the podocyte, was significantly recovered with GAP-43 overexpression. In contrast, the migration ability in LPS-treated podocyte was reduction after GAP-43 overexpressing. Moreover, overexpression of GAP-43 attenuated podocyte apoptosis by up-regulating the ratio of Bcl-2/Bax with LPS treatment. Finally, Plaue and Rcan1 which are downstream target gene of NFATc1 decreased with overexpression of GAP-43 podocytes. We concluded that GAP-43 attenuated podocyte injury by inhibiting calcineurin/NFATc1 signaling. The findings may provide a promising treatment for podocyte injury-related diseases.
... Importantly, mutations or genetic deletions of NEPHRIN and NEPH1 lead to dysfunctional podocytes which, in turn, results in loss of renal filtration function (2)(3)(4)(5). While several studies suggest that the extracellular domains of NEPHRIN and NEPH1 have a structure-based function where their spatial arrangement provides integrity to the slit diaphragm, their intracellular domains were shown to initiate signaling cascades leading to actin cytoskeletal changes in podocytes (1, 6,7). This suggests that NEPHRIN and NEPH1 undergo activation that is driven by phosphorylation prior to initiation of downstream signaling. ...
... Since SHP-2 binds NEPHRIN in a phosphorylation-dependent manner (8), we investigated whether NEPH1 phosphorylation also enhanced SHP-2 binding. Similar to NEPHRIN, phosphorylation of NEPH1 can be accomplished either by the treatment of cells (stable HEK293 NEPH1overexpressing cells) with pervanadate (6,9), or by co-expressing with FYN kinase. In Fig. 1C we show that FYN kinase significantly increases NEPH1 and SHP-2 binding. ...
... Since SHP-2 appeared to be a potent phosphatase for NEPH1, we hypothesized that the phosphorylation (ligand-induced) of endogenous NEPH1 would be suppressed in the presence of SHP-2. We therefore generated stable SHP-2 knockdown (KD) human podocytes that are known to endogenously express NEPH1, and tested the level of NEPH1 phosphorylation following exposure to various growth factors using a NEPH1specific phosphoantibody (6,13,14). Phosphorylation of endogenous NEPH1 was only visible in SHP-2 knockdown podocytes treated with HGF (Fig. 1D). Fig. 1C, FYN kinase increases binding of SHP-2 to NEPH1. ...
HGF phosphorylation of NEPHRIN and NEPH1 in podocyte repair 2 ABSTRACT 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 (MET) receptor in a ligand-dependent fashion through engagement of their extracellular domains by HGF. Further, we demonstrate SH2 domain-containing protein tyrosine phosphatase-2 (SHP-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, though NEPHRIN binding was 20-fold higher than that of NEPH1. Additionally, 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.
... Since SHP-2 bound NEPHRIN in a phosphorylation-dependent manner [16], we investigated whether NEPH1 phosphorylation also enhanced SHP-2 binding. Similar to NEPHRIN, the phosphorylation of NEPH1 either via treatment of podocytes with pervanadate [17][18][19], or by co-expressing with FYN kinase, significantly increased NEPH1 and SHP-2 interaction ( Fig. 1B and C). By mixing recombinant purified proteins, we further demonstrated a direct interaction between the cytoplasmic domain of NEPH1 and SHP-2 ( Fig. 1D). ...
... Thus, to evaluate phosphorylation of these proteins, we constructed stable SHP-2 knockdown (KD) cultured human podocytes that are known to endogenously express NEPH1 [23][24][25][26]. These podocytes were screened against various growth factors and the phosphorylation of NEPH1 was evaluated using a phospho NEPH1 antibody [19,26,27]. ...
... HGF induces podocytes recovery from injury in the in vitro and in vivo models of podocyte injury: Since HGF functions as an injury-induced effector for tissue repair, we hypothesized that HGF is involved in podocyte repair following injury. To test this, cultured human podocytes overexpressing NEPH1 were injured with PS (protamine sulphate), as described previously [19,24,31,32], which resulted in severe actin cytoskeleton disorganization ( Fig. 5A-B). Interestingly, addition of exogenous recombinant HGF resulted in significant recovery of actin cytoskeleton (Fig. 5A-B). ...
When activated, slit diaphragm proteins NEPHRIN and NEPH1 enable signaling pathways leading to podocyte actin cytoskeleton reorganization, which is critical for podocyte recovery from injury. However, the mechanisms through which these proteins are activated remain unknown. This study presents a novel concept showing ligand-induced activation of NEPHRIN and NEPH1. We first identified phosphatase SHP-2, which directly dephosphorylated these proteins. We next identified HGF, a known SHP-2 modulator, as a rapid inducer of NEPHRIN and NEPH1 phosphorylation. Using baculovirus expressed recombinant purified proteins, SPR (surface plasma resonance), molecular modeling and peptide binding approaches, we show that HGF directly binds NEPHRIN and NEPH1 extracellular domains. Further, using cultured podocytes and Drosophila nephrocytes, we demonstrate that while HGF treatment repaired injured podocytes, the addition of inhibitory NEPH1 or NEPHRIN peptides blocked HGF-induced recovery. Overall, this study shows novel activation and deactivation mechanisms for NEPHRIN and NEPH1 that are required for their function.
... Phosphorylated NEPH1 and nephrin coordinate distinct signaling pathway through binding to different SH2 domain proteins Grb2 and NCK. NEPH1 is phosphorylated in several injured podocyte models [48,100,101]. Since inhibiting NEPH1 phosphorylation protects podocyte from injury, it is assumed that inhibiting NEPH1 signaling is therapeutically significant in preventing podocyte damage [48]. ...
Dysfunction of slit diaphragm, a cell–cell junction of glomerular podocytes, is involved in the development of proteinuria in several glomerular diseases. Slit diaphragm should be a target of a novel therapy for proteinuria. Nephrin, NEPH1, P-cadherin, FAT, and ephrin-B1 were reported to be extracellular components forming a molecular sieve of the slit diaphragm. Several cytoplasmic proteins such as ZO-1, podocin, CD2AP, MAGI proteins and Par-complex molecules were identified as scaffold proteins linking the slit diaphragm to the cytoskeleton. In this article, new insights into these molecules and the pathogenic roles of the dysfunction of these molecules were introduced. The slit diaphragm functions not only as a barrier but also as a signaling platform transfer the signal to the inside of the cell. For maintaining the slit diaphragm function properly, the phosphorylation level of nephrin is strictly regulated. The recent studies on the signaling pathway from nephrin, NEPH1, and ephrin-B1 were reviewed. Although the mechanism regulating the function of the slit diaphragm had remained unclear, recent studies revealed TRPC6 and angiotensin II-regulating mechanisms play a critical role in regulating the barrier function of the slit diaphragm. In this review, recent investigations on the regulation of the slit diaphragm function were reviewed, and a strategy for the establishment of a novel therapy for proteinuria was proposed.
... Podocytes are an important cellular component of the three-layered system containing a fenestrated endothelium and glomerular basement membrane that together constitute the glomerular filtration system [1][2][3]. Podocytes have a unique architecture, consisting of a cell body that attaches to the glomerular basement membrane, and primary, secondary, and tertiary processes that wrap around the capillary in an interdigitating fashion [2]. ...
... Many glomerular diseases, including nephrotic syndromes, diabetic nephropathy, and focal segmental glomerulosclerosis (FSGS), affect podocyte structure and function, leading to proteinuria and total loss of renal function [2,4]. Studies performed in various in vitro and in vivo disease models suggest that podocytes respond dynamically to injury through changes in their actin cytoskeleton, leading to a process known as podocyte effacement, which is characterized by the flattening of podocytes and loss of slit-diaphragm [3,5]. Although these pathological events cannot be recapitulated under in vitro conditions, the molecular processes associated with these changes have been studied using cultured human podocytes, which have served as an excellent tool to investigate the molecular Int. ...
... Sci. 2020, 21, 274 2 of 11 and cellular mechanisms of podocyte pathogenesis [3]. Despite significant advancements in podocyte biology, the molecular mechanisms involved in podocyte pathogenesis remain poorly understood, and are the major stumbling block in identifying novel therapeutics to preserve and prevent podocyte loss during glomerular injury. ...
Podocytes have a unique structure that supports glomerular filtration function, and many glomerular diseases result in loss of this structure, leading to podocyte dysfunction and ESRD (end stage renal disease). These structural and functional changes involve a complex set of molecular and cellular mechanisms that remain poorly understood. To understand the molecular signature of podocyte injury, we performed transcriptome analysis of cultured human podocytes injured either with PAN (puromycin aminonucleoside) or doxorubicin/adriamycin (ADR). The pathway analysis through DE (differential expression) and gene-enrichment analysis of the injured podocytes showed Tumor protein p53 (P53) as one of the major signaling pathways that was significantly upregulated upon podocyte injury. Accordingly, P53 expression was also up-regulated in the glomeruli of nephrotoxic serum (NTS) and ADR-injured mice. To further confirm these observations, cultured podocytes were treated with the P53 inhibitor pifithrin-α, which showed significant protection from ADR-induced actin cytoskeleton damage. In conclusion, signaling pathways that are involved in podocyte pathogenesis and can be therapeutically targeted were identified by high-throughput transcriptomic analysis of injured podocytes.
