cell culture normal rat kidney fibroblast cell line  (ATCC)

Journal: Diabetes, Metabolic Syndrome and Obesity

Article Title: Claudin-2 Mediates the Proximal Tubular Epithelial Cell–Fibroblast Crosstalk via Paracrine CTGF

doi: 10.2147/DMSO.S432173

Figure Lengend Snippet: Knockdown of Cldn2 in PTECs enhances fibroblast activation and proliferation. ( A ) Representative immunostaining images showing the nuclear localization of proliferating cell nuclear antigen (PCNA) in activated fibroblasts. Cells were immune-stained for α-SMA (red) and PCNA (green) and counterstained with DAPI (blue). White arrows indicate positive staining. Magnification ×400, scale bar =100µm. ( B ) Quantitative analysis of PCNA + /α-SMA + cells showed knockdown of Cldn2 in PTECs markedly ameliorated the activation and proliferation in fibroblasts by co-culturing with PTECs in NG for 48 hours. (C and D) Representative images and quantitative analysis show knockdown of Cldn2 in PTECs promotes fibroblast proliferation, as demonstrated by EdU incorporation. (E and F) Western blot revealed the expression levels of α-SMA and collagen I in NRK49F cells in 5.5mM D-glucose (NG) medium± co-culture with NRK52E cells ±si- Cldn2 for 48 hours. Representative blots and quantitative analysis of α-SMA and collagen I are shown above. Data are the mean ± SEM (n=3). ** P <0.01, vs NRK52E-siNC+NRK49F+NG.

Article Snippet: The normal rat kidney fibroblast cell (NRK49F) and rat renal tubule epithelial cells (NRK52E) were obtained from ProCell Corporation (Wuhan, China) and cultured in RPMI-1640 medium (Corning, NY, USA).

Techniques: Knockdown, Activation Assay, Immunostaining, Staining, Western Blot, Expressing, Co-Culture Assay

Journal: Diabetes, Metabolic Syndrome and Obesity

Article Title: Claudin-2 Mediates the Proximal Tubular Epithelial Cell–Fibroblast Crosstalk via Paracrine CTGF

doi: 10.2147/DMSO.S432173

Figure Lengend Snippet: Overexpression of Cldn2 reverses high glucose-induced fibroblast activation and proliferation. ( A and B ) Immunocytochemistry revealed the nuclear localization of proliferating cell nuclear antigen (PCNA) in activated-fibroblasts (NRK49Fcells) in 5.5mM D-glucose (NG) medium or 30mM D-glucose (HG) medium ± co-culture with NRK52E ± pcDNA3.1- Cldn2 . Cells were immune-stained for α-SMA (red) and PCNA (green) and counterstained with DAPI (blue). White Arrows indicate positive staining. Magnification ×400, scale bar =100µm. ( C and D ) Representative images and quantitative analysis show HG stimulated fibroblast proliferation, and co-culture with PTECs exacerbated this process. And overexpressed Cldn2 in PTECs inhibited HG-induced fibroblast proliferation. ( E and F ) Western blot revealed the expression levels of α-SMA and collagen I in NRK49F cells in 5.5mM D-glucose (NG) medium or 30mM D-glucose (HG) medium ± co-culture with NRK52E cells ± pcDNA3.1- Cldn2 for 48 hours. Representative blots and quantitative analysis of α-SMA and collagen I are shown above. Data are expressed as mean data ± SEM (n = 3), ** P <0.01, vs NRK49F+NG; ## P <0.01, vs NRK49F+HG; && P <0.01, vs NRK52E-pcDNA3.1-NC+NRK49F+HG.

Article Snippet: The normal rat kidney fibroblast cell (NRK49F) and rat renal tubule epithelial cells (NRK52E) were obtained from ProCell Corporation (Wuhan, China) and cultured in RPMI-1640 medium (Corning, NY, USA).

Techniques: Over Expression, Activation Assay, Immunocytochemistry, Co-Culture Assay, Staining, Western Blot, Expressing

Journal: Diabetes, Metabolic Syndrome and Obesity

Article Title: Claudin-2 Mediates the Proximal Tubular Epithelial Cell–Fibroblast Crosstalk via Paracrine CTGF

doi: 10.2147/DMSO.S432173

Figure Lengend Snippet: Claudin-2 regulates CTGF in PTECs. ( A and B ) Western blot and quantitative analysis showed that the CTGF protein expression increased time-dependent when NRK52E cells were exposed to HG for 24 hours, 48 hours, and 72 hours, respectively. Values represent mean ± SEM, n = 3, * P < 0.05, ** P < 0.01, vs NG; # P < 0.05, ## P < 0.01 vs HG (24 h), & P <0.05 vs HG (48 h). ( C ) Concentrations of CTGF in the culture supernatants of PTECs after interference. Experiments were performed in triplicate. ** P <0.01, vs NRK52E-si-NC +NG; ## P <0.01, vs NRK52E-pcDNA3.1-NC+HG. ( D – F ) NRK52E cells were transduced with control (si-NC) or si- Cldn2 followed by co-culture with NRK49F cells in 5.5mM D-glucose (NG) medium for 48 hours. Western blot revealed the CTGF and Claudin-2 expression levels in NRK52E cells in 5.5mM D-glucose (NG) medium ± co-culture with NRK49F cells ± si- Cldn2. Cldn2 siRNA knockdown efficiency was confirmed by Western blot analyses. Representative blots and quantitative analysis of Claudin-2 and CTGF are shown above. Data are expressed as the mean ± S.E.M. ** P <0.01, vs NRK52E-si-NC+NRK49F+NG. (G and H and I) Western blot revealed the CTGF and Claudin-2 expression levels in NRK52E cells in 5.5mM D-glucose (NG) medium or 30mM D-glucose (HG) medium ±co-culture with NRK49F cells ± pcDNA3.1- Cldn2 for 48 hours. The overexpression efficiency of pcDNA3.1- Cldn2 plasmid was confirmed by Western blot analyses. Representative blots and quantitative analysis of Claudin-2 and CTGF are shown above. ** P <0.01, vs NRK49F+NG; ## P <0.01, vs NRK49F+HG; && P <0.01, vs NRK52E-pcDNA3.1-NC+NRK49F+HG.

Article Snippet: The normal rat kidney fibroblast cell (NRK49F) and rat renal tubule epithelial cells (NRK52E) were obtained from ProCell Corporation (Wuhan, China) and cultured in RPMI-1640 medium (Corning, NY, USA).

Techniques: Western Blot, Expressing, Transduction, Control, Co-Culture Assay, Knockdown, Over Expression, Plasmid Preparation

Journal: Diabetes, Metabolic Syndrome and Obesity

Article Title: Claudin-2 Mediates the Proximal Tubular Epithelial Cell–Fibroblast Crosstalk via Paracrine CTGF

doi: 10.2147/DMSO.S432173

Figure Lengend Snippet: Claudin-2 deficiency induced tubular epithelial CTGF is involved in fibroblast activation and proliferation. ( A and B ) Immunocytochemistry revealed the nuclear localization of proliferating cell nuclear antigen (PCNA) in activated-fibroblasts in 5.5mM D-glucose (NG) medium or 30mM D-glucose (HG) medium ± co-culture with NRK52E cells ± si- Cldn2 ± pcDNA3.1- Cldn2 ± si- Ctgf ± pcDNA3.1- Ctgf . Cells were immune-stained for α-SMA (red) and PCNA (green) and counterstained with DAPI (blue). White arrows indicate positive staining. Magnification ×400, scale bar =100µm. ( C and D ) Representative image and quantitative analysis show the effect of interfering with Claudin-2 and CTGF protein expression in PTECs on fibroblast proliferation in 5.5mM D-glucose (NG) medium or 30mM D-glucose (HG) medium. Data are the mean ± SEM (n=3). ** P <0.01, vs NRK52E-si- Cldn2 -si-NC+NRK49F+NG; ## P <0.01, vs NRK52E-pcDNA3.1- Cldn2 -pcDNA3.1-NC+HG.

Article Snippet: The normal rat kidney fibroblast cell (NRK49F) and rat renal tubule epithelial cells (NRK52E) were obtained from ProCell Corporation (Wuhan, China) and cultured in RPMI-1640 medium (Corning, NY, USA).

Techniques: Activation Assay, Immunocytochemistry, Co-Culture Assay, Staining, Expressing

Journal: Diabetes, Metabolic Syndrome and Obesity

Article Title: Claudin-2 Mediates the Proximal Tubular Epithelial Cell–Fibroblast Crosstalk via Paracrine CTGF

doi: 10.2147/DMSO.S432173

Figure Lengend Snippet: Claudin-2 deficiency induced tubular epithelial CTGF facilitates fibroblasts to product matrix protein. ( A ) Western blot demonstrated CTGF inhibition in NRK52E-si- Cldn2 cells decreased α-SMA and collage I expression in NRK49F cells, when the cells were co-cultured in 5.5mM D-glucose (NG) medium. Overexpression of CTGF in NRK52E-pcDNA3.1- Cldn2 cells increased α-SMA and collage I expression in NRK49F cells, when the cells were co-cultured in 30mM D-glucose (HG) medium. ( B and C ) The interference efficiency of Claudin-2 and CTGF protein expression in NRK52E cells were confirmed by Western blot analyses. Quantitative analysis of Claudin-2 and CTGF are shown above. ( D and E ) Quantitative analysis of α-SMA and collagen I in NRK49F cells are shown above. Data are expressed as mean data ± SEM (n = 3), ** P <0.01, NRK52E-si- Cldn2 -si-NC+NRK49F+NG; ## P <0.01, vs NRK52E-pcDNA3.1- Cldn2 -pcDNA3.1-NC+HG.

Article Snippet: The normal rat kidney fibroblast cell (NRK49F) and rat renal tubule epithelial cells (NRK52E) were obtained from ProCell Corporation (Wuhan, China) and cultured in RPMI-1640 medium (Corning, NY, USA).

Techniques: Western Blot, Inhibition, Expressing, Cell Culture, Over Expression