Mineralocorticoid Receptor Blockade Mitigates Loop Diuretic-Exacerbated Plasma Volume Contraction and Target-Organ Fibrosis in Angiotensin II-Induced Hypertension

Figures

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  Figure 1. Experimental design, systolic blood pressure (SBP), diastolic blood pressure (DBP), urine output, water intake, body weight, and changes in plasma volume (ΔPV). (A) Experimental design. Plasma volume (PV) was measured in all rats on the first experimental day (Day 1). Seven days after PV measurement (Day 8), Ang II was continuously infused (800 ng/kg/min) using an osmotic pump. Seven days after pump implantation (Day 15), oral administration of furosemide (50 mg/kg/day) or furosemide plus spironolactone (100 mg/kg/day) was initiated. On Day 35 (28 days after pump implantation), PV was measured again immediately before euthanasia. Rats were allocated into six groups according to the treatment protocol: Sham, Ang II, SF, AF, SFS, and AFS, with six rats in each group. (B) Comparison of SBP and DBP among the experimental groups. Values are expressed as mean ± SD. Sham vs. Ang II, ** p < 0.01, *** p < 0.001, **** p < 0.0001; Sham vs. AF, # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001; Ang II vs. AFS, §§ p < 0.01, §§§ p < 0.001, §§§§ p < 0.0001; n = 6 per group. (C) Comparison of urine output, water intake, and body weight among the experimental groups. Values are expressed as…

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  Figure 2. Representative H&E-stained images of the heart, kidney, and aorta. The upper two rows show the heart (upper: ×100; lower: ×200), the middle two rows show the renal cortex and medulla (upper: ×100; lower: ×100), and the lower two rows show the aorta (upper: ×40; lower: ×100).

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  Figure 3. Representative Masson’s trichrome staining images and quantitative analysis of fibrotic area. (A) Representative Masson’s trichrome-stained images of the heart, kidney, and aorta. The upper two rows show the heart (upper: ×100; lower: ×200), the middle two rows show the renal cortex and medulla (upper: ×100; lower: ×100), and the lower two rows show the aorta (upper: ×40; lower: ×100). (B) Quantitative analysis of the fibrotic area in cardiac microscopic fields acquired at ×200 magnification. A total of 16 microscopic fields per group were obtained from tissue sections of two rats. Each data point represents one microscopic field, and values are shown as mean ± SD. Statistical comparisons were performed using nested one-way ANOVA, with the individual animal as the biological experimental unit and microscopic fields nested within-animal. Sham vs. AF, ** p < 0.01. (C) Quantitative analysis of the fibrotic area in the renal cortex and medulla using microscopic fields acquired at ×100 magnification. Twenty cortical and ten medullary microscopic fields per group were obtained from tissue sections of two rats. Each data point represents one microscopic field, and values are shown as…

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  Figure 4. Representative eNOS immunohistochemical images and quantitative analysis of eNOS staining intensity in the heart. (A) Representative eNOS IHC images of the heart. Low-magnification images are shown at ×10, and high-magnification images are shown at ×100. (B) Quantitative analysis of eNOS staining intensity in the heart. A total of 12 microscopic fields per group were obtained from tissue sections of two rats. Each data point represents one microscopic field, and values are shown as mean ± SD. Statistical comparisons were performed using nested one-way ANOVA, with the individual animal as the biological experimental unit and microscopic fields nested within-animal. Sham vs. AF, * p < 0.05.

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  Figure 5. Renal eNOS immunohistochemistry and quantitative assessment of glomerular structural alterations. (A) Representative eNOS immunohistochemical images of superficial cortical and juxtamedullary glomeruli. Low-magnification images are shown at ×10, and high-magnification images are shown at ×400. (B) Quantitative analysis of eNOS staining intensity in superficial cortical and juxtamedullary glomeruli. Thirty superficial cortical glomeruli and fifteen juxtamedullary glomeruli per group were obtained from tissue sections of two rats. Each data point represents one glomerulus, and values are shown as mean ± SD. Separate nested one-way ANOVAs were performed for superficial cortical and juxtamedullary glomeruli, with the individual animal as the biological experimental unit and glomeruli nested within-animal. Superficial cortical glomeruli: Sham vs. Ang II, **** p < 0.0001; Sham vs. AF, **** p < 0.0001; AF vs. AFS, ### p < 0.001. No statistically significant differences were detected among the prespecified comparisons in juxtamedullary glomeruli. (C) Quantitative analysis of the glomerular-to-Bowman’s capsule ratio in superficial cortical glomeruli. Seventy glomeruli per group were…

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  Figure 6. Expression of fibrosis-related proteins in the heart, kidney, and aorta determined by Western blot analysis. For panels (B–D): (1) PDGFRβ; (2) RGS5; (3) TGFβ1; (4) MMP-2; and (5) MMP-3. (A) Representative Western blot images of fibrosis-related proteins in the heart, kidney, and aorta. (B) Quantitative analysis of fibrosis-related protein expression in the heart. (C) Quantitative analysis of fibrosis-related protein expression in the kidney. (D) Quantitative analysis of fibrosis-related protein expression in the aorta. Values are expressed as mean ± SD. Each data point represents one individual rat. Asterisks indicate significant differences between the Sham group and the Ang II, SF, or AF groups; hash symbols indicate significant differences between the Ang II and AF or AFS groups, as well as between the AF and AFS groups. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001; # p < 0.05, ## p < 0.01, ### p < 0.001, and #### p < 0.0001.

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  Figure 7. Representative PDGFRβ immunohistochemical images and quantitative analysis of PDGFRβ staining intensity in the heart and renal glomeruli. (A) Representative PDGFRβ immunohistochemical images of the heart. Low-magnification images are shown at ×10, and high-magnification images are shown at ×100. (B) Quantitative analysis of PDGFRβ staining intensity in the heart. Fifteen microscopic fields per group were obtained from tissue sections of two rats. Each data point represents one microscopic field, and values are shown as mean ± SD. Statistical comparisons were performed using nested one-way ANOVA, with the individual animal as the biological experimental unit and microscopic fields nested within-animal. Sham vs. AF, * p < 0.05. (C) Representative PDGFRβ immunohistochemical images of superficial cortical glomeruli and juxtamedullary glomeruli. Low-magnification images are shown at ×10, and high-magnification images are shown at ×400. (D) Quantitative analysis of PDGFRβ staining intensity in superficial cortical and juxtamedullary glomeruli. Thirty superficial cortical glomeruli and twenty juxtamedullary glomeruli per group were obtained from tissue sections of two rats. Each data…

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  Figure 8. Representative Western blot images and quantitative analysis of mineralocorticoid receptor (MR), ion transport-related proteins, and inflammatory proteins in the heart, kidney, and aorta. For panels (B–D): (1) MR; (2) Na+/K+-ATPase; (3) NCX1; (4) TNF-α; and (5) NF-κB. (A) Representative Western blot images of MR, Na+/K+-ATPase, NCX1, TNF-α, and NF-κB in the heart, kidney, and aorta. (B) Quantitative analysis of protein expression in the heart. (C) Quantitative analysis of protein expression in the kidney. (D) Quantitative analysis of protein expression in the aorta. Values are expressed as mean ± SD. Each data point represents one individual rat. Asterisks indicate significant differences between the Sham group and the Ang II, SF, or AF groups; hash symbols indicate significant differences between the Ang II and AF or AFS groups, as well as between the AF and AFS groups. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001; # p < 0.05, ## p < 0.01 and ### p < 0.001.