Antiproteinuric and Hyperkalemic Mechanisms Activated by Dual Versus Single Blockade of the RAS in Renovascular Hypertensive Rats

Figures

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  FIGURE 1. Dual RAS blockade, but not losartan or enalapril alone, normalizes blood pressure in renovascular hypertensive rats. Blood pressure was measured using tail-cuff plethysmography in two-kidney one-clip (2K-1C) renovascular-hypertensive rats and controls (2K). Experiments were conducted (A) before and (B) 14 days after treatment with enalapril (2K-1C + E, 20 mg/kg/day), losartan (2K-1C + L, 30 mg/kg/day), enalapril plus losartan (2K-1C + E + L, 30 and 20 mg/kg/day, respectively), or vehicle (2K and 2K-1C, water: 1 ml/kg). The values represent individual measurements and the means ± SEM. **P < 0.01, ***P < 0.001, and ****P < 0.0001 vs. 2K; ###P < 0.001 and ####P < 0.0001 vs. 2K-1C; &&P < 0.01 vs. 2K-1C + E; $$P < 0.01 vs. 2K-1C + L.

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  FIGURE 2. Dual RAS blockade, but not losartan or enalapril alone, normalizes proteinuria and albuminuria in renovascular hypertensive rats. Two-kidney one-clip (2K-1C) renovascular-hypertensive rats treated with enalapril (2K-1C + E, 20 mg/kg/day), losartan (2K-1C + L, 30 mg/kg/day), enalapril plus losartan (2K-1C + E + L, 30 and 20 mg/kg/day, respectively), or vehicle (2K-1C) and vehicle-treated control rats (2K) were placed into metabolic cages for 24-h urine collection. (A) Urinary protein excretion was measured using a commercially available kit based on the pyrogallol red-molybdate method. (B) Urinary albumin concentration was determined by ELISA. The values represent individual measurements and the means ± SEM. *P < 0.05,***P < 0.001, and ****P < 0.0001 vs. 2K; ####P < 0.0001 vs. 2K-1C; &P < 0.05 vs. 2K-1C + E; $$P < 0.01 vs. 2K-1C + L. (C) Profile of urine proteins excreted by the five groups of rats. The 24-h urine samples (volume equivalent to 5.0 μg of creatinine) were subjected to 10% SDS-PAGE. Following electrophoresis, the gels were silver stained. The urinary excretion of proteins of a size smaller than albumin (low molecular weight proteins) and higher than albumin (high…

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  FIGURE 3. Effect of dual versus single RAS blockade on the expression of components of the glomerular filtration barrier in the non-clipped kidney of renovascular hypertensive rats. Samples isolated from the non-clipped kidney of renovascular-hypertensive rats treated with enalapril (2K-1C + E, 20 mg/kg/day), losartan (2K-1C + L, 30 mg/kg/day), enalapril plus losartan (2K-1C + E + L, 30 and 20 mg/kg/day, respectively), or vehicle (2K-1C) and vehicle-treated control rats (2K) containing equal amounts of membrane proteins (20 μg for podocin and nephrin and 5 μg for actin) were subjected to SDS-PAGE, after which the proteins were transferred to a PVDF membrane and incubated with primary antibodies. Actin was used as a loading control. Representative immunoblots of electrophoresed renal membrane proteins and graphical representation of the relative expression of (A) podocin and (B) nephrin. The values represent individual measurements and the means ± SEM. *P < 0.05 and ****P < 0.0001 vs. 2K; #P < 0.05, ##P < 0.01, and ####P < 0.0001 vs. 2K-1C; &&P < 0.01 vs. 2K-1C + E; $$P < 0.01 vs. 2K-1C + L.

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  FIGURE 4. Effect of dual versus single RAS blockade on the expression of selected key components of the proximal tubular endocytic machinery in the non-clipped kidney of renovascular hypertensive rats. Samples isolated from the non-clipped kidney of renovascular-hypertensive rats treated with enalapril (2K-1C + E, 20 mg/kg/day), losartan (2K-1C + L, 30 mg/kg/day), enalapril plus losartan (2K-1C + E + L, 30 and 20 mg/kg/day, respectively), or vehicle (2K-1C) and vehicle-treated control rats (2K) containing equal amounts of membrane proteins (10 μg for megalin, 20 μg for cubilin and ClC-5 nephrin and 5 μg for actin) were subjected to SDS-PAGE, after which the proteins were transferred to a PVDF membrane and incubated with primary antibodies. Actin was used as a loading control. Representative immunoblots of electrophoresed renal membrane proteins and graphical representation of the relative expression of (A) megalin, (B) cubilin, and (C) ClC-5. The values represent individual measurements and the means ± SEM. **P < 0.01, ***P < 0.001, and ****P < 0.0001 vs. 2K; #P < 0.05, ##P < 0.01, ###P < 0.001, and ####P < 0.0001 vs. 2K-1C; &&P < 0.01 and &&&P < 0.001 vs. 2K-1C + E; $$P < 0.01 vs. 2K-1C +…

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  FIGURE 5. Effect of dual versus single RAS blockade on fibrosis of the non-clipped kidney of renovascular hypertensive rats. (A) Representative images of Picrosirius Red staining non-clipped kidney sections (400 × magnification) from renovascular-hypertensive rats treated with enalapril (2K-1C + E, 20 mg/kg/day), losartan (2K-1C + L, 30 mg/kg/day), enalapril plus losartan (2K-1C + E + L, 30 and 20 mg/kg/day, respectively), or vehicle (2K-1C) and vehicle-treated control rats (2K). The scale bar shows 50 μm. (B) Quantification of Picrossiuris Red staining kidney sections. The levels of non-clipped kidney collagen were expressed as percent of the total cross-section area. (C) The concentration of TGF-β was measured in non-clipped kidneys homogenate by ELISA. The values represent individual measurements and the means ± SEM. **P < 0.01, ***P < 0.001, and ****P < 0.0001 vs. 2K; ####P < 0.0001 vs. 2K-1C; &P < 0.05 and &&P < 0.01 vs. 2K-1C + E.

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  FIGURE 6. Dual RAS blockade, but not losartan or enalapril alone, induces hyperkalemia in renovascular hypertensive rats. (A) The plasma potassium concentration (Plasma K+) and (B) the Fractional excretion of potassium (FE K+) were determined in normotensive (2K) and renal hypertensive (2K-1C) rats treated for 14 days with enalapril (2K-1C + E, 20 mg/kg/day), losartan (2K-1C + L, 30 mg/kg/day), enalapril plus losartan (2K-1C + E + L, 30 and 20 mg/kg/day, respectively), or vehicle (2K and 2K-1C, water: 1 ml/kg). The values represent individual measurements and the means ± SEM. *P < 0.05 and ****P < 0.0001 vs. 2K, ####P < 0.0001 vs. 2K-1C; &&P < 0.01 vs. 2K-1C + E; $$P < 0.01 vs. 2K-1C + L.

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  FIGURE 7. Effect of dual versus single RAS blockade on the expression of total and cleaved ENaC subunits in the non-clipped kidney of renovascular hypertensive rats. Samples isolated from the non-clipped kidney of renovascular-hypertensive rats treated with enalapril (2K-1C + E, 20 mg/kg/day), losartan (2K-1C + L, 30 mg/kg/day), enalapril plus losartan (2K-1C + E + L, 30 and 20 mg/kg/day, respectively), or vehicle (2K-1C) and vehicle-treated control rats (2K) containing equal amounts of membrane proteins (75 μg for α-ENaC, β-ENaC, and γ-ENaC and 20 μg for actin) were subjected to SDS-PAGE, after which the proteins were transferred to a PVDF membrane and incubated with primary antibodies. Actin was used as a loading control. Representative immunoblots of electrophoresed renal membrane proteins and graphical representation of the relative expression of (A) Full-length (FL) α-ENaC, (B) cleaved α-ENaC, (C) β-ENaC, (D) cleaved γ-ENaC. The values represent individual measurements and the means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 vs. 2K; ##P < 0.01, ###P < 0.001, and ####P < 0.0001 vs. 2K-1C; &&&P < 0.001 and &&&&P < 0.0001 vs. 2K-1C + E; $$P < 0.01 and $$$$P < 0.0001…