Obesity-induced arterial redox imbalance involving mitochondrial NOX4, endothelial dysfunction, and ER stress underlie kidney injury compensated by enhanced mitochondrial bioenergetics

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  Fig. 1. HFD-induced obesity causes renal structural abnormalities and kidney injury. (A–C) Kidney function measurements showing reduced urine output (A), increased protein excretion (B) and reduced cystatin C plasma values indicative of decrease GFR (C). n = 7 HFD rats compared to 6 STD. (D–J) Morphometric histopathological study showing increased kidney weight (D) but reduced nº of glomeruli (E upper panel, F) and glomerular hypertrophy (glomerulomegaly) (E lower panel) illustrated by the increased glomerular area (G) and volume (I), and increased Bowman space area (H) without changes in the number of cells of the glomerular tuft (J) in HFD kidneys compared to STD. Measurements were taken from the average of 5 cortical fields, or 10 glomeruli cut at the vascular pole for each kidney from 6 STD and 6 HFD rats. (K) Masson's trichrome staining revealing glomerular and interstitial fibrotic changes in kidney of HFD compared to STD, depicted by the increased blue staining within the glomerular tuft and tubulointerstitial compartment. (Left) Quantified collagen content expressed as a percentage of the total glomerular area. P < 0.05; p < 0.01 Student's t-Test for impaired observations.

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  Fig. 2. NOX1-derived ROS are involved in NO-mediated endothelial dysfunction of kidney preglomerular arteries from obese rats (A) Isometric force recordings showing the relaxations to the endothelial agonist ACh in preglomerular renal arteries from STD (upper) compared to HFD rats (down). (B) Average relaxations to ACh in arteries from STD and HFD rats. (C–F) Average relaxations to ACh in renal arteries after treatment with the nitric oxide (NO) synthase (NOS) inhibitor, L-NG-nitro-arginine (L-NOARG) (C–D), and with the NOX1 inhibitor NoxA1 ds-tat (E–F). Results are expressed as a percentage of the increase in tension induced by Phe (1 μM). Data are presented as the mean ± SEM of 7–9 arteries of STD and 8–9 arteries of HFD. Significant differences between means were analyzed by Student's t-test for unpaired observations. ∗p < 0.05, ∗∗p < 0.01 versus STD.

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  Fig. 3. Augmented peroxynitrite and preserved exogenous NO-mediated relaxations in kidney preglomerular from HFD-induced obese rats. (A) Nitrotyrosine immunostaining for peroxynitrite was enhanced in renal cortex, mostly in the glomerular tufts and endothelial lining of renal arterioles from HFD (right) compared to STD (left) rats. Sections are representative of n = 3 STD and n = 3 HFD rats. (B) Average relaxations to the NO donor SNAP in preglomerular arteries from STD and HFD rats. Data are presented as the mean ± SEM of arteries of 4 STD and 4 HFD.

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  Fig. 4. Mitochondrial ROS imbalance contributes to renal endothelial dysfunction in HFD-induced obesity (A–B) Basal mitochondrial ROS (O2•-) measured by MitoSOX fluorescence, and (C–F) basal H2O2 (C,D) and NADPH-stimulated H2O2 (E,F) production measured by AmplexRed fluorescence in renal arteries (A–E) and cortex (B–F) from STD and HFD rats. Data are presented as mean ± SEM from 24 to 25 arteries and 26–27 cortex samples form 6 STD and 6 HFD (A, B); 24–26 arteries and 23–25 cortical samplex from 9 STD and 9 HFD rats (C,D); 6 arteries and 6 cortex samples of 4 STD and 4 HFD (E,F). Significant differences between means were analyzed by Student's t-test for unpaired observations. ∗∗p < 0.01; ∗∗∗p < 0.001 versus STD group (A–D) and One-way ANOVA followed by Bonferroni test (E,F). ∗∗∗p < 0.001 vs basal levels, ††p < 0.01vs NADPH-stimulated; (G,H) Average relaxations to ACh in the absence and after treatment with the mitochondrial antioxidant mitoTempo in control STD (G) and HFD (H) renal arteries. Results are expressed as a percentage of the pre-contraction induced by Phe (0.1–0.3 μM). Data are presented as the mean ± SEM of 6–8 arteries from 6 STD and 6 HFD rats. Significant differences…

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  Fig. 5. Obesity induced by HFD downregulates PGC-1α and NOX4 in kidney tissues (A) Immunofluorescence for NOX4 protein (red) is distributed throughout the endothelial lining of the renal arteries in STD (upper panels) and HFD (lower panels) groups. The endothelial cell layer is visualized using the anti-eNOS marker (green). Double immunofluorescence labeling for eNOS and NOX4 shows colocalization in the renal endothelium (yellow). Nuclei are stained with DAPI (blue). Scale bars indicate 25 μm. Sections are representative of n = 3 STD and n = 3 HFD rats. (B–G) Western blot analysis of NOX4, PGC-1α and MnSOD expression in samples of preglomerular renal arteries (B–D) and cortex (E–G) showing that expression of NOX4 and PGC-1α was decreased in both renal arteries and cortex of HFD rats, while MnSOD was not changed in renal arteries, but increased in renal cortex from obese animals. Data are shown as the mean ± SEM of 4–7 STD and 4–7 HFD rats. Significant differences between means were analyzed by using unpaired t-test. ∗P < 0.05, ∗∗P < 0.01 versus STD.

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  Fig. 6. Obesity enhances mitochondrial bioenergetics in kidney preglomerular arteries (A–G) The MitoStress test was performed on freshly isolated preglomerular arterial segments using Seahorse technology to measure the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in STD and HFD rats. Sequential injections of oligomycin, FCCP, and rotenone/antimycin A were administered to assess mitochondrial function (A). Renal arteries from HFD rats exhibited increased basal respiration (A and B), ATP production (A and C), maximal respiration (A and D), proton leak (A and E), non-mitochondrial respiration (A and F), and ECAR (G) compared to STD. Results are expressed as pmol O2/min (OCR) or mpH/min (ECAR) values and referred to the arterial segment surface in mm2. (H) Western Blot analysis showing increased expression of mitochondrial ECT complex IV (COX-IV). Data are shown as means ± SEM of 7–8 STD and 7–9 HFD arteries from n = 7 rats. Significant differences between means were analyzed by using unpaired t-test. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗p < 0.001 versus STD.

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  Fig. 7. ER stress markers in kidney tissues of HFD-induced obese rats Western blot analysis showing the expression of ERS markers in kidney preglomerular arteries (A–D) and cortex (E–J) from HFD-fed rats. The expression of pPERK (A), peIF2α (B) and pIRE1α (D) was increased in renal arteries from HFD rats, while ATF6α (C) was decreased. Protein expression of BiP (E), peIF2α (G), ATF6α (H), pIRE1α (I) were significantly augmented, while CHOP showed a trend to increase (J) in renal cortex from HFD rats. Data are shown as the mean ± SEM of 4–7 STD and 5–7 HFD rats. Significant differences between means were analyzed by using unpaired t-test. ∗P < 0.05, ∗∗∗P < 0.001 versus STD.

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  Fig. 8. Vascular dysfunction in kidney preglomerular arteries from ER stress rats (A) Contractions to the α1-adrenergic agonist Phe, (B) relaxations to the endothelial agonist ACh and (C) the β-adrenoceptor agonist isoprenaline in renal arteries from control and the ERS inductor tunicamycin in vivo-treated rats. (D) Average relaxations to ACh in presence of the NOS inhibitor L-NOARG in control and tunicamycin-treated rats. (E–F) Effect of apocynin on ACh-induced relaxations in renal arteries of (E) control and (F) tunicamycin-treated rats incubated with the NOX inhibitor apocynin. Contractile results are expressed as a percentage of KPSS (A), relaxation is expressed as percentage of the increase in tension induced by Phe (0.1–0.3 μM) (B–F). Data are presented as the mean ± SEM of 6–28 arteries of control and 6–30 of tunicamycin-treated rats. Significant differences between means were analyzed by Student's t-test for paired observations. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 versus control. (G–H) ROS production measured by lucigenin-enhanced chemiluminescence, under basal, NADPH-stimulated and NADPH-induced in presence of the NOX inhibitor apocynin conditions in renal arteries (G) and…

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  Fig. 9. Reduced NOX4 expression and lower H2O2 levels contribute to renal endothelial dysfunction in tunicamycin-induced ER stress. (A–B) Average relaxations to ACh after treatment with the mitochondrial antioxidant mitoTempo in renal arteries of control (A) and tunicamycin treated rats (B). The results are expressed as a percentage of the pre-contraction induced by Phe (0.1–0.5 μM). Data are presented as the mean ± SEM from 10 to 12 arteries from 6 control and 7 ER stress rats. Significant differences between means were analyzed by Student's t-test for paired observations. ∗p < 0.05; ∗∗p < 0.01 versus arteries without MitoTempo. (C and G) Mitochondrial ROS (O2•-) measured by MitoSOX fluorescence, and (D and H) H2O2 production measured by AmplexRed in renal arteries (C–D) and cortex (G–H) from control and tunicamycin treated rats. Data are presented as mean ± SEM from 13 to 14 arteries and 14–16 cortex samples from 4 control and 4 tunicamycin-treated rats. Significant differences between means were analyzed by Student's t-test for unpaired observations. ∗p < 0.05 ∗∗p < 0.01 versus control. (E-F and I-J) Western blot analysis of NOX4 and MnSOD expression in samples of preglomerular renal…

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  Fig. 10. ER stress enhances mitochondrial respiration in kidney preglomerular arteries (A–C) The MitoStress test was performed in freshly isolated preglomerular artery segments using Seahorse technology to measure the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in control and tunicamycin treated rats. Sequential injections of oligomycin, FCCP, and rotenone/antimycin A were administered to assess mitochondrial function (A). Renal arteries from ERS rats exhibited increased basal respiration, oxygen consumed for ATP production (A and B), unchanged maximal respiration (A and D), reduced spare respiratory capacity (A and E) and increased ECAR (G) glycolytic activity, compared to controls. Results are expressed as pmol O2/min (OCR) or mpH/min (ECAR) values and referred to the arterial segment surface in mm2. Data are shown as means ± SEM of 10–12 control and 10 tunicamycin treated arteries from n = 9 rats from each group. Significant differences between means were analyzed by using unpaired t-test. ∗P < 0.05, ∗∗P < 0.01 versus tunicamycin-induced ERs stress rats.

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