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

Takamura, Sayuri; Fukusumi, Yoshiyasu; Zhang, Ying; Narita, Ichiei; Kawachi, Hiroshi

doi:10.1016/j.ajpath.2019.10.015

Cited by 12 publications

(29 citation statements)

References 52 publications

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“…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

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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.

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Section: Discussionsupporting

confidence: 91%

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

Hatje

Rinschen

Wedekind

et al. 2020

Preprint

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“…Hartleben et al reported that Par-3 and aPKC are expressed at podocyte slit diaphragm and NEPH1-nephrin complex binds to the Par-complex [72]. Recently, Takamura et al demonstrated that Par-3 binds to nephrin, and Par-6 binds to ephrin-B1, another transmembrane protein at slit diaphragm [73]. The mice administered with a dominant-negative aPKC construct showed significant proteinuria, and the loss of foot process architecture was detected in the isolated glomeruli treated with an inhibitor of aPKC.…”

Section: Multiple Scaffold Proteins Participate In the Link Of Slit Diaphragm To The Cytoskeletonmentioning

confidence: 99%

“…The nephrin-binding ephrin-B1 at slit diaphragm transfers the signals nephrin detected to the inside of the cell via different ways from nephrin-mediated signaling [44]. If ephrin-B1 is phosphorylated, nephrin and Par-6 were dissociated from ephrin-B1 [44,73]. Phosphorylation of ephrin-B1 promoted mobility of podocyte through activation of JNK.…”

Section: Slit Diaphragm Functions As a Signaling Platformmentioning

confidence: 99%

“…The Par complex is essential for the establishment of cell polarity and the formation of the tight junction. It is reported that the tight link between the Par complex to the slit diaphragm components is critical for maintaining the structure of the slit diaphragm [72,73]. Reinforcement of the linking between the transmembrane proteins and the Par complex molecules is also a rational strategy.…”

Section: Future Lines Of Study: a Strategy For The Establishment Of A Novel Therapy For Proteinuriamentioning

confidence: 99%

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New insight into podocyte slit diaphragm, a therapeutic target of proteinuria

Kawachi

Fukusumi

2020

Clin Exp Nephrol

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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.

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“…As long-established cell lines in literature, mesangial cells and podocytes are important biological samples to determine the best reference gene -many researchers in the field are focused on these structures (42)(43)(44)(45)(46)(47) and the data provided by our work could potentially influence many studies, providing support to avoid wrong interpretation of results and its influence in downstream analysis and further conclusions.…”

Section: Discussionmentioning

confidence: 87%

Reference genes for mesangial cell and podocyte qPCR gene expression studies under high-glucose and renin-angiotensin-system blocker conditions

Hosni

Anauate

Boim

2021

Preprint

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BackgroundReal-time PCR remains currently the gold standard method for gene expression studies. Identification of the best reference gene is a key point in performing high quality qPCR, providing strong support for results, as well as performing as a source of bias when inappropriately chosen. Mesangial cells and podocytes, as essential cell lines to study diabetic kidney disease (DKD) physiopathology, demand accurate analysis of the reference genes used so far to enhance validity of gene expression studies, especially regarding high glucose (HG) and DKD treatments, with angiotensin II receptor blockers (e.g. Losartan) being the most commonly used. This study aimed to evaluate the suitability and define the most stable reference gene for mesangial cells and podocytes studies of an in vitro DKD model of disease and its treatment.MethodsFive software packages (RefFinder, NormFinder, GeNorm, Bestkeeper, and DataAssist) and the comparative ΔCt method were selected to analyze six different candidate genes: HPRT, ACTB, PGAM-1, GAPDH, PPIA, and B2M. RNA was extracted and cDNA was synthesized from immortalized mouse mesangial cells and podocytes cultured in 4 groups: control (n=5; 5mM glucose), mannitol (n=5; 30mM, as osmotic control), HG (n=5; 30mM glucose), and HG + losartan (n=5; 30mM glucose and 10-4 mM of losartan). Real-time PCR was performed according to MIQE guidelines.ResultsWe identified that the use of 2 genes is the best combination for qPCR normalization for both mesangial cell and podocytes. For mesangial cells, the combination of HPRT and ACTB presented higher stability values. For podocytes, HPRT and GAPDH showed the best results.ConclusionThis analysis provides support for the use of HPRT and ACTB as reference genes in mouse mesangial cell studies of gene expression via real-time PCR technique, while for podocytes, HPRT and GAPDH should be chosen.

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

Cited by 12 publications

References 52 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

New insight into podocyte slit diaphragm, a therapeutic target of proteinuria

Reference genes for mesangial cell and podocyte qPCR gene expression studies under high-glucose and renin-angiotensin-system blocker conditions

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