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: Pflugers Archiv : European journal of physiology

Article Title: Apolipoprotein L1 (APOL1) cation current in HEK-293 cells and in human podocytes

doi: 10.1007/s00424-022-02767-8

Figure Lengend Snippet: A. Immunoblot detection of APOL1 in podocytes treated in the absence (-INFγ, rightmost lane) or presence of INFγ (Ctrl, leftmost lane). APOL1 is not induced by IFNγ in three clonal populations derived from Celprogen human primary podocytes (C1KO, C2KO, C3KO) in which the APOL1 gene was subjected to CRISPR-mediated inactivation. β-actin served as loading control. B. Preservation of differentiated phenotype in Celprogen podocytes. Upper panels: immunoblot of nephrin in lysates of HEK-293 cells transiently overexpressing nephrin (lane 1), in SV40LgT-immortalized human podocytes (lane 2), in Celprogen primary human podocytes (lane 3) and in podocyte outgrowths from human glomeruli. Lower panels: immunoblots of WT1 and podocin in HEK-293 cells transiently overexpressing WT1 or podocin (lane 1), in SV40LgT-immortalized human podocytes (lane 2) and in Celprogen primary human podocytes (lane 3). β-actin served as loading control for both upper and lower panels. C. Whole cell IV curve (linear ramp from +100 (at left) to −100 mV (at right)) recorded from a representative human primary podocyte incubated 18 h in the absence (lower trace) or presence of IFNγ (10 ng/mL, upper trace). D. Whole cell IV curve (linear ramp from +100 (at left) to −100 mV (at right), recorded first in the absence (red trace) and then after 2 min in the presence of anti-APOL1 aa 1–238 antibody (black trace +Ab), and finally after 3 min of antibody washout (black trace “washout”). Seal resistances at the end of each period were 9.7 GΩ (-Ab), 8.3 GΩ (+Ab) and 4.0 GΩ (washout). E. Capacitance-normalized whole cell currents measured at −60 mV in normal human primary podocytes incubated 24 h in the absence (n=11, left three datasets) or presence of 10 ng/ml IFNγ (n=19, right three datasets); *, p<0.016 vs. minus IFNγ. Each cell was recorded first in the absence and then in the subsequent presence of anti-APOL1 N-terminal domain antibody for 2 min (2 μg/ml, +Ab; n=6 −IFNγ and n=11 +IFNγ), followed by 3 min antibody washout (w/o, n=3 −IFNγ and n 5 +IFNγ). Among the 19 IFNγ-pretreated cells, open circles represent cells for which patch seals did not survive the antibody exposure, black circles connected by black lines depict cells for which patch seals did not survive antibody washout, and black circles connected by red lines show cells for which patch seals survived all three recording conditions. #, p= 0.135, vs. −IFNγ before antibody; ##, p=0.008 vs. +IFNγ before antibody; one-way ANOVA on ranks with Dunnett’s post-test.

Article Snippet: Human primary podocytes (Celprogen, Torrance, CA) were grown per supplier’s recommendations in Celprogen human podocyte primary cell culture complete growth medium and subcultured every 48 h on Celprogen podocyte cell culture extracellular matrix.

Techniques: Western Blot, Derivative Assay, CRISPR, Preserving, Incubation

Journal: Pflugers Archiv : European journal of physiology

Article Title: Apolipoprotein L1 (APOL1) cation current in HEK-293 cells and in human podocytes

doi: 10.1007/s00424-022-02767-8

Figure Lengend Snippet: A. Representative on-cell current traces measured at −Vp = +50 mV in podocytes previously treated 24 h with vehicle (upper trace) or with IFNγ (10 ng/mL, lower trace). B. I-V curve of the predominant unitary conductance class (28 pS) of on-cell patch currents in a representative IFNγ-treated Celprogen human podocyte. C. NPo of predominant unitary current class in native Celprogen podocytes (WT, middle pair of bars) and in two Celprogen knockout cell lines (KO #1 and KO #2), each treated 24 h in the absence (left unfilled bars of each pair) and presence of IFNγ (right bars of each pair; data points overlap in all bars). Values are means ± s.e.m. for indicated (n). *, p<0.05 for WT+IFNγ vs −IFNγ, and for WT+IFNγ vs each KO+IFNγ; unpaired two-way t-tests.

Article Snippet: Human primary podocytes (Celprogen, Torrance, CA) were grown per supplier’s recommendations in Celprogen human podocyte primary cell culture complete growth medium and subcultured every 48 h on Celprogen podocyte cell culture extracellular matrix.

Techniques: Knock-Out

Journal: Pflugers Archiv : European journal of physiology

Article Title: Apolipoprotein L1 (APOL1) cation current in HEK-293 cells and in human podocytes

doi: 10.1007/s00424-022-02767-8

Figure Lengend Snippet: A. NPo of unitary currents measured at −Vp = +50 mV in IFNγ-pretreated Celprogen podocytes treated without (n=14, white bar, data points overlap) or with 2 μg/ml anti-APOL1 N-terminal domain antibody (n=7, black bar, data points overlap) or nonspecific rabbit IgG (n=6, gray bar) in the pipette solution. Steady-state values at 2–3 min after achievement of seal. *, p=0.003 vs “none”; unpaired two-tailed t-test. B. NPo of unitary currents measured at −Vp = +50 mV in IFNγ-pretreated Celprogen podocytes (n=6) exposed to 3 μM recombinant N-terminal fragment of Serum Response-Associated (SRA) of Trypanosoma brucei rhodesiense in the pipette solution, and recorded immediately after establishment of a gigohm seal (gray bar) and ~110 s after seal establishment (black bar; data points overlap). *, p<0.041 vs ~0 sec; unpaired two-tailed t-test. Gigohm resistances were maintained throughout the recording periods.

Article Snippet: Human primary podocytes (Celprogen, Torrance, CA) were grown per supplier’s recommendations in Celprogen human podocyte primary cell culture complete growth medium and subcultured every 48 h on Celprogen podocyte cell culture extracellular matrix.

Techniques: Transferring, Two Tailed Test, Recombinant

Journal: Pflugers Archiv : European journal of physiology

Article Title: Apolipoprotein L1 (APOL1) cation current in HEK-293 cells and in human podocytes

doi: 10.1007/s00424-022-02767-8

Figure Lengend Snippet: A. Immunoblot detection of APOL1 in podocytes treated in the absence (-INFγ, rightmost lane) or presence of INFγ (Ctrl, leftmost lane). APOL1 is not induced by IFNγ in three clonal populations derived from Celprogen human primary podocytes (C1KO, C2KO, C3KO) in which the APOL1 gene was subjected to CRISPR-mediated inactivation. β-actin served as loading control. B. Preservation of differentiated phenotype in Celprogen podocytes. Upper panels: immunoblot of nephrin in lysates of HEK-293 cells transiently overexpressing nephrin (lane 1), in SV40LgT-immortalized human podocytes (lane 2), in Celprogen primary human podocytes (lane 3) and in podocyte outgrowths from human glomeruli. Lower panels: immunoblots of WT1 and podocin in HEK-293 cells transiently overexpressing WT1 or podocin (lane 1), in SV40LgT-immortalized human podocytes (lane 2) and in Celprogen primary human podocytes (lane 3). β-actin served as loading control for both upper and lower panels. C. Whole cell IV curve (linear ramp from +100 (at left) to −100 mV (at right)) recorded from a representative human primary podocyte incubated 18 h in the absence (lower trace) or presence of IFNγ (10 ng/mL, upper trace). D. Whole cell IV curve (linear ramp from +100 (at left) to −100 mV (at right), recorded first in the absence (red trace) and then after 2 min in the presence of anti-APOL1 aa 1–238 antibody (black trace +Ab), and finally after 3 min of antibody washout (black trace “washout”). Seal resistances at the end of each period were 9.7 GΩ (-Ab), 8.3 GΩ (+Ab) and 4.0 GΩ (washout). E. Capacitance-normalized whole cell currents measured at −60 mV in normal human primary podocytes incubated 24 h in the absence (n=11, left three datasets) or presence of 10 ng/ml IFNγ (n=19, right three datasets); *, p<0.016 vs. minus IFNγ. Each cell was recorded first in the absence and then in the subsequent presence of anti-APOL1 N-terminal domain antibody for 2 min (2 μg/ml, +Ab; n=6 −IFNγ and n=11 +IFNγ), followed by 3 min antibody washout (w/o, n=3 −IFNγ and n 5 +IFNγ). Among the 19 IFNγ-pretreated cells, open circles represent cells for which patch seals did not survive the antibody exposure, black circles connected by black lines depict cells for which patch seals did not survive antibody washout, and black circles connected by red lines show cells for which patch seals survived all three recording conditions. #, p= 0.135, vs. −IFNγ before antibody; ##, p=0.008 vs. +IFNγ before antibody; one-way ANOVA on ranks with Dunnett’s post-test.

Article Snippet: This result suggests that neither APOL2 nor APOL6, which along with APOL1 are induced by IFNγ treatment of immortalized human podocytes [ 39 ] and which both mediate ion channel activity in lipid bilayers [ 41 ], contribute significantly to IFNγ-stimulated current measured by on-cell patch recording in Celprogen primary human G0 podocytes.

Techniques: Western Blot, Derivative Assay, CRISPR, Preserving, Incubation

Journal: Pflugers Archiv : European journal of physiology

Article Title: Apolipoprotein L1 (APOL1) cation current in HEK-293 cells and in human podocytes

doi: 10.1007/s00424-022-02767-8

Figure Lengend Snippet: A. Representative on-cell current traces measured at −Vp = +50 mV in podocytes previously treated 24 h with vehicle (upper trace) or with IFNγ (10 ng/mL, lower trace). B. I-V curve of the predominant unitary conductance class (28 pS) of on-cell patch currents in a representative IFNγ-treated Celprogen human podocyte. C. NPo of predominant unitary current class in native Celprogen podocytes (WT, middle pair of bars) and in two Celprogen knockout cell lines (KO #1 and KO #2), each treated 24 h in the absence (left unfilled bars of each pair) and presence of IFNγ (right bars of each pair; data points overlap in all bars). Values are means ± s.e.m. for indicated (n). *, p<0.05 for WT+IFNγ vs −IFNγ, and for WT+IFNγ vs each KO+IFNγ; unpaired two-way t-tests.

Article Snippet: This result suggests that neither APOL2 nor APOL6, which along with APOL1 are induced by IFNγ treatment of immortalized human podocytes [ 39 ] and which both mediate ion channel activity in lipid bilayers [ 41 ], contribute significantly to IFNγ-stimulated current measured by on-cell patch recording in Celprogen primary human G0 podocytes.

Techniques: Knock-Out

Journal: Pflugers Archiv : European journal of physiology

Article Title: Apolipoprotein L1 (APOL1) cation current in HEK-293 cells and in human podocytes

doi: 10.1007/s00424-022-02767-8

Figure Lengend Snippet: A. NPo of unitary currents measured at −Vp = +50 mV in IFNγ-pretreated Celprogen podocytes treated without (n=14, white bar, data points overlap) or with 2 μg/ml anti-APOL1 N-terminal domain antibody (n=7, black bar, data points overlap) or nonspecific rabbit IgG (n=6, gray bar) in the pipette solution. Steady-state values at 2–3 min after achievement of seal. *, p=0.003 vs “none”; unpaired two-tailed t-test. B. NPo of unitary currents measured at −Vp = +50 mV in IFNγ-pretreated Celprogen podocytes (n=6) exposed to 3 μM recombinant N-terminal fragment of Serum Response-Associated (SRA) of Trypanosoma brucei rhodesiense in the pipette solution, and recorded immediately after establishment of a gigohm seal (gray bar) and ~110 s after seal establishment (black bar; data points overlap). *, p<0.041 vs ~0 sec; unpaired two-tailed t-test. Gigohm resistances were maintained throughout the recording periods.

Article Snippet: This result suggests that neither APOL2 nor APOL6, which along with APOL1 are induced by IFNγ treatment of immortalized human podocytes [ 39 ] and which both mediate ion channel activity in lipid bilayers [ 41 ], contribute significantly to IFNγ-stimulated current measured by on-cell patch recording in Celprogen primary human G0 podocytes.

Techniques: Transferring, Two Tailed Test, Recombinant