MilliporeSigma
  • VEGF-A inhibition ameliorates podocyte apoptosis via repression of activating protein 1 in diabetes.

VEGF-A inhibition ameliorates podocyte apoptosis via repression of activating protein 1 in diabetes.

American journal of nephrology (2015-01-13)
Xiaoyan Bai, Jian Geng, Xiao Li, Fang Yang, Jianwei Tian
ABSTRACT

Vascular endothelial growth factor-A (VEGF-A) upregulation and podocyte apoptosis have been documented in diabetes. This study was designed to investigate whether inhibiting VEGF-A could ameliorate podocyte apoptosis in diabetes and the underlying mechanisms. In vitro, small interfering RNAs (siRNAs) of VEGF-A and activator protein 1 (AP-1, c-fos and c-jun), bevacizumab (VEGF-A inhibitor) and SP600125 (AP-1 inhibitor) were added to high glucose (30 mM) induced podocytes. Luciferase reporter assay was used to investigate whether AP-1 was a direct target of VEGF-A. In vivo, bevacizumab and SP600125 were administered to 12-week-old streptozotocin-induced male Sprague Dawley rats. The level of VEGF-A, c-fos, c-jun and bcl-2 were examined using immunostaining and Western blot analysis. Podocyte apoptosis was detected using the terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling (TUNEL) assay, electron microscopy and flow cytometry. Silencing VEGF-A or AP-1 upregulated bcl-2 and ameliorated podocyte apoptosis. Silencing VEGF-A decreased the level of c-fos and c-jun and bevacizumab and SP600125 treatment attenuated podocyte apoptosis. Luciferase reporter activity of VEGF-A-3'-UTR constructs was significantly provoked when stimulated with TGF-β1. In diabetic rat kidneys, VEGF-A co-localized with bcl-2 in podocytes. With bevacizumab and SP600125 treatment, the level of VEGF-A and AP-1 decreased while bcl-2 increased. Podocyte apoptotic rate was reduced with condensed podocyte nuclei less frequently observed. The urine albumin excretion rate (UAER) and albumin/creatinine were improved. This study demonstrates VEGF-A inhibition ameliorates podocyte apoptosis by regulating AP-1 and bcl-2 signaling. AP-1 is a direct target of VEGF-A and a novel player in podocyte apoptosis.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Glutaric dialdehyde solution, 50 wt. % in H2O, FCC
Sigma-Aldrich
Osmium tetroxide solution, 4 wt. % in H2O
Sigma-Aldrich
Osmium tetroxide, ReagentPlus®, 99.8%
Sigma-Aldrich
Osmium tetroxide solution, 2.5 wt. % in tert-butanol
Sigma-Aldrich
Osmium tetroxide, ACS reagent, ≥98.0%
Sigma-Aldrich
Osmium tetroxide, Sealed ampule.
Sigma-Aldrich
Glutaraldehyde solution, Grade II, 25% in H2O
Sigma-Aldrich
Glutaraldehyde solution, 50% in H2O, suitable for photographic applications
Sigma-Aldrich
Glutaraldehyde solution, 50 wt. % in H2O
Sigma-Aldrich
Osmium tetroxide solution, suitable for electron microscopy, 4% in H2O
Sigma-Aldrich
Glutaraldehyde solution, Grade I, 25% in H2O, specially purified for use as an electron microscopy fixative
Sigma-Aldrich
Glutaraldehyde solution, Grade I, 70% in H2O, specially purified for use as an electron microscopy fixative or other sophisticated use
Sigma-Aldrich
Glutaraldehyde solution, Grade I, 50% in H2O, specially purified for use as an electron microscopy fixative or other sophisticated use
Sigma-Aldrich
Osmium tetroxide solution, suitable for electron microscopy, 2% in H2O
Sigma-Aldrich
Glutaraldehyde solution, Grade I, 8% in H2O, specially purified for use as an electron microscopy fixative or other sophisticated use
Sigma-Aldrich
Os EnCat® 40, extent of labeling: 0.3 mmol/g Os loading
Sigma-Aldrich
Streptozocin, ≥75% α-anomer basis, ≥98% (HPLC), powder
Sigma-Aldrich
SP600125, ≥98% (HPLC)