Loss of endothelial glucocorticoid receptor accelerates diabetic nephropathy

Figures

• 

  Fig. 1. Loss of endothelial GR results in a fibrogenic phenotype in the kidneys of diabetic mice. a Immunofluorescence analysis was performed in the kidneys of control and diabetic CD-1 and C57BL/6 mice using FITC-labeled GR, rhodamine-labeled CD31 and DAPI (blue). Representative merged images are shown; original magnification ×400. Scale bar 50 µm. N = 7 biologically independent mice/group combined from three separate experiments. Quantification of GR/CD31 double-labeled cells is shown. Data are mean ± SEM. b Western blot and qPCR analysis of GR protein and mRNA levels in isolated endothelial cells from the kidneys of control and diabetic CD-1 and C57BL/6 mice. Endothelial cells were isolated and cultured from six biologically independent mice/group. Three independent experiments were performed. Densitometry analysis of six samples/group is shown and is normalized to β-actin. mRNA expression from six independent cell culture samples was analyzed and normalized to 18S. Two independent experiments were performed in triplicate. Data are mean ± SEM. For all panels, two-way ANOVA was used for the analysis of statistical significance. Significance *p < 0.05, **p < 0.01, ***p < 0.001. Source…

• 

  Fig. 2. Loss of endothelial GR worsens renal fibrosis in diabetic mice. a Schematic diagram, showing induction of diabetes in GR fl/fl; Tie1 Cre− (Control). GR fl/fl; Tie1 Cre+ (GRECKO), Apoe;−/−GR fl/fl; Tie1 Cre− (Apoe−/−) and Apoe;−/−GR fl/fl; Tie1 Cre+ (DKO) mice. Five doses of STZ (50 mg/kg/day IP) were injected to induce diabetes and renal fibrosis. b–i Physiological parameters including body weight, blood glucose, kidney weight/body weight, heart weight/body weight, liver weight/body weight, albumin-to-creatinine ratio (ACR), plasma triglycerides, and plasma cholesterol were measured. N = 6 biologically independent mice/group from two separate experiments. j Masson trichome and Sirius red staining in kidneys of nondiabetic and diabetic control, GRECKO, Apoe−/−, and DKO mice were analyzed. Representative images are shown; original magnification ×300. Relative area of fibrosis (%) and relative collagen (%) were measured using the ImageJ program. N = 7 biologically independent mice/group from two separate experiments. Scale bar 50 µm. Data are mean ± SEM. For all panels, two-way ANOVA was used for the analysis of statistical significance. Significance *p < 0.05, **p < 0.01, ***p <…

• 

  Fig. 3. IL-6 neutralization rescues renal fibrosis in diabetes. a Plasma IL-6 levels in nondiabetic and diabetic mice were measured by cytokine array (Luminex). N = 5 biologically independent mice/group. b Measurement of IL-6 in media of cultured endothelial cells isolated from the indicated groups. Endothelial cells were isolated and cultured from six biologically independent mice per group and were analyzed in triplicate. Data are mean ± SEM. c IL-6 neutralization in diabetic GRECKO and diabetic DKO mice. Representative images are shown; original magnification ×300. Relative area of fibrosis (%) and relative collagen (%) were measured using the ImageJ program. N = 7 biologically independent mice/group. Scale bar 50 μm. Data are mean ± SEM. For all panels, one-way ANOVA with Tukey post-test was used for the analysis of statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided in the source data file.

• 

  Fig. 4. Upregulation of Wnt signaling and fibrogenic markers with loss of endothelial GR. a Western blot analysis of vimentin, HIF1α, Snail1, active β-catenin, CPT1a, and PPARα in isolated endothelial cells from the kidneys of nondiabetic and diabetic control and GRECKO mice. Endothelial cells were isolated and cultured from six biologically independent mice/group. Densitometry calculations from six samples/group combined from three independent experiments are shown and are normalized to β-actin or HSP90 as indicated. Representative blots are shown. Data are mean ± SEM. b Western blot analysis of αSMA, fibronectin, active β-catenin and β-catenin in isolated endothelial cells from the kidneys of diabetic control, GRECKO, Apoe−/−, and DKO mice. Endothelial cells were isolated and cultured from six biologically independent mice/group. Densitometry calculations from six samples/group combined from three independent experiments are shown and are normalized to β-actin or HSP90 as indicated. Data are mean ± SEM. Representative blots are shown. c Immunofluorescence analysis of αSMA/CD31 and TGFβR1/CD31 was performed in the kidneys of nondiabetic and diabetic control, GRECKO, Apoe−/−, and DKO…

• 

  Fig. 5. Wnt inhibitor partially abrogates renal fibrosis in diabetic GRECKO and DKO mice. a Schematic diagram showing the treatment protocol of Wnt inhibition in diabetic control and GRECKO mice. b Physiological parameters including body weight, blood glucose, and kidney weight/body weight were analyzed. N = 6 biologically independent mice/group combined from two experiments. c Masson trichrome, Sirius red, and PAS staining as well as immunohistochemical analysis of β-catenin levels in the kidneys of wnti-treated diabetic control and GRECKO mice. Representative images are shown; original magnification ×300. Relative area of fibrosis (%) and relative collagen deposition (%) were measured using the ImageJ program. N = 8 biologically independent mice/group combined from three experiments were analyzed. Scale bar 50 μm. Data are mean ± SEM. For all panels, one-way ANOVA with Tukey post-test was used for the analysis of statistical significance. Significance *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as source data file. Components of this figure were created using Servier Medical Art templates, which are licensed under a Creative Commons Attribution 3.0 Unported License;…

• 

  Fig. 6. Metabolic reprogramming by loss of endothelial GR loss worsens diabetic kidney disease. a Radiolabeled 14C-palmitate uptake analysis in isolated endothelial cells from kidneys of nondiabetic and diabetic control, GRECKO, Apoe−/−, and DKO mice. CPM of each sample were counted. Data were normalized to μg protein. Fold activity is presented. Endothelial cells isolated and cultured from six biologically independent mice/group were performed in triplicate and analyzed for 14C-palmitate uptake analysis. Data are mean ± SEM. b Radiolabeled [14C]palmitate oxidation and [14CO2]release were measured in isolated endothelial cells from kidneys of nondiabetic and diabetic control, GRECKO, Apoe−/−, and DKO mice. CPM of each sample was counted. Data were normalized to μg protein. Fold activity is presented. Endothelial cells isolated and cultured from six biologically independent mice/group were performed in triplicate and analyzed for 14C-palmitate uptake analysis. Data are mean ± SEM. c Body weight, blood glucose, and kidney weight/body weight were measured at the end of these experiments. N = 6 biologically independent mice from two experiments. Ex vivo radiolabeled [14C]palmitate oxidation…

• 

  Fig. 7. GR loss in endothelial cells reprograms central metabolism in renal tubular cells. a Conditioned media experimental design. HUVECs were transfected with scrambled or GR siRNA; after 6 h, the medium was changed and cells were incubated for 96 h. The subsequently harvested media was transferred to HK-2 cells. b Representative Western blotting analysis of E-cadherin, αSMA, TGFβR1, and active β-catenin expression. N = 7 independent experiments. Representative blots from seven biologically independent samples/group were combined from four independent experiments. Densitometric analysis relative to β-actin is shown. c Immunofluorescence analysis of E-cadherin and α-SMA expression in conditioned medium-treated TECs. For each slide, images of six different fields of view at Å ~ 400 magnification were evaluated. N = 7 independent samples/group. Scale bar 30 μm. d [14C]palmitate oxidation measured by [14CO2]release. CPM were counted and normalized to the protein in the well. N = 8 independent samples/group. e Oxygen consumption rate (OCR) in conditioned medium-treated TECs; each data-point represents the mean of eight independent samples. Three independent experiments were performed. OCR…

• 

  Fig. 8. Graphical representation of the role of GR in endothelial cell homeostasis. GR is crucial for endothelial cell homeostasis and its loss causes homeostatic disruption through release of the pro-inflammatory cytokine IL-6 and aberrant activation of Wnt signaling which lead to defective fatty acid oxidation and associated mesenchymal activation. Components of this figure were created using Servier Medical Art templates, which are licensed under a Creative Commons Attribution 3.0 Unported License; https://smart.servier.com.