Renin–Angiotensin System Activation Drives Early Cardiorenal Dysfunction in Normotensive Prediabetes

Figures

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  FIGURE 1. Computational assessment of the interplay among protein targets involved in the renin–angiotensin system and those implicated in the pathology of prediabetes and its complications of nephropathy and autonomic neuropathy. (A) Protein–protein interaction network of all protein targets in the four categories; (B) Overlap in protein targets among prediabetes and its complications; (C) Protein–protein interaction network among insulin and the proteins in the renin–angiotensin system. ACE, angiotensin‐converting enzyme; AGT, angiotensin‐II; INS, insulin.

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  FIGURE 2. Daily calorie intake, metabolic, and glycemic manifestations of high calorie (HC) feeding. (A) Daily calorie intake (KCal) of control vs. HC‐fed rats for 12 weeks, (B) Body weight (g) measured at week 12 before sacrifice, (C) Fat:lean ratio, in control and HC‐fed rats, calculated using fat and lean tissue densities obtained via nuclear magnetic resonance at week 12, (D) Fasting blood glucose levels (BGL, mg/dL) measured using tail vein blood drop at week 12 after an overnight fast, (E) Glycated haemoglobin (HbA1c%) at week 12 for control and HC‐fed rats. (Control: n = 8, HC: n = 8, Control‐Ang: n = 7, HC‐Ang: n = 8, Control‐CAP: n = 7, HC‐CAP: n = 7). Values are presented as mean ± SEM. Statistical significance was determined using unpaired student t‐test for A, C, and E and one‐way ANOVA followed by Tukey post hoc test for B and D. * denotes p < 0.05.

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  FIGURE 3. Non‐invasive blood pressure and cardiac function. (A) Systolic blood pressure (mmHg) obtained using CODA tail cuff, (B) ejection fraction (%) and (C) fractional shortening (%) assessed using echocardiography performed at prior to sacrifice at week 12. Statistical significance was determined using one‐way ANOVA followed by Tukey post hoc test. * denotes p < 0.05.

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  FIGURE 4. Representative tracings of parasympathetic responses to phenylephrine. Reflex bradycardic responses to blood pressure increments at increasing doses of the vasopressor, phenylephrine (PE) in (A) Control, (B) Control‐Ang, (C) Control‐CAP, (D) HC, (E) HC‐Ang, and (F) HC‐CAP rats.

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  FIGURE 5. Parasympathetic baroreceptor sensitivity assessed by invasive hemodynamic workup. (A) Changes in mean arterial pressure (ΔMAP, mmHg) and (B) Changes in heart rate (ΔHR, BPM) in response to increasing doses of phenylephrine (PE), (C) linear regression of ΔHR vs. ΔMAP, and (D) parasympathetic baroreceptor sensitivity, as the slope of the regression line in the different rat groups. (Control: n = 8, HC: n = 8, Control‐Ang: n = 7, HC‐Ang: n = 8, Control‐CAP: n = 7, HC‐CAP: n = 7). Values are presented as mean ± SEM. Statistical significance was determined by one‐way ANOVA followed by Tukey's post hoc test for the comparison of baroreceptor sensitivity slopes and two‐way repeated measures ANOVA followed by Sidak's post hoc test for the dose‐effect curves (A & B). * and # denote p < 0.05 versus control and HC rats, respectively.

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  FIGURE 6. Representative tracings of sympathetic responses to sodium nitroprusside. Reflex tachycardic responses to blood pressure drops at increasing doses of the vasodepressor, sodium nitroprusside (SNP) in (A) Control, (B) Control‐Ang, (C) Control‐CAP, (D) HC, (E) HC‐Ang, and (F) HC‐CAP rats.

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  FIGURE 7. Sympathetic baroreceptor sensitivity assessed by invasive hemodynamic workup. (A) Changes in mean arterial pressure (ΔMAP, mmHg) and (B) Changes in heart rate (ΔHR, BPM) in response to increasing doses of sodium nitroprusside (SNP), (C) linear regression of ΔHR vs. ΔMAP, and (D) sympathetic baroreceptor sensitivity, as the slope of the regression line in the different rat groups. (Control: n = 8, HC: n = 8, Control‐Ang: n = 7, HC‐Ang: n = 8, Control‐CAP: n = 7, HC‐CAP: n = 7). Values are presented as mean ± SEM. Statistical significance was determined by one‐way ANOVA followed by Tukey's post hoc test for the comparison of baroreceptor sensitivity slopes (D) and two‐way repeated measures ANOVA followed by Sidak's post hoc test for the dose‐effect curves (A & B).

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  FIGURE 8. Examination of vascular active tone (μm) in middle cerebral arterioles by ex vivo pressure myography in Control (n = 5), HC (n = 5), Control‐Ang (n = 4), and HC‐Ang (n = 5) rats. Active tone was calculated as the difference of vascular diameters in calcium‐containing and calcium‐free buffer at the corresponding pressure points. Representative tracings are shown on the right. Values are presented as mean ± SEM. Statistical significance was determined by two‐way repeated measures ANOVA followed by Sidak's post hoc test for the different comparisons in each panel. * denotes p < 0.05.

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  FIGURE 9. Examination of left renal artery endothelium‐dependent vasodilation. Change in perfusion pressure (mmHg) in response to increasing doses of carbachol (CCh, M), with and without L‐NAME and diclofenac in (A) Control and HC (n = 5 each), (B) Control + Ang (n = 4) and HC + Ang (n = 5), and (C) Control + CAP and HC + CAP rats (n = 4 each). Representative tracings are shown on the right. Values are presented as mean ± SEM. Statistical significance was determined by two‐way repeated measures ANOVA followed by Sidak's post hoc test for the different comparisons in each panel. * denotes p < 0.05.

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  FIGURE 10. Quantification of protein concentration (ng/mg protein) of ACE1 in (A) PVAT and (B) PVAT of control and HC‐fed rats using ELISA. Values are presented as mean ± SEM. Statistical significance was determined by student t‐test for the different comparisons in each panel. * denotes p < 0.05.

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  FIGURE 11. Examination of (A) thoracic aorta medial thickness using Haematoxylin and Eosin stain and (B) brainstem inflammation by immunological staining for IBA‐1. (n = 3/group) Representative micrographs are shown on the left. Scale represents 50 μm. Values are presented as mean ± SEM. Statistical significance was determined by one‐way ANOVA followed by Tukey's post hoc test. * and # denote p < 0.05 versus control and HC rats, respectively.

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  FIGURE 12. Assessment of (A) glomerular morphology and tubular fibrosis using Periodic Acid Schiff's and (B) Masson Trichrome stains, respectively (n = 3/group). Representative micrographs are shown on the left. Scale represents 50 μm. Values are presented as mean ± SEM. Statistical significance was determined by one‐way ANOVA followed by Tukey's post hoc test. * denotes p < 0.05.