Combined endothelin a blockade and chlorthalidone treatment in a rat model of metabolic syndrome

Abstract

Experiments determined whether the combination of endothelin A (ETA) receptor antagonist [ABT-627, atrasentan; (2R,3R,4S)-4-(1,3-benzodioxol-5-yl)-1-[2-(dibutylamino)-2-oxoethyl]-2-(4-methoxyphenyl)pyrrolidine-3-carboxylic acid] and a thiazide diuretic (chlorthalidone) would be more effective at lowering blood pressure and reducing renal injury in a rodent model of metabolic syndrome compared with either treatment alone. Male Dahl salt-sensitive rats were fed a high-fat (36% fat), high-salt (4% NaCl) diet for 4 weeks. Separate groups of rats were then treated with vehicle (control), ABT-627 (ABT; 5 mg/kg per day, in drinking water), chlorthalidone (CLTD; 5 mg/kg per day, in drinking water), or both ABT plus CLTD. Mean arterial pressure (MAP) was recorded continuously by telemetry. After 4 weeks, both ABT and CLTD severely attenuated the development of hypertension, whereas the combination further reduced MAP compared with ABT alone. All treatments prevented proteinuria. CLTD and ABT plus CLTD significantly reduced nephrin (a podocyte injury marker) and kidney injury molecule-1 (a tubulointerstitial injury marker) excretion. ABT, with or without CLTD, significantly reduced plasma 8-oxo-2'-deoxyguanosine, a measure of DNA oxidation, whereas CLTD alone had no effect. All treatments suppressed the number of ED1(+) cells (macrophages) in the kidney. Plasma tumor necrosis factor receptors 1 and 2 were reduced only in the combined ABT and CLTD group. These results suggest that ABT and CLTD have antihypertensive and renal-protective effects in a model of metabolic syndrome that are maximally effective when both drugs are administered together. The findings support the hypothesis that combined ETA antagonist and diuretic treatment may provide therapeutic benefit for individuals with metabolic syndrome consuming a Western diet.

Fig. 1.

Systolic blood pressure (BP), mean arterial pressure, diastolic BP, heart rate, and pulse pressure measured by telemetry in conscious DS rats maintained on a HF/HS diet during a 4-week course of study. Separate groups were given vehicle, the ET_A receptor antagonist, ABT-627 (ABT), or the diuretic, CLTD. Group effects are indicated as *P < 0.05 versus control; ^†P < 0.05 versus ABT alone. n = 6 each group. Comparisons between individual groups on individual days are indicated in the inset table (P < 0.05 for specific days indicated).

Fig. 2.

Determination of glomerular injury as assessed by measurement of proteinuria (A), albuminuria (B), and nephrinuria (C) in DS rats on a HF/HS diet treated with vehicle, ABT, CLTD, and ABT + CLTD for 4 weeks. Tubulointerstitial injury was assessed by measuring urinary KIM-1 excretion (D). *P < 0.05 versus control; ^†P < 0.05 versus ABT alone.

Fig. 3.

Qualitative assessment of vascular injury (A), glomerular necrosis (B), and tubulointerstitial injury (C) in control, ABT-, CLTD-, and ABT + CLTD–treated rats after 4 weeks of treatment.

Fig. 4.

Histologic images depicting vascular injury (A) (H&E, 400×), glomerular necrosis (B) (periodic acid-Schiff [PAS], 400×), and tubulointerstitial injury (C) (Trichrome, 100×) in control, ABT-, CLTD-, and ABT + CLTD–treated rats.

Fig. 5.

Quantification of ED-1 (A) and CD3 (B)-positive staining cells in the renal cortex of control, ABT-, CLTD-, and ABT + CLTD–treated rats (field: 400 × 400 µm). *P < 0.05 versus control; ^†P < 0.05 versus ABT alone.

Fig. 6.

Representative immunostaining images of T cells (CD3⁺) and macrophages (ED-1) in kidney cortical sections from control, ABT-, CLTD-, and ABT + CLTD–treated rats.