Journal: Scientific Reports

Article Title: C1-Ten is a PTPase of nephrin, regulating podocyte hypertrophy through mTORC1 activation

doi: 10.1038/s41598-017-12382-8

Figure Lengend Snippet: C1-Ten induces dephosphorylation of nephrin in a PTPase-dependent manner. Expression level of C1-Ten in ( a,b ) the kidney of db/db mice or ( c ) human podocyte cell line under high glucose (HG) condition. ( a ) Expression levels of C1-Ten were measured by Western blotting in whole kidney lysates of db/m and db/db mice. Data are means ± SEM (n = 3 mice per group). ( b ) Localization of endogenous C1-Ten was observed in the kidneys of db/m and db/db mice. Podocyte colocalization was revealed by immunofluorescence of C1-Ten (green), synaptopodin (red) and Hoechst (blue). Original magnification: X400 (scale bar, 50 µm) ( c ) Fully differentiated human podocytes were serum-starved for 18 h, then incubated in medium with normal glucose (NG) or HG for 24 h. Level of C1-Ten protein was measured. Data are means ± SEM (n = 3). ( d,e ) Effect of C1-Ten depletion on the HG-mediated reduction of nephrin phosphorylation. ( d ) C1-Ten knockdown was performed in human podocytes, which were then transfected with 50 nM of control or TNS2 siRNA (siC1-Ten), then stimulated with NG or HG for 24 h. Expression levels of C1-Ten were measured by Western blotting in total cell lysates. ( e ) Remaining cells lysates were subjected to immunoprecipitation with anti-nephrin antibodies. Immunoprecipitates were analyzed by immunoblotting with phosphotyrosine (pY), PI3K regulatory subunit α (p85α), and nephrin antibodies. Data are means ± SEM (n = 3). ( f ) Effect of C1-Ten on Fyn-induced nephrin phosphorylation. 293NPHS cells were cotransfected with WT-Fyn and either FLAG vector, FLAG C1-Ten WT or FLAG C1-Ten CS. GFP nephrin was immunoprecipitated from cell lysates and subjected to immunoblotting with phosphotyrosine (pY), PI3K regulatory subunit α (p85α), and nephrin antibodies. Data are means ± SEM (n = 3). *P < 0.05.

Article Snippet: Anti-Akt1/2 (sc-1619), anti-pY20 (sc-508), anti-pY99 (sc-7020), anti-synaptopodin (sc-21537), anti-GFP (sc-9996) and polyclonal anti-nephrin (sc-28192) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: De-Phosphorylation Assay, Expressing, Western Blot, Immunofluorescence, Incubation, Phospho-proteomics, Knockdown, Transfection, Control, Immunoprecipitation, Plasmid Preparation

Journal: Scientific Reports

Article Title: C1-Ten is a PTPase of nephrin, regulating podocyte hypertrophy through mTORC1 activation

doi: 10.1038/s41598-017-12382-8

Figure Lengend Snippet: Nephrin competes with IRS-1, sequesters PI3K from IRS-1. ( a,b ) The preferential substrate of C1-Ten when IRS-1 and nephrin coexist. ( a ) FLAG C1-Ten CS was introduced with HA IRS-1 or GFP Nephrin into HEK293 cells, then immunoprecipitated with anti-FLAG beads and immunoblotted with IRS-1, GFP or FLAG. Data are means ± SEM (n = 3). ( b ) Nephrin knockdown was performed in human podocytes, which were transfected with 50 nM of control or NPHS1 siRNA (siNephrin). Cell lysates were subjected to immunoprecipitation with anti-IRS-1 antibodies. Immunoprecipitates were analyzed by immunoblotting with C1-Ten and IRS-1. Expression levels of nephrin, IRS-1 and actin were measured by Western blotting in total cell lysates. Data are means ± SEM (n = 3). ( c ) IRS-1 Y612 phosphorylation and PI3K interaction were monitored by increasing FLAG nephrin. HEK293 cells were transfected with FLAG nephrin. HEK293 cells were serum-starved for 18 h, then treated with 10 nM of insulin for 5 min, and subjected to immunoprecipitation with anti-IRS-1 antibody. The immunoprecipitates were subjected to immunoblotting with pY612 IRS-1, PI3K regulatory subunit α (p85α) or total IRS-1. Data are means ± SEM (n = 3). ( d ) Effect of nephrin on the insulin signaling. 293VEC or 293NPHS cells were serum-starved for 18 h, then treated with 10 nM insulin. Data are means ± SEM (n = 3). *P <0.05; **P <0.01.

Article Snippet: Anti-Akt1/2 (sc-1619), anti-pY20 (sc-508), anti-pY99 (sc-7020), anti-synaptopodin (sc-21537), anti-GFP (sc-9996) and polyclonal anti-nephrin (sc-28192) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Immunoprecipitation, Knockdown, Transfection, Control, Western Blot, Expressing, Phospho-proteomics

Journal: Scientific Reports

Article Title: C1-Ten is a PTPase of nephrin, regulating podocyte hypertrophy through mTORC1 activation

doi: 10.1038/s41598-017-12382-8

Figure Lengend Snippet: C1-Ten PTPase activates mTORC1. ( a ) Effect of nephrin Y1138F mutant on IRS-1-mediated mTORC1 activation. HEK293 cells were transfected with GFP vector, GFP nephrin WT, or GFP nephrin Y1138F for 24 h. The cells were serum-starved for 18 h, then treated with 10 nM insulin for 30 min. mTORC1 activation was measured by immunoblotting of phospho- and total- S6K1. Data are means ± SEM (n = 3). ( b ) Effect of C1-Ten overexpression on mTORC1 activation. Human podocytes were transduced with Adenovirus (Ad) GFP, Ad C1-Ten WT or Ad C1-Ten CS for 48 h. Protein expression of phospho- and total-S6K1 were detected by Western blotting. Data are means ± SEM (n = 3). ( c ) Effect of C1-Ten knockdown on the HG-mediated mTORC1 activation. mTORC1 activation was presented by immunoblotting of phospho- and total- S6K1. Data are means ± SEM (n = 3). *P <0.05.

Article Snippet: Anti-Akt1/2 (sc-1619), anti-pY20 (sc-508), anti-pY99 (sc-7020), anti-synaptopodin (sc-21537), anti-GFP (sc-9996) and polyclonal anti-nephrin (sc-28192) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Mutagenesis, Activation Assay, Transfection, Plasmid Preparation, Western Blot, Over Expression, Transduction, Expressing, Knockdown

Journal: Scientific Reports

Article Title: C1-Ten is a PTPase of nephrin, regulating podocyte hypertrophy through mTORC1 activation

doi: 10.1038/s41598-017-12382-8

Figure Lengend Snippet: C1-Ten PTPase causes kidney dysfunction in vivo . ( a ) GFP expression measured using Western blot analysis of whole kidney lysates. ( b ) Localization of adenovirus-mediated transgene expression. Mice were infected with adenovirus, then sacrificed 7 d later and their kidneys stained with GFP, phospho S6 (red) and Hoechst (blue). Original magnification: X400 (scale bar, 50 µm) ( c ) Effect of C1-Ten overexpression on the nephrin dephosphorylation and mTORC1 activation were measured in adenovirus-infected-whole kidney lysates. Immunoprecipitation was performed with anti-nephrin antibody, and the immunoprecipitates were analyzed by immunoblotting with phosphotyrosine (pY), PI3K regulatory subunit α (p85α), and nephrin antibodies. mTORC1 activation was also measured by immunoblotting of phospho- and total- S6K1. ( d and e ) Morphological changes of glomeruli in adenovirus-infected kidneys. ( d ) Adenovirus-infected kidneys stained with periodic acid Schiff (PAS) reagent for renal pathology. Upper image: magnification X100 (scale bar, 100 µm); lower image: magnification X400 (scale bar, 20 µm). ( e ) After PAS staining, glomerular volume was analyzed using Meta Morph image analysis (20 glomeruli per animal). ( f ) Adenovirus-infected kidney podocytes were analyzed by transmission electron microscopy (TEM). TEM analysis revealed effaced podocyte foot process in the kidneys treated with C1-Ten WT (asterisks). Original magnification: X30000 (scale bar, 1 µm) ( g ) Urinary albumin excretion for 24 h was measured using ELISA. Data are means ± SEM (n = 3–5 mice per group). *P <0.05.

Article Snippet: Anti-Akt1/2 (sc-1619), anti-pY20 (sc-508), anti-pY99 (sc-7020), anti-synaptopodin (sc-21537), anti-GFP (sc-9996) and polyclonal anti-nephrin (sc-28192) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: In Vivo, Expressing, Western Blot, Infection, Staining, Over Expression, De-Phosphorylation Assay, Activation Assay, Immunoprecipitation, Transmission Assay, Electron Microscopy, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: C1-Ten is a PTPase of nephrin, regulating podocyte hypertrophy through mTORC1 activation

doi: 10.1038/s41598-017-12382-8

Figure Lengend Snippet: Graphical summary. The balance of mTORC1 activity in normal podocytes is maintained by competition between nephrin and IRS-1 to PI3K. Under high glucose condition, upregulated C1-Ten acts as a PTPase at the nephrin-PI3K binding site and renders PI3K for IRS-1, thereby activating mTORC1. By activating mTORC1, excessive C1-Ten contributes to development of podocyte dysfunctions such as hypertrophy and proteinuria.

Article Snippet: Anti-Akt1/2 (sc-1619), anti-pY20 (sc-508), anti-pY99 (sc-7020), anti-synaptopodin (sc-21537), anti-GFP (sc-9996) and polyclonal anti-nephrin (sc-28192) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activity Assay, Binding Assay

Journal: Aging (Albany NY)

Article Title: Inhibiting NLRP3 signaling in aging podocytes improves their life- and health-span

doi: 10.18632/aging.204897

Figure Lengend Snippet: Podocyte health was improved upon inhibition/absence of NLRP3. ( A ) Comparison of the transcripts for canonical podocyte genes ( Wt1 , Nphs1 , Nphs2 , Synpo , Cdkn1c , Vegfa ) using the normalized mRNA-seq read counts from isolated podocytes of middle-aged saline-treated (red bars) and MCC950-treated mice (blue bars) compared to their young counterparts (grey bars). ( B – Z ) Immunostaining of kidneys from young, saline- and MCC95-treated middle-aged as well as middle-aged Nlrp3 null mice for WT-1/Wt1 ( B – F ), Nephrin/Nphs1 ( G – K ), Podocyin/Nphs2 ( L – P ), Synaptopodin/Synpo ( Q – U ) and VEGFA ( V – Z ). Representative images are shown; scale bars represent 25 μm. Staining intensities were quantified and depicted in the graphs as % tuft area.

Article Snippet: The digital images were imported into Visiopharm software (Hoersholm, Denmark) and its Image Analysis Deep Learning module was trained to detect glomeruli and assess immunohistochemical staining-positivity for Collagen IV, p57, Nephrin, Synaptopodin and ERG.

Techniques: Inhibition, Comparison, Isolation, Saline, Immunostaining, Staining