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A. Immunofluorescence analysis of CD31/VEGFR1 and CD31/VEGFR2 in kidneys of nondiabetic and diabetic control and emut mice. FITC-labeled-VEGFR1, VEGFR2; Rhodamine labeled-CD31 and DAPI-nucleus blue. Representative images are shown. N=6/group. Original magnification 40x. Scale bar 50 μm. B. Immunofluorescence analysis of CD31/DPP-4 and CD31/β1-integrin in kidneys of nondiabetic and diabetic control and emut mice. FITC-DPP-4, β1-integrin; Rhodamine-CD31 and DAPI-blue. Representative images are shown. N=6/group. Original magnification 40x. Scale bar 50 μm. C. Conditioned media (CM) experimental design. Three independent sets of experiments were analyzed . D. Immunofluorescence analysis of αSMA expression, DPP-4 and <t>p-SMAD3</t> in conditioned media-treated TECs. For each slide, images of six different fields of view at 400x magnification were evaluated. FITC-DPP-4 and p-smad3l Rhodamine-αSMA; DAPI-blue. N = 3 independent samples/group. Scale bar, 30 μm. E. TGFβRI:TGFβRII and DPP-4:β1-integrin proximity was analyzed by Duo-link proximity ligation assay in isolated cells of CM media-treated isolated tubules (from diabetic control and diabetic emut). Representative images are shown. Three independent sets of experiments were analyzed. For each slide, images of six different fields of view at 400x magnification were evaluated. Scale bar 30 μm. F. Cellular ATP measurement. Six independent samples were analyzed. G. [ 14 C]palmitate uptake and [14C]palmitate oxidation as measured by [ 14 CO 2 ]release. CPM was counted and normalized to the protein in the well. N = 5 independent samples/group. H. Relative mRNA levels of FSP-1, Vimentin, TGFβR1, DPP-4, FASN, CPT1a, PPARα determined by qRT-PCR in conditioned media-treated isolated TECs. N = 6 independent replicates per group. Data are presented as mean ± SEM. Student’s t test was used for the analysis of statistical significance.
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A. Immunofluorescence analysis of CD31/VEGFR1 and CD31/VEGFR2 in kidneys of nondiabetic and diabetic control and emut mice. FITC-labeled-VEGFR1, VEGFR2; Rhodamine labeled-CD31 and DAPI-nucleus blue. Representative images are shown. N=6/group. Original magnification 40x. Scale bar 50 μm. B. Immunofluorescence analysis of CD31/DPP-4 and CD31/β1-integrin in kidneys of nondiabetic and diabetic control and emut mice. FITC-DPP-4, β1-integrin; Rhodamine-CD31 and DAPI-blue. Representative images are shown. N=6/group. Original magnification 40x. Scale bar 50 μm. C. Conditioned media (CM) experimental design. Three independent sets of experiments were analyzed . D. Immunofluorescence analysis of αSMA expression, DPP-4 and <t>p-SMAD3</t> in conditioned media-treated TECs. For each slide, images of six different fields of view at 400x magnification were evaluated. FITC-DPP-4 and p-smad3l Rhodamine-αSMA; DAPI-blue. N = 3 independent samples/group. Scale bar, 30 μm. E. TGFβRI:TGFβRII and DPP-4:β1-integrin proximity was analyzed by Duo-link proximity ligation assay in isolated cells of CM media-treated isolated tubules (from diabetic control and diabetic emut). Representative images are shown. Three independent sets of experiments were analyzed. For each slide, images of six different fields of view at 400x magnification were evaluated. Scale bar 30 μm. F. Cellular ATP measurement. Six independent samples were analyzed. G. [ 14 C]palmitate uptake and [14C]palmitate oxidation as measured by [ 14 CO 2 ]release. CPM was counted and normalized to the protein in the well. N = 5 independent samples/group. H. Relative mRNA levels of FSP-1, Vimentin, TGFβR1, DPP-4, FASN, CPT1a, PPARα determined by qRT-PCR in conditioned media-treated isolated TECs. N = 6 independent replicates per group. Data are presented as mean ± SEM. Student’s t test was used for the analysis of statistical significance.
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A. Immunofluorescence analysis of CD31/VEGFR1 and CD31/VEGFR2 in kidneys of nondiabetic and diabetic control and emut mice. FITC-labeled-VEGFR1, VEGFR2; Rhodamine labeled-CD31 and DAPI-nucleus blue. Representative images are shown. N=6/group. Original magnification 40x. Scale bar 50 μm. B. Immunofluorescence analysis of CD31/DPP-4 and CD31/β1-integrin in kidneys of nondiabetic and diabetic control and emut mice. FITC-DPP-4, β1-integrin; Rhodamine-CD31 and DAPI-blue. Representative images are shown. N=6/group. Original magnification 40x. Scale bar 50 μm. C. Conditioned media (CM) experimental design. Three independent sets of experiments were analyzed . D. Immunofluorescence analysis of αSMA expression, DPP-4 and <t>p-SMAD3</t> in conditioned media-treated TECs. For each slide, images of six different fields of view at 400x magnification were evaluated. FITC-DPP-4 and p-smad3l Rhodamine-αSMA; DAPI-blue. N = 3 independent samples/group. Scale bar, 30 μm. E. TGFβRI:TGFβRII and DPP-4:β1-integrin proximity was analyzed by Duo-link proximity ligation assay in isolated cells of CM media-treated isolated tubules (from diabetic control and diabetic emut). Representative images are shown. Three independent sets of experiments were analyzed. For each slide, images of six different fields of view at 400x magnification were evaluated. Scale bar 30 μm. F. Cellular ATP measurement. Six independent samples were analyzed. G. [ 14 C]palmitate uptake and [14C]palmitate oxidation as measured by [ 14 CO 2 ]release. CPM was counted and normalized to the protein in the well. N = 5 independent samples/group. H. Relative mRNA levels of FSP-1, Vimentin, TGFβR1, DPP-4, FASN, CPT1a, PPARα determined by qRT-PCR in conditioned media-treated isolated TECs. N = 6 independent replicates per group. Data are presented as mean ± SEM. Student’s t test was used for the analysis of statistical significance.
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A. Immunofluorescence analysis of CD31/VEGFR1 and CD31/VEGFR2 in kidneys of nondiabetic and diabetic control and emut mice. FITC-labeled-VEGFR1, VEGFR2; Rhodamine labeled-CD31 and DAPI-nucleus blue. Representative images are shown. N=6/group. Original magnification 40x. Scale bar 50 μm. B. Immunofluorescence analysis of CD31/DPP-4 and CD31/β1-integrin in kidneys of nondiabetic and diabetic control and emut mice. FITC-DPP-4, β1-integrin; Rhodamine-CD31 and DAPI-blue. Representative images are shown. N=6/group. Original magnification 40x. Scale bar 50 μm. C. Conditioned media (CM) experimental design. Three independent sets of experiments were analyzed . D. Immunofluorescence analysis of αSMA expression, DPP-4 and <t>p-SMAD3</t> in conditioned media-treated TECs. For each slide, images of six different fields of view at 400x magnification were evaluated. FITC-DPP-4 and p-smad3l Rhodamine-αSMA; DAPI-blue. N = 3 independent samples/group. Scale bar, 30 μm. E. TGFβRI:TGFβRII and DPP-4:β1-integrin proximity was analyzed by Duo-link proximity ligation assay in isolated cells of CM media-treated isolated tubules (from diabetic control and diabetic emut). Representative images are shown. Three independent sets of experiments were analyzed. For each slide, images of six different fields of view at 400x magnification were evaluated. Scale bar 30 μm. F. Cellular ATP measurement. Six independent samples were analyzed. G. [ 14 C]palmitate uptake and [14C]palmitate oxidation as measured by [ 14 CO 2 ]release. CPM was counted and normalized to the protein in the well. N = 5 independent samples/group. H. Relative mRNA levels of FSP-1, Vimentin, TGFβR1, DPP-4, FASN, CPT1a, PPARα determined by qRT-PCR in conditioned media-treated isolated TECs. N = 6 independent replicates per group. Data are presented as mean ± SEM. Student’s t test was used for the analysis of statistical significance.
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A. Immunofluorescence analysis of CD31/VEGFR1 and CD31/VEGFR2 in kidneys of nondiabetic and diabetic control and emut mice. FITC-labeled-VEGFR1, VEGFR2; Rhodamine labeled-CD31 and DAPI-nucleus blue. Representative images are shown. N=6/group. Original magnification 40x. Scale bar 50 μm. B. Immunofluorescence analysis of CD31/DPP-4 and CD31/β1-integrin in kidneys of nondiabetic and diabetic control and emut mice. FITC-DPP-4, β1-integrin; Rhodamine-CD31 and DAPI-blue. Representative images are shown. N=6/group. Original magnification 40x. Scale bar 50 μm. C. Conditioned media (CM) experimental design. Three independent sets of experiments were analyzed . D. Immunofluorescence analysis of αSMA expression, DPP-4 and p-SMAD3 in conditioned media-treated TECs. For each slide, images of six different fields of view at 400x magnification were evaluated. FITC-DPP-4 and p-smad3l Rhodamine-αSMA; DAPI-blue. N = 3 independent samples/group. Scale bar, 30 μm. E. TGFβRI:TGFβRII and DPP-4:β1-integrin proximity was analyzed by Duo-link proximity ligation assay in isolated cells of CM media-treated isolated tubules (from diabetic control and diabetic emut). Representative images are shown. Three independent sets of experiments were analyzed. For each slide, images of six different fields of view at 400x magnification were evaluated. Scale bar 30 μm. F. Cellular ATP measurement. Six independent samples were analyzed. G. [ 14 C]palmitate uptake and [14C]palmitate oxidation as measured by [ 14 CO 2 ]release. CPM was counted and normalized to the protein in the well. N = 5 independent samples/group. H. Relative mRNA levels of FSP-1, Vimentin, TGFβR1, DPP-4, FASN, CPT1a, PPARα determined by qRT-PCR in conditioned media-treated isolated TECs. N = 6 independent replicates per group. Data are presented as mean ± SEM. Student’s t test was used for the analysis of statistical significance.

Journal: bioRxiv

Article Title: Metabolic reprogramming by endothelial ANGPTL4 depletion protects against diabetic kidney disease

doi: 10.1101/2025.05.08.652142

Figure Lengend Snippet: A. Immunofluorescence analysis of CD31/VEGFR1 and CD31/VEGFR2 in kidneys of nondiabetic and diabetic control and emut mice. FITC-labeled-VEGFR1, VEGFR2; Rhodamine labeled-CD31 and DAPI-nucleus blue. Representative images are shown. N=6/group. Original magnification 40x. Scale bar 50 μm. B. Immunofluorescence analysis of CD31/DPP-4 and CD31/β1-integrin in kidneys of nondiabetic and diabetic control and emut mice. FITC-DPP-4, β1-integrin; Rhodamine-CD31 and DAPI-blue. Representative images are shown. N=6/group. Original magnification 40x. Scale bar 50 μm. C. Conditioned media (CM) experimental design. Three independent sets of experiments were analyzed . D. Immunofluorescence analysis of αSMA expression, DPP-4 and p-SMAD3 in conditioned media-treated TECs. For each slide, images of six different fields of view at 400x magnification were evaluated. FITC-DPP-4 and p-smad3l Rhodamine-αSMA; DAPI-blue. N = 3 independent samples/group. Scale bar, 30 μm. E. TGFβRI:TGFβRII and DPP-4:β1-integrin proximity was analyzed by Duo-link proximity ligation assay in isolated cells of CM media-treated isolated tubules (from diabetic control and diabetic emut). Representative images are shown. Three independent sets of experiments were analyzed. For each slide, images of six different fields of view at 400x magnification were evaluated. Scale bar 30 μm. F. Cellular ATP measurement. Six independent samples were analyzed. G. [ 14 C]palmitate uptake and [14C]palmitate oxidation as measured by [ 14 CO 2 ]release. CPM was counted and normalized to the protein in the well. N = 5 independent samples/group. H. Relative mRNA levels of FSP-1, Vimentin, TGFβR1, DPP-4, FASN, CPT1a, PPARα determined by qRT-PCR in conditioned media-treated isolated TECs. N = 6 independent replicates per group. Data are presented as mean ± SEM. Student’s t test was used for the analysis of statistical significance.

Article Snippet: Rabbit polyclonal anti-smad3 (#9513) antibody was obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Immunofluorescence, Control, Labeling, Expressing, Proximity Ligation Assay, Isolation, Quantitative RT-PCR