Volume 19, Issue 8, Pages 1631-1639 (May 2017) Malate and Aspartate Increase L-Arginine and Nitric Oxide and Attenuate Hypertension  Entai Hou, Na Sun, Fuchang Zhang, Chenyang Zhao, Kristie Usa, Mingyu Liang, Zhongmin Tian  Cell Reports  Volume 19, Issue 8, Pages 1631-1639 (May 2017) DOI: 10.1016/j.celrep.2017.04.071 Copyright © 2017 The Author(s) Terms and Conditions

Cell Reports 2017 19, 1631-1639DOI: (10.1016/j.celrep.2017.04.071) Copyright © 2017 The Author(s) Terms and Conditions

Figure 1 Levels of L-Arginine, Citrulline, Aspartate, Glutamate, and NO Metabolites in the Renal Cortex and Medulla of SS Rats and SS.13BN Rats (A) L-arginine, citrulline, aspartate, and glutamate levels in the renal cortex. (B) L-arginine, citrulline, aspartate, and glutamate levels in the renal medulla. (C) NO metabolites. Data are presented as mean values ± SEM; n = 4. ∗p < 0.05; ∗∗p < 0.01, compared to SS.13BN rats. Cell Reports 2017 19, 1631-1639DOI: (10.1016/j.celrep.2017.04.071) Copyright © 2017 The Author(s) Terms and Conditions

Figure 2 Activities or Protein Abundance of Enzymes Involved in Malate Metabolism, L-Arginine Regeneration, and NO Production in the Renal Cortex and Medulla of SS and SS.13BN Rats (A) Fumarase (FH) activity. (B) Malate dehydrogenase (MDH) activity. (C) Aspartate transaminase (AST) activity. (D) Western blot analysis of protein abundance of argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL). (E) ASS activity. (F) Nitric oxide synthase (NOS) activity. Data are presented as mean values ± SEM; n = 4. ∗p < 0.05; ∗∗p < 0.01, compared to SS.13BN rats. Cell Reports 2017 19, 1631-1639DOI: (10.1016/j.celrep.2017.04.071) Copyright © 2017 The Author(s) Terms and Conditions

Figure 3 Fumarase Knockdown in HK-2 Human Kidney Epithelial Cells Resulted in Decreased Levels of Malate, Aspartate, and NO (A) Fumarase (FH) activity in HK-2 cells treated with control (NC) and fumarase siRNA (FH-siRNA). (B) Western blot analysis of fumarase protein abundance. (C) Malate dehydrogenase (MDH) activity. (D) Aspartate transaminase (AST) activity. (E) ASS activity. (F) NOS activity. (G) Levels of fumarate, malate, aspartate, and L-arginine. (H) NO levels measured by a nitrite assay. (I) NO levels measured by a fluorescent dye. Data are presented as mean values ± SEM; n = 6. ∗p < 0.05; ∗∗p < 0.01, compared to NC. Cell Reports 2017 19, 1631-1639DOI: (10.1016/j.celrep.2017.04.071) Copyright © 2017 The Author(s) Terms and Conditions

Figure 4 Fumarase Knockdown in HUVEC Human Endothelial Cells Resulted in Decreased Levels of L-Arginine and NO (A) Fumarase (FH) activity in HUVECs treated with control (NC) and fumarase siRNA (FH-siRNA). (B) Western blot analysis of fumarase protein abundance. (C) MDH activity. (D) AST activity. (E) ASS activity. (F) Levels of fumarate, malate, aspartate, and L-arginine. (G) NO levels measured by a fluorescent dye. (H) NOS activity. Data are presented as mean values ± SEM; n = 6. ∗p < 0.05; ∗∗p < 0.01, compared to NC. Cell Reports 2017 19, 1631-1639DOI: (10.1016/j.celrep.2017.04.071) Copyright © 2017 The Author(s) Terms and Conditions

Figure 5 Aspartate Supplementation in SS Rats Attenuated Salt-Induced Hypertension and Increased L-Arginine and NO (A) MAP of 7- to 9-week-old SS rats on a 0.4% salt (normal salt; NS) or an 8% salt (high salt; HS) diet with or without aspartate (Asp) supplementation by gavage (250 mg/kg/day). (B) Levels of aspartate and L-arginine in the renal cortex and medulla. (C) Levels of NO metabolite nitrite in the renal cortex and medulla. (D) Levels of NO metabolite nitrite in the serum. (E) ASS activity in the renal cortex and medulla. (F) NOS activity in the renal cortex and medulla. (G) Fumarase (FH) activity in the renal cortex and medulla. Data are presented as mean values ± SEM; n = 5–7. ∗p < 0.05 and ∗∗p < 0.01, between the HS + Asp group and the HS + water group; #p < 0.05 and ##p < 0.01, between the NS + Asp group and the NS + water group. Cell Reports 2017 19, 1631-1639DOI: (10.1016/j.celrep.2017.04.071) Copyright © 2017 The Author(s) Terms and Conditions

Figure 6 Malate Supplementation in SS Rats Attenuated Salt-Induced Hypertension and Increased Aspartate, L-Arginine, and NO (A) Mean arterial blood pressure (MAP) of 7- to 9-week-old SS rats on a 0.4% salt (normal salt, NS) or an 8% salt (high salt, HS) diet with or without malate supplementation by gavage (600 mg/kg/day). (B) Levels of malate, aspartate, and L-arginine in the renal cortex and medulla. (C) Levels of NO metabolite nitrite in the renal cortex and medulla. (D) Levels of NO metabolite nitrite in the serum. (E) ASS activity in the renal cortex and medulla. (F) NOS activity in the renal cortex and medulla. (G) Fumarase (FH) activity in the renal cortex and medulla. Data are presented as mean values ± SEM; n = 5–6. ∗p < 0.05 and ∗∗p < 0.01, between the HS + malate group and the HS + water group; #p < 0.05 and ##p < 0.01, between the NS + malate group and the NS + water group. Cell Reports 2017 19, 1631-1639DOI: (10.1016/j.celrep.2017.04.071) Copyright © 2017 The Author(s) Terms and Conditions