Diabetes and Hypertension Differentially Affect Renal Catecholamines and Renal Reactive Oxygen Species

FIGURE 1. Markers of renal injury: albuminuria (A; geometric means ± geometric SD), plasma cystatin C (B), tubulointerstitial area (C), urinary kidney injury molecule-1 (KIM-1) (D), mesangial expansion (E). Diabetes resulted in significant differences for albuminuria, plasma cystatin C, KIM-1, and mesangial expansion. While hypertensive diabetic mice had much greater albuminuria there were no significant differences between the two diabetic groups for the other measures of renal injury assessed. Cont., non-diabetic control; Diab., diabetic; Normot., normotensive BPN/3J mice; Hypert., hypertensive BPH/2J mice. n = 5–10/group except for control normotensive animals: n = 3 urinary KIM-1, n = 4 albuminuria. 2-factor ANOVA p-values: I, interaction; D, relative to diabetic status; S, relative to strain. T-test p < 0.05: ∗ vs. Cont. Normot.; † vs. Cont. Hypert.; ‡ vs. Diab; Mesangial area Cont. Hypert. vs. Diab. Hypert. p = 0.07.

FIGURE 2. Mean arterial pressure (MAP; A,B), systolic BP (sBP; C,D), and diastolic BP (dBP; E,F), 24 h recordings were taken from conscious mice implanted with telemetric blood pressure probes after 10 weeks of study (24 h averages: B,D,F). Diabetic hypertensive mice maintained a high blood pressure despite polyuria and polydipsia. n = 3–9/group. Mean ± SEM. 2-factor ANOVA p-values: I, interaction; D, relative to diabetic status; S, relative to strain. t-test p < 0.05: ∗ vs Cont. Normot.; ‡ vs Diab. Normot.

FIGURE 3. Immunohistochemical localization in the renal cortex of the rate limiting enzyme in catecholamine production, tyrosine hydroxylase (TH; a–d; brown stain) in non-diabetic control and diabetic normotensive and hypertensive mice. Periodic acid Schiff’s (PAS) stain (pink) was used as a counterstain to distinguish proximal (PTs) and distal tubule (DTs) populations. Quantification of proximal tubules (e) and distal tubules (f) shows non-diabetic control hypertensive mice had a greater percentage area of staining for TH in proximal tubules compared to diabetic hypertensive animals. There were no significant differences between groups for the percentage area stained for TH in distal tubules. Renal cortical catecholamine content (g) for DOPA, dopamine, and norepinephrine. Note change in scale for norepinephrine. Hypertensive animals had significantly greater amounts of norepinephrine and dopamine. Diabetic animals had significantly less dopamine and norepinephrine content. Diabetic hypertensive mice retained significantly greater levels of dopamine and norepinephrine as compared to diabetic normotensive mice. n = 6–9/group. 2-factor ANOVA p-values: I=interaction, D=relative to diabetic…

FIGURE 4. Western blot analysis (a,b) confirmed significantly greater levels of monoamine oxidase A (MAO-A) (60 kDa) in the renal cortex of diabetic hypertensive animals after correction for β-actin. Immunohistochemical staining for MAO-A (c–f) was significantly greater in the proximal (PTs; g) and distal tubules (DTs) of hypertensive mice (h). (b) n = 3/group; (g,h) n = 7–8/group. 2-way ANOVA p-values: I, interaction; D, relative to diabetic status; S, relative to strain. t-test p < 0.05: † vs. Cont. Hypert.; ‡ vs. Diab. Normot. Cont-, non-diabetic control; Diab., diabetic; Normot., normotensive BPN/3J mice; Hypert., hypertensive BPH/2J mice. Scale bar: 100 μm. See Supplementary Data for whole blots.

FIGURE 5. Measures of renal cortical oxidant and anti-oxidant systems. Assessment of frozen sections for superoxide by dihydroethidium (DHE; a–e) found significantly greater fluorescence intensity in diabetic animals relative to non-diabetic control hypertensive mice (e). Assessment of renal cortical cytosolic and mitochondrial fractions for hydrogen peroxide production (f,g) found hypertensive animals had significantly greater mitochondrial levels irrespective of diabetic status. Catalase activity (h,i) was significantly greater in the cortical mitochondrial and cytosolic fraction of diabetic normotensive animals as compared to non-diabetic normotensive mice. The cytosolic fraction of diabetic hypertensive mice had significantly less catalase activity than diabetic normotensive mice. DHE n = 5–6/group except control normotensive n = 3; Hydrogen peroxide production and catalase activity n = 6-8/group. Normot. Cont- non-diabetic control; Diab., diabetic; Normot., normotensive BPN/3J mice; Hypert., hypertensive BPH/2J mice. 2-way ANOVA p-values: I, interaction; D, relative to diabetic status; S, relative to strain. t-test p < 0.05: ∗ vs. Cont. Normot.; † vs. Cont. Hypert.; ‡ vs. Diab.…

FIGURE 6. Working hypothesis relating to changes in catecholamines and oxidation in the kidney cortex of mice with hypertension and diabetes with respect to monoamine oxidase (MAO-A). Dotted lines represent theorized outcomes. (–): no change; ↑: increase in levels; ↑↑: large increase in levels; ↓: decrease in levels. PCT, proximal convoluted tubules; MAO, monoamine oxidase levels; sod2- superoxide dismutase 2 (MnSOD) gene transcript levels; TH, tyrosine hydroxylase protein levels. Large bottom arrows: relation to albuminuria and the overall level of kidney injury seen.