Nephrin-Binding Ephrin-B1 at the Slit Diaphragm Controls Podocyte Function through the JNK Pathway

Fukusumi, Yoshiyasu; Zhang, Ying; Yamagishi, Ryohei; Oda, Kanako; Watanabe, Toru; Matsui, Katsuyuki; Kawachi, Hiroshi

doi:10.1681/asn.2017090993

Cited by 26 publications

(51 citation statements)

References 44 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We found the expression of the Ephb4 -Efnb2 system in adult endothelial cells, an expression described in the context of the developing kidney (Takahashi et al, 2001) and in the recovery from glomerulonephritis (Ephb4 on podocytes (Wnuk et al, 2012)). In line with our findings, localization of Efnb1 on podocytes has been reported (Hashimoto et al, 2007), and an involvement in diabetic nephropathy through an interaction with Partitioning-defective (Par)-6 (Takamura et al, 2020) and in JNK pathway regulation (Fukusumi et al, 2018) have been demonstrated.…”

Section: Discussionsupporting

confidence: 91%

Tripartite separation of glomerular cell-types and proteomes from reporter-free mice

Hatje

Rinschen

Wedekind

et al. 2020

Preprint

View full text Add to dashboard Cite

Purpose: The kidney glomerulus comprises a syncytium of podocytes, mesangial and endothelial cells, which jointly determine glomerular filtration barrier function, and thereby kidney and cardiovascular health. The understanding of this intricate functional unit and its intracellular communication beyond the transcriptome requires bulk isolation of these cell-types from glomeruli for subsequent biochemical investigations. Therefore, we developed a globally applicable tripartite isolation method for murine mesangial and endothelial cells and podocytes (timMEP). Methods: Glomerular cells were separated via a novel FACS-sort depending on a cell-specific antibody labeling in wildtype mice or based on a combination of transgenic fluorescent protein expression and antibody labeling in mT/mG mice. The purity of isolated cell-types was validated by qPCR and immunoblot. The proteome of podocytes, mesangial and endothelial cells was determined and compared between species, ages and gender of wildtype and mT/mG mice. The method was also applied to the podocyte-targeting immunologic injury model of THSD7A-associated membranous glomerulonephritis. Results: TimMEP enabled protein-biochemical analyses of podocytes, mesangial and endothelial cells derived from a single reporter free mouse. Proteomic analyses allowed the first characterization of podocyte, endothelial and mesangial proteomes of individual mice. Marker proteins for mesangial and endothelial proteins were determined, and protein-based interaction and intraglomerular cell communication networks were elucidated. Interestingly, analyses revealed significant cell-type specific proteome differences between mouse strains, artefacts induced by reporters, and alterations depending on gender and age. Within the glomerulus, timMEP resolved a fine-tuned initial stress response exclusively in podocytes after exposure to anti-THSD7A antibodies, which was not detectable using conventional analyses in whole glomeruli. Conclusion: Globally applicable timMEP abolishes the need for costly, time- and animal- consuming breeding of mice to glomerular cell-type reporters. TimMEP enables glomerular cell-type resolved investigations at the transcriptional and protein biochemical level in health and disease, while avoiding reporter-based artefacts, paving the way towards the comprehensive and systematic characterization of glomerular cell-type biology.

show abstract

Section: Discussionsupporting

confidence: 91%

Tripartite separation of glomerular cell-types and proteomes from reporter-free mice

Hatje

Rinschen

Wedekind

et al. 2020

Preprint

View full text Add to dashboard Cite

show abstract

“…NPHS1 knockout mice subjected to podocyte injury failed to recover from foot process effacement as well as the persistence of proteinuria [31]. Ephrin-B1, a membrane-bound protein, bound to and interacted with nephrin by immunoprecipitation assay [33]. 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 [33].…”

Section: Discussionmentioning

confidence: 99%

“…Ephrin-B1, a membrane-bound protein, bound to and interacted with nephrin by immunoprecipitation assay [33]. 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 [33]. The interaction of nephrin and ephrin-B1 maintains the structure and barrier function of the slit diaphragm [33].…”

Section: Discussionmentioning

confidence: 99%

“…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 [33]. The interaction of nephrin and ephrin-B1 maintains the structure and barrier function of the slit diaphragm [33]. It suggested that nephrin was required to maintain slit diaphragm integrity and slit diaphragm-mediated signaling, and it played an important role for the maintenance of podocyte function, production of proteinuria and pathogenesis of FSGS.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

A comprehensive analysis of NPHS1 gene mutations in patients with sporadic focal segmental glomerulosclerosis

et al. 2019

View full text Add to dashboard Cite

Background Focal segmental glomerulosclerosis (FSGS) is still one of the common causes of refractory nephrotic syndrome. Nephrin, encoded by podocyte-specific NPHS1 gene, participated in the pathogenesis of FSGS. The sites of NPHS1 mutations in FSGS is not clarified very well. In this study, we investigated the specific mutations of NPHS1 gene in Chinese patients with sporadic FSGS. Methods A total of 309 patients with sporadic FSGS were collected and screened for NPHS1 mutations by second-generation sequencing. The variants were compared with those extracted from 2504 healthy controls in the 1000 Genomes Project. The possible pathogenic roles of missense variants were predicted by three different software. We also compared these candidate causal mutations with those summarized from the previous studies. Results Thirty-two genetic mutations of NPHS1 gene were identified in FSGS patients, including 12 synonymous mutations, 17 missense mutations, 1 splicing mutation, and 2 intron mutations, of which c.G3315A (p.S1105S) was the most common variant (261/309). A novel missense mutation c.G2638 T (p.V880F) and a novel splicing mutation 35830957 C > T were identified in FSGS patients. The frequencies of the four synonymous mutations (c.C294T [p.I98I], c.C2223T [p.T741 T], c.C2289T [p.V763 V], c.G3315A [p.S1105S]) were much higher in FSGS patients than in controls. The frequencies of the four missense mutations (c.G349A [p.E117K], c.G1339A [p.E447K], c.G1802C [p.G601A], c.C2398T [p.R800C]) were much higher and one (c.A3230G [p.N1077S]) was lower in FSGS patients than in controls. Five missense mutations, c.C616A (p.P206T), c.G1802C (p.G601A), c.C2309T (p.P770L), c.G2869C (p.V957 L), and c.C3274T (p.R1092C), were predicted to be pathogenic mutations by software analysis. Conclusions NPHS1 gene mutations were quite common in sporadic FSGS patients. We strongly recommend mutation analysis of the NPHS1 gene in the clinical management of FSGS patients.

show abstract

“…12 Podocyte-specific ephrin-B1 conditional knockout (CKO) mice display the alteration of podocyte morphology, disarrangement of the slit diaphragm molecules, and proteinuria. 13 The report also showed that ephrin-B1 interacts with nephrin, a key molecule of the slit diaphragm, via their extracellular domains. These findings showed that ephrin-B is essential for maintenance of the slit diaphragm.…”

mentioning

confidence: 95%

Partitioning-Defective-6–Ephrin-B1 Interaction Is Regulated by Nephrin-Mediated Signal and Is Crucial in Maintaining Slit Diaphragm of Podocyte

Takamura

Fukusumi

Zhang

et al. 2020

The American Journal of Pathology

Self Cite

View full text Add to dashboard Cite

Ephrin-B1 plays a critical role at slit diaphragm. Partitioning-defective (Par)-6 is down-regulated in podocyte of ephrin-B1 knockout mouse, suggesting that Par-6 is associated with ephrin-B1. Par polarity complex, consisting of Par-6, Par-3, and atypical protein kinase C, is essential for tight junction formation. In this study, the expression of Par-6 was analyzed in the normal and nephrotic syndrome model rats, and the molecular association of Par-6, Par-3, ephrin-B1, and nephrin was assessed with the human embryonic kidney 293 cell expression system. Par-6 was concentrated at slit diaphragm. Par 6 interacted with ephrin-B1 but not with nephrin, and Par-3 interacted with nephrin but not with ephrin-B1. The complexes of Par-6eephrin-B1 and Par-3enephrin were linked via extracellular sites of ephrin-B1 and nephrin. The Par-6eephrin-B1 complex was delinked from the Par-3enephrin complex, and Par-6 and ephrin-B1 were clearly down-regulated already at early phase of nephrotic model. The alteration of Par-6/ephrin-B1 advanced that of Par-3/nephrin. Stimulation to nephrin phosphorylated not only nephrin but also ephrin-B1, and consequently inhibited the interaction between ephrin-B1 and Par-6. Par-6 appeared at presumptive podocyte of early developmental stage and moved to basal area at capillary loop stage to participate in slit diaphragm formation at the final stage. Par-6eephrin-B1 interaction is crucial for formation and maintenance of slit diaphragm of podocyte.

show abstract

Nephrin-Binding Ephrin-B1 at the Slit Diaphragm Controls Podocyte Function through the JNK Pathway

Cited by 26 publications

References 44 publications

Tripartite separation of glomerular cell-types and proteomes from reporter-free mice

Tripartite separation of glomerular cell-types and proteomes from reporter-free mice

A comprehensive analysis of NPHS1 gene mutations in patients with sporadic focal segmental glomerulosclerosis

Partitioning-Defective-6–Ephrin-B1 Interaction Is Regulated by Nephrin-Mediated Signal and Is Crucial in Maintaining Slit Diaphragm of Podocyte

Contact Info

Product

Resources

About