Combination of sacubitril/valsartan and blockade of the PI3K pathway enhanced kidney protection in a mouse model of cardiorenal syndrome

Figures

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  Figure 1. Effects of ARNI and valsartan treatment on BW, BP, and HR in mice with cardiorenal injury. (A) Change in BW, (B) rate of BW gain, (C) change in systolic BP, (D) increase in systolic BP, and (E) change in HR during 4 weeks after treatment in the CTL, ANS-vehicle, ANS-ARNI, ANS-VAL M, and ANS-VAL H groups. Values are expressed as the mean ± standard error (five or six mice/group). *P < 0.05, **P < 0.01, ***P < 0.001 vs. the CTL group; #P < 0.05, ###P < 0.001 vs. the ANS-vehicle group; †P < 0.05, ††P < 0.01 vs. the ANS-ARNI group; $P < 0.05, $$$P < 0.001 vs. the ANS-VAL M group. CTL, control; ANS, angiotensin II infusion, nephrectomy, and salt loading; ARNI, angiotensin receptor-neprilysin inhibitor; VAL M, moderate dose of valsartan; VAL H, high dose of valsartan; BW, body weight; BP, blood pressure; HR, heart rate.

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  Figure 2. Effects of an ARNI and valsartan treatment on cardiac fibrosis and function in mice with cardiorenal injury. (A) Representative images of the heart stained with picrosirius red (bars = 100 μm). (B) Quantitative analysis of the fibrotic area in the heart. (C–H) Relative mRNA expression of Col1, Col3a, TGF-β, TNF-α, Nppa, and Nppb in the heart. (I) Cardiac function over 4 weeks and (J) change in rate of cardiac function from baseline to 4 weeks. Values are expressed as the mean ± standard error (five or six mice/group). *P < 0.05, **P < 0.01, ***P < 0.001 vs. the CTL group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. the ANS-vehicle group. CTL, control; ANS, angiotensin II infusion, nephrectomy, and salt loading; ARNI, angiotensin receptor-neprilysin inhibitor; VAL M, moderate dose of valsartan; VAL H, high dose of valsartan; FS, fractional shortening.

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  Figure 3. Effects of ARNI and valsartan treatment on kidney function and glomerular size in mice with cardiorenal injury. Kidney function as determined by (A) plasma creatinine concentration, (B) creatinine clearance, and (C) urine albumin. (D) Representative images of kidneys stained with PAS (bars = 20 μm) and (E) quantitative analysis of the glomerular area. Values are expressed as the mean ± standard error (five or six mice per group). *P < 0.05, ***P < 0.001 vs. the CTL group; #P < 0.05, ###P < 0.001 vs. the ANS-vehicle group. CTL, control; ANS, angiotensin II infusion, nephrectomy, and salt loading; ARNI, angiotensin receptor-neprilysin inhibitor; VAL M, moderate dose of valsartan; VAL H, high dose of valsartan; PAS, periodic acid–Schiff.

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  Figure 4. Effects of ARNI and valsartan treatment on kidney fibrosis in mice with cardiorenal injury. (A) Representative images of kidneys stained with picrosirius red (bars = 100 μm). (B) Quantitative analysis of the fibrotic area in the kidneys. (C–G) Relative mRNA expression of Col1, Col3a, α-SMA, angiotensinogen, and AT1a in the kidneys. (H) Plasma aldosterone levels in each group. Values are expressed as the mean ± standard error (five or six mice/group). *P < 0.05, **P < 0.01, ***P < 0.001 vs. the CTL group; #P < 0.05, ##P < 0.01 vs. the ANS-vehicle group. CTL, control; ANS, angiotensin II infusion, nephrectomy, and salt loading; ARNI, angiotensin receptor-neprilysin inhibitor; VAL M, moderate dose of valsartan; VAL H, high dose of valsartan; SMA, smooth muscle actin; AT1aR, angiotensin II receptor, type 1a.

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  Figure 5. Molecular association of ARNI and valsartan treatment in mice with cardiorenal injury via kidney transcriptomic analysis. (A) Heatmaps of DEGs in CTL, ANS-vehicle, ANS-ARNI, and ANS-VAL M groups. (B) Intersection of genes between up-regulated genes in CTL vs. ANS-vehicle groups and down-regulated genes in ANS-vehicle vs. ANS-ARNI or ANS-vehicle vs. ANS-VAL M groups. (C) KEGG pathway and (D) GO analysis of genes and DEGs that showed improvement only in the ANS-VAL M group. (E) Representative images of kidneys stained with P-PI3K and P-AKT (bars = 100 μm). (F) Kidney P-PI3K/PI3K and P-AKT/AKT protein expression ratio in ANS-ARNI and ANS-VAL M groups by western blotting analysis. CTL, control; ANS, angiotensin II infusion, nephrectomy, and salt loading; ARNI, angiotensin receptor-neprilysin inhibitor; VAL M, moderate dose of valsartan; DEGs, differentially expressed genes; KEGG, Kyoto Encyclopedia of Genes and Genomes; GO, gene ontology; P-PI3K, phospho-phosphoinositide 3-kinase; P-AKT, phospho-AKT.

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  Figure 6. Effects of supplemental PI3K inhibitor treatment with ARNI and valsartan treatment on heart and kidneys in mice with cardiorenal injury. Kidney function was determined by (A) plasma creatinine and (B) urine albumin concentrations. (C) Representative images of kidneys (upper panel, stained with PAS, bars = 20 μm; lower panel, stained with PSR, bars = 100 μm). Quantitative analysis of (D) glomerular and (E) fibrotic areas. (F, G) Relative mRNA expression of Col1 and Col3a in kidneys. (H) Representative images of the hearts stained with PSR (bars = 50 μm). (I) Quantitative analysis of cardiac fibrotic area. (J–L) Relative mRNA expression of Col1, Col3a, and TGF-β in the heart. Values are expressed as the mean ± standard error (six mice per group). *P < 0.05, vs. the ANS-ARNI + Ly294002 group. VAL M, moderate dose of valsartan; PI3K, phosphoinositide 3-kinase; PAS, periodic acid–Schiff; PSR, picrosirius red.