human primary podocytes  (Celprogen Inc)

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: INF2 formin variants linked to human inherited kidney disease reprogram the transcriptome, causing mitotic chaos and cell death

doi: 10.1007/s00018-024-05323-y

Figure Lengend Snippet: Effect of INF2 R218Q in human primary podocytes. A Images of podocytes expressing wt INF2 and INF2 R218Q stained for p53. B Percentage of cells positive for nuclear p53. C Images of INF2 R218Q mitotic podocytes displaying abnormal spindles. Cells were stained for α-tubulin. Chromosomes were visualized using DAPI. D Images of podocytes expressing wt INF2 or INF2 R218Q for 48 h that were treated or not with 100 nM LatB for 24 h. Cells were stained for F-actin. E Percentage of cells displaying nuclear abnormalities. More than 300 cells were examined; three independent experiments. Nuclei were visualized with DAPI. Scale bars, 10 μm ( A , D ), 5 μm ( C ). **, p < 0.01; ***, p < 0.001. F Schematic depicting the effect of pathogenic INF2. Abnormal mitosis leads to cell death during mitosis or results in cells with nuclear abnormalities that eventually die. The ER is represented in green, chromosomes in brown, and microtubules in blue

Article Snippet: Human primary podocytes (Innoprot, # P10669) were grown on poly-L-lysine coated dishes using epithelial cell medium (Innoprot, #P60106) with 2% FBS.

Techniques: Expressing, Staining

Journal: Renal Failure

Article Title: IGFBP2 and IGFBP4 interact to activate complement pathway in diabetic kidney disease

doi: 10.1080/0886022X.2024.2440528

Figure Lengend Snippet: The crosstalk effect among HK-2 cells, THP-1 cells, and primary human renal podocytes was investigated as follows: (A) the serum from DKD mice (10%) was added to HK-2 cells for culture for 24 h, after which relevant indicators were detected. The supernatant from HK-2 cells was then transferred to THP-1 cells to assess the functional changes in THP-1 cells, and subsequently, the THP-1 cell supernatant was added to primary human renal podocytes to observe their functional alterations. (B) The levels of IGFBP2 and IGFBP4 following HK-2 cell stimulation were measured. (C) The polarization of THP-1 cells was assessed using flow cytometry, where CD86% served as the marker for M1 polarization and CD206% indicated M2 polarization. (D) ELISA was employed to detect changes in complement proteins C3, C4B, C5, and C9 in THP-1 cells. (E) The levels of MAC and MBL in THP-1 cells were also measured using ELISA. (F) Podocyte proliferation was evaluated using the CCK-8 assay. (G) Apoptosis of primary human renal podocytes was analyzed through flow cytometry. (H) The expression of reactive oxygen species (ROS) in primary human renal podocytes was visualized by immunofluorescence, with green fluorescence indicating the intensity of ROS expression. Data are presented as mean ± SD ( n = 3; * p < .05, ** p < .01, *** p < .001).

Article Snippet: The primary human renal podocytes were purchased from Wuhan Punosay Life Technology Co., Ltd. (CP-H075) (Wuhan, China).

Techniques: Functional Assay, Cell Stimulation, Flow Cytometry, Marker, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Expressing, Immunofluorescence, Fluorescence

Journal: Renal Failure

Article Title: IGFBP2 and IGFBP4 interact to activate complement pathway in diabetic kidney disease

doi: 10.1080/0886022X.2024.2440528

Figure Lengend Snippet: Effects of exogenous addition of IGFBP2 and IGFBP4 on THP-1 cells and primary human renal podocytes. (A) Following the addition of recombinant IGFBP2 and IGFBP4 proteins to THP-1 cells, the supernatant was collected for the assessment of related indices, which were subsequently applied to primary human renal podocytes to observe functional changes. (B) Different concentrations of recombinant IGFBP2 and IGFBP4 (50 ng/mL, 100 ng/mL, 200 ng/mL, and 400 ng/mL) were found to stimulate the levels of complement proteins C3, C4B, C5, C9, as well as MAC and MBL in THP-1 cells. (C) The effects of individually adding IGFBP2h or IGFBP4 recombinant protein, or their combined addition, on the levels of complement proteins C3, C4B, C5, C9, MAC, and MBL in THP-1 cells were assessed. (D) Flow cytometry analysis was conducted to determine the impact of IGFBP2h or IGFBP4 recombinant protein, either alone or in combination, on the polarization of THP-1 cells. (E) The influence of THP-1 cell supernatant on podocyte proliferation was evaluated using the CCK-8 assay. (F) The effect of THP-1 cell supernatant on podocyte apoptosis was measured through flow cytometry. (G) Immunofluorescence was utilized to observe the effect of THP-1 cell supernatant on ROS expression in primary human renal podocytes. (H) The impact of THP-1 cell supernatant on podocyte morphology was also assessed. Data are presented as mean ± SD. n = 3; * p < .05, ** p < .01, *** p < .001.

Article Snippet: The primary human renal podocytes were purchased from Wuhan Punosay Life Technology Co., Ltd. (CP-H075) (Wuhan, China).

Techniques: Recombinant, Functional Assay, Flow Cytometry, CCK-8 Assay, Immunofluorescence, Expressing

Journal: Kidney360

Article Title: AMPK-SP1–Guided Dynein Expression Represents a New Energy-Responsive Mechanism and Therapeutic Target for Diabetic Nephropathy

doi: 10.34067/KID.0000000000000392

Figure Lengend Snippet: Hyperglycemia induces dynein gene expression via an AMPK/SP1-dependent mechanism. (A) Hyperglycemia-responsive dynein genes share binding motif for SP1 TF. SP1 binding motifs (red) identified in genes encoding mouse dynein subunits, using SwissRegulon tools. The promoter sequences for each individual dynein gene and their GenBank locations are highlighted in orange. ChIP primers (gray) were designed close to the SP1 binding motif and the translation starting sites (blue). (B) Workflow of crosslinking SP1 ChIP. (C) Hypothesis and research design of hyperglycemia-induced dynein gene expression via an AMPK/SP1 axis. Hyperglycemia suppresses AMPK and disinhibits SP1, which subsequently initiates the transcription of dynein subunits. (D) The fold enrichment (=2 (Ct IgG−Ct SP1) ) of dynein gene locus sequences immunoprecipitated with SP1 was quantified by ChIP-qPCR. The values were normalized to that of NG. The PCR products were confirmed by running an agarose gel. (E) SP1 activity reflected by the nuclear location of phosphorylated SP1 in podocytes with different treatments, related to the unchanged total SP1 (NG; HG; CC, an AMPK inhibitor; AICAR: an AMPK agonist). Medium containing 0.3% DMSO served as a negative control for chemical intervention. (F) Relative quantification of dynein gene transcription using Gapdh as a housekeeping gene was normalized to that of NG and was compared in a heatmap. (G) Protein levels of representative dynein subunits in podocytes with different treatments were examined using Western blot. The corresponding AMPK activity was expressed as the Thr 172 phosphorylated to total AMPK ratio (AMPK-p/AMPK). The log OD values against the β -actin housekeeping protein were normalized to that of NG. n =3, * P < 0.05 versus NG; ^ P < 0.05 SP1 siRNA versus control siRNA. AICAR, 5-aminoimidazole-4-carboxamide ribonucleoside; AMPK, AMP-activated protein kinase; CC, compound C; ChIP, chromatin immunoprecipitation; HG, high glucose; NG, normal glucose; OD, optical density; qPCR, quantitative PCR; siRNA, small interfering RNA; SP1, specificity protein 1; TF, transcription factor.

Article Snippet: Primary human podocytes were purchased from Celprogen Biotechnology Company (Celprogen 36036-08), cultured in precoated flasks with Human Podocyte Cell Culture Extracellular Matrix (Celprogen E36036-08) and maintained in complete medium with serum (M36036).

Techniques: Expressing, Binding Assay, Immunoprecipitation, Agarose Gel Electrophoresis, Activity Assay, Negative Control, Western Blot, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Small Interfering RNA

Journal: Kidney360

Article Title: AMPK-SP1–Guided Dynein Expression Represents a New Energy-Responsive Mechanism and Therapeutic Target for Diabetic Nephropathy

doi: 10.34067/KID.0000000000000392

Figure Lengend Snippet: MIT restored SP1-mediated dynein expression and dynein-mediated nephrin homeostasis in STZ-induced diabetic mice. Type 1 diabetes was induced in 8-week-old male C57BL/6J mice by single high dose injection of STZ (150 mg/kg, i.p.). Two weeks after the STZ injection, MIT diluted in NS was given via i.p. injection at the dose of 0.25 mg/kg, twice weekly for a total of eight doses. Vehicle controls received NS injections. Nondiabetic mice that received MIT injections were included in this study to exclude nephrotoxicity of MIT at this regimen. In STZ-induced diabetic mice, MIT restored SP1 activity (reflected by immunostaining of Thr 453 phosphorylated SP1 costained with podocyte nuclear marker WT1 (A) reduced nephrin protein (B) and dynein-mediated mistrafficking of nephrin (reflected by increased Dynll1 colocalizing with nephrin, C), and the upregulated expression of dynein subunits (costained with podocyte cytosol marker INF2, D). Mean fluorescence intensity of dynein subunits per podocyte was quantified for comparison. n =15 (three glomeruli or three interstitial areas per section×five mice). * P < 0.05 versus nondiabetic mice, ^ P < 0.05 versus STZ-induced diabetic mice treated with NS. Scale bar: 20 μ m. INF2, inverted formin 2; i.p., intraperitoneal; MIT, mithramycin; NS, normal saline; STZ, streptozotocin.

Article Snippet: Primary human podocytes were purchased from Celprogen Biotechnology Company (Celprogen 36036-08), cultured in precoated flasks with Human Podocyte Cell Culture Extracellular Matrix (Celprogen E36036-08) and maintained in complete medium with serum (M36036).

Techniques: Expressing, Injection, Activity Assay, Immunostaining, Marker, Fluorescence, Comparison, Saline

Journal: Kidney360

Article Title: AMPK-SP1–Guided Dynein Expression Represents a New Energy-Responsive Mechanism and Therapeutic Target for Diabetic Nephropathy

doi: 10.34067/KID.0000000000000392

Figure Lengend Snippet: MIT attenuated podocytopathy and slowed the development of overt nephrosis in STZ-induced diabetic mice. Masson trichrome staining (A, scale bar 50 μ m) and SEM of mouse kidney tissue (B, arrowhead: microvillus transformation, asterisk: podocyte detachment, X: foot process effacement). (C) MIT reduced the urine albumin-to-creatinine ratio in STZ-induced diabetic mice but did not cause albuminuria in nondiabetic mice ( n =5). (D) The histologic and ultrastructural features of the kidney are quantified as glomerular area, percentages of interstitial fibrosis, percentages of mesangial expansion per glomerulus, and percentages of glomerular capillaries covered by irregular foot processes. n =15 (three glomeruli or three interstitial areas per section×five mice), * P < 0.05 versus nondiabetic mice, ^ P < 0.05 versus STZ-induced diabetic mice treated with NS. SEM, scanning electron microscopy.

Article Snippet: Primary human podocytes were purchased from Celprogen Biotechnology Company (Celprogen 36036-08), cultured in precoated flasks with Human Podocyte Cell Culture Extracellular Matrix (Celprogen E36036-08) and maintained in complete medium with serum (M36036).

Techniques: Staining, Transformation Assay, Electron Microscopy

Journal: Kidney360

Article Title: AMPK-SP1–Guided Dynein Expression Represents a New Energy-Responsive Mechanism and Therapeutic Target for Diabetic Nephropathy

doi: 10.34067/KID.0000000000000392

Figure Lengend Snippet: HG impaired nephrin proteosis via AMPK/SP1-regulated dynein expression. (A) Coimmunostaining of nephrin and Dynll1 in podocytes cultured under different conditions: NG (NG+0.3% DMSO); HG (HG+0.3% DMSO); HG in the presence of MIT (0.1 μ M MIT); or AICAR (0.5 mM). (B–D) Nephrin expressed on podocyte surface underwent crosslink-induced endocytosis induced by fluorophore-labeled antinephrin. The postendocytic trafficking of nephrin in HG-cultured podocytes in the presence of MIT or Ciliobrevin D (50 µ M) versus control cells (HG+DMSO) was visualized using live cell imaging and analyzed using the TrackMate and KymographClear plugins of Fiji/ImageJ software. With TrackMate (B), faster retrograde trafficking of nephrin (from surface membrane to cytosol) is displayed in warmer colors, and slower tracks are displayed in cooler colors. In the Kymograph (C), the retrograde, static, and anterograde (from cytosol to surface membrane) tracking events are displayed in red, blue, and green. (D) The fraction and velocities of the trafficking events were quantified using KymographDirect software. * P < 0.05 versus HG+DMSO.

Article Snippet: Primary human podocytes were purchased from Celprogen Biotechnology Company (Celprogen 36036-08), cultured in precoated flasks with Human Podocyte Cell Culture Extracellular Matrix (Celprogen E36036-08) and maintained in complete medium with serum (M36036).

Techniques: Expressing, Cell Culture, Labeling, Live Cell Imaging, Software, Membrane

Journal: Kidney360

Article Title: AMPK-SP1–Guided Dynein Expression Represents a New Energy-Responsive Mechanism and Therapeutic Target for Diabetic Nephropathy

doi: 10.34067/KID.0000000000000392

Figure Lengend Snippet: Hyperglycemia induces dynein gene expression via an AMPK/SP1-dependent mechanism. (A) Hyperglycemia-responsive dynein genes share binding motif for SP1 TF. SP1 binding motifs (red) identified in genes encoding mouse dynein subunits, using SwissRegulon tools. The promoter sequences for each individual dynein gene and their GenBank locations are highlighted in orange. ChIP primers (gray) were designed close to the SP1 binding motif and the translation starting sites (blue). (B) Workflow of crosslinking SP1 ChIP. (C) Hypothesis and research design of hyperglycemia-induced dynein gene expression via an AMPK/SP1 axis. Hyperglycemia suppresses AMPK and disinhibits SP1, which subsequently initiates the transcription of dynein subunits. (D) The fold enrichment (=2 (Ct IgG−Ct SP1) ) of dynein gene locus sequences immunoprecipitated with SP1 was quantified by ChIP-qPCR. The values were normalized to that of NG. The PCR products were confirmed by running an agarose gel. (E) SP1 activity reflected by the nuclear location of phosphorylated SP1 in podocytes with different treatments, related to the unchanged total SP1 (NG; HG; CC, an AMPK inhibitor; AICAR: an AMPK agonist). Medium containing 0.3% DMSO served as a negative control for chemical intervention. (F) Relative quantification of dynein gene transcription using Gapdh as a housekeeping gene was normalized to that of NG and was compared in a heatmap. (G) Protein levels of representative dynein subunits in podocytes with different treatments were examined using Western blot. The corresponding AMPK activity was expressed as the Thr 172 phosphorylated to total AMPK ratio (AMPK-p/AMPK). The log OD values against the β -actin housekeeping protein were normalized to that of NG. n =3, * P < 0.05 versus NG; ^ P < 0.05 SP1 siRNA versus control siRNA. AICAR, 5-aminoimidazole-4-carboxamide ribonucleoside; AMPK, AMP-activated protein kinase; CC, compound C; ChIP, chromatin immunoprecipitation; HG, high glucose; NG, normal glucose; OD, optical density; qPCR, quantitative PCR; siRNA, small interfering RNA; SP1, specificity protein 1; TF, transcription factor.

Article Snippet: Primary human podocytes were purchased from Celprogen Biotechnology Company (Celprogen 36036-08), cultured in precoated flasks with Human Podocyte Cell Culture Extracellular Matrix (Celprogen E36036-08) and maintained in complete medium with serum (M36036).

Techniques: Expressing, Binding Assay, Immunoprecipitation, Agarose Gel Electrophoresis, Activity Assay, Negative Control, Western Blot, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Small Interfering RNA

Journal: Kidney360

Article Title: AMPK-SP1–Guided Dynein Expression Represents a New Energy-Responsive Mechanism and Therapeutic Target for Diabetic Nephropathy

doi: 10.34067/KID.0000000000000392

Figure Lengend Snippet: MIT restored SP1-mediated dynein expression and dynein-mediated nephrin homeostasis in STZ-induced diabetic mice. Type 1 diabetes was induced in 8-week-old male C57BL/6J mice by single high dose injection of STZ (150 mg/kg, i.p.). Two weeks after the STZ injection, MIT diluted in NS was given via i.p. injection at the dose of 0.25 mg/kg, twice weekly for a total of eight doses. Vehicle controls received NS injections. Nondiabetic mice that received MIT injections were included in this study to exclude nephrotoxicity of MIT at this regimen. In STZ-induced diabetic mice, MIT restored SP1 activity (reflected by immunostaining of Thr 453 phosphorylated SP1 costained with podocyte nuclear marker WT1 (A) reduced nephrin protein (B) and dynein-mediated mistrafficking of nephrin (reflected by increased Dynll1 colocalizing with nephrin, C), and the upregulated expression of dynein subunits (costained with podocyte cytosol marker INF2, D). Mean fluorescence intensity of dynein subunits per podocyte was quantified for comparison. n =15 (three glomeruli or three interstitial areas per section×five mice). * P < 0.05 versus nondiabetic mice, ^ P < 0.05 versus STZ-induced diabetic mice treated with NS. Scale bar: 20 μ m. INF2, inverted formin 2; i.p., intraperitoneal; MIT, mithramycin; NS, normal saline; STZ, streptozotocin.

Article Snippet: Primary human podocytes were purchased from Celprogen Biotechnology Company (Celprogen 36036-08), cultured in precoated flasks with Human Podocyte Cell Culture Extracellular Matrix (Celprogen E36036-08) and maintained in complete medium with serum (M36036).

Techniques: Expressing, Injection, Activity Assay, Immunostaining, Marker, Fluorescence, Comparison, Saline

Journal: Kidney360

Article Title: AMPK-SP1–Guided Dynein Expression Represents a New Energy-Responsive Mechanism and Therapeutic Target for Diabetic Nephropathy

doi: 10.34067/KID.0000000000000392

Figure Lengend Snippet: MIT attenuated podocytopathy and slowed the development of overt nephrosis in STZ-induced diabetic mice. Masson trichrome staining (A, scale bar 50 μ m) and SEM of mouse kidney tissue (B, arrowhead: microvillus transformation, asterisk: podocyte detachment, X: foot process effacement). (C) MIT reduced the urine albumin-to-creatinine ratio in STZ-induced diabetic mice but did not cause albuminuria in nondiabetic mice ( n =5). (D) The histologic and ultrastructural features of the kidney are quantified as glomerular area, percentages of interstitial fibrosis, percentages of mesangial expansion per glomerulus, and percentages of glomerular capillaries covered by irregular foot processes. n =15 (three glomeruli or three interstitial areas per section×five mice), * P < 0.05 versus nondiabetic mice, ^ P < 0.05 versus STZ-induced diabetic mice treated with NS. SEM, scanning electron microscopy.

Article Snippet: Primary human podocytes were purchased from Celprogen Biotechnology Company (Celprogen 36036-08), cultured in precoated flasks with Human Podocyte Cell Culture Extracellular Matrix (Celprogen E36036-08) and maintained in complete medium with serum (M36036).

Techniques: Staining, Transformation Assay, Electron Microscopy

Journal: Kidney360

Article Title: AMPK-SP1–Guided Dynein Expression Represents a New Energy-Responsive Mechanism and Therapeutic Target for Diabetic Nephropathy

doi: 10.34067/KID.0000000000000392

Figure Lengend Snippet: HG impaired nephrin proteosis via AMPK/SP1-regulated dynein expression. (A) Coimmunostaining of nephrin and Dynll1 in podocytes cultured under different conditions: NG (NG+0.3% DMSO); HG (HG+0.3% DMSO); HG in the presence of MIT (0.1 μ M MIT); or AICAR (0.5 mM). (B–D) Nephrin expressed on podocyte surface underwent crosslink-induced endocytosis induced by fluorophore-labeled antinephrin. The postendocytic trafficking of nephrin in HG-cultured podocytes in the presence of MIT or Ciliobrevin D (50 µ M) versus control cells (HG+DMSO) was visualized using live cell imaging and analyzed using the TrackMate and KymographClear plugins of Fiji/ImageJ software. With TrackMate (B), faster retrograde trafficking of nephrin (from surface membrane to cytosol) is displayed in warmer colors, and slower tracks are displayed in cooler colors. In the Kymograph (C), the retrograde, static, and anterograde (from cytosol to surface membrane) tracking events are displayed in red, blue, and green. (D) The fraction and velocities of the trafficking events were quantified using KymographDirect software. * P < 0.05 versus HG+DMSO.

Article Snippet: Primary human podocytes were purchased from Celprogen Biotechnology Company (Celprogen 36036-08), cultured in precoated flasks with Human Podocyte Cell Culture Extracellular Matrix (Celprogen E36036-08) and maintained in complete medium with serum (M36036).

Techniques: Expressing, Cell Culture, Labeling, Live Cell Imaging, Software, Membrane