Volume 65, Issue 4, Pages 1311-1319 (April 2004) Augmented arginine uptake, through modulation of cationic amino acid transporter-1, increases GFR in diabetic rats  Idit F. Schwartz, Adrian Iaina, Yishayahu Benedict, Yoram Wollman, Tamara Chernichovski, Eli Brasowski, Faina Misonzhnik, Amir Ben- Dor, Miriam Blum, Yoram Levo, Doron Schwartz  Kidney International  Volume 65, Issue 4, Pages 1311-1319 (April 2004) DOI: 10.1111/j.1523-1755.2004.00508.x Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 1 Effects of glomerular arginine uptake in diabeti rats. (A) Sodium independence and cis-inhibition by lysine. Uptake of radiolabeled arginine ([3H]L-arginine) by freshly harvested glomeruli from diabetic animals in the presence of either 140mmol/L sodium chloride (Na) or 140mmol/L choline chloride and lysine or methionine 10mmol/L. Data are presented as the mean ± SEM of four different experiments. *P < 0.05 vs. control. (B) Concentration dependence of L-arginine. Uptake of [3H]-arginine was measured for 4 minutes in freshly harvested glomeruli from diabetic rats over a range of concentrations (0 to 1mmol/L). Data are presented as the mean ± SEM of four different experiments. Kidney International 2004 65, 1311-1319DOI: (10.1111/j.1523-1755.2004.00508.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 2 Uptake of radiolabeled arginine ([3H]L-arginine) by freshly harvested glomeruli. (A) Uptake from controls (CTL), diabetes (D), uninephrectomy (Neph), diabetes + lysine (D + L), and control + lysine (CTL + L). Data are presented as the mean ± SEM of at least five different experiments. *P < 0.05 vs control. (B) Uptake from diabetic rats pretreated with different concentrations of lysine in the drinking water. Data are presented as the mean ± SEM of four different experiments *P < 0.05 vs. control. Kidney International 2004 65, 1311-1319DOI: (10.1111/j.1523-1755.2004.00508.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 3 Glomerular filtration rate (GFR) indexed by creatinine clearance, in controls (CTL), control + lysine (C + L 0.5%), diabetic (D), and diabetes + lysine (D + L 0.5%)(N = 6). Results shown are mean ± SEM. *P < 0.05 vs. controls. Kidney International 2004 65, 1311-1319DOI: (10.1111/j.1523-1755.2004.00508.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 4 Cationic amino acid transporter-1 (CAT-1), CAT-2, and glyceraldehyde-3 phosphate dehydrogenase (GAPDH) cDNA of glomeruli. (A) Ethidium-stained agarose electrophoresis gel showing polymerase chain reaction (PCR)-amplified cDNA harvested from control (CTL) and diabetic (D) rats. These blots are representative of three different experiments. See text for definition of groups. (B) Relative amount of CAT-1 and CAT-2 mRNA quantitated by densitometry and expressed as CAT/GAPDH ratio of the same experiments shown in (A). Data are presented as the mean ± SEM of three different experiments. *P < 0.05 vs. control. Kidney International 2004 65, 1311-1319DOI: (10.1111/j.1523-1755.2004.00508.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 5 Effect of diabetes on steady-state cationic amino acid transporter-1 (CAT-1) mRNA in rat glomeruli. (A) Northern blot analysis of glomerular CAT-1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA from all experimental groups. These blots are representative of three different experiments. Abbreviations are: CTL, control; CTL + L, normal rats treated with lysine; D, diabetic rats; D+L, diabetic rats treated with lysine. (B) Graphic representation of relative changes in mRNA levels. The ratio of the signal of the 7.9kb CAT-1 mRNA over the signal of GAPDH mRNA was plotted against the appropriate experimental group. *P < 0.05 vs. control (mean ± SEM of three different experiments). Kidney International 2004 65, 1311-1319DOI: (10.1111/j.1523-1755.2004.00508.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 6 Immunostaining for cationic amino acid transporter-1 (CAT-1) protein. Glomeruli from control rats (A), rats treated with lysine (B), diabetic rats (C), and diabetic rats treated with lysine (D) (details of the protocol are described in the Methods section). CAT-1 staining was increased in the capillary endothelial cells of diabetic rats either treated or not treated with lysine. Kidney International 2004 65, 1311-1319DOI: (10.1111/j.1523-1755.2004.00508.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 7 Evaluations of arginase-1, arginase-2, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA. (A) Ethidium-stained agarose electrophoresis gel showing polymerase chain reaction (PCR)-amplified from freshly harvested glomeruli of control and diabetic rats. (B) Relative amount of arginase-1 and arginase-2 mRNA quantitated by densitometry and expressed as arginase/GAPDH ratio of the same experiments shown in (A). Data are presented as the mean ± SEM of three different experiments. *P < 0.05 vs. control. Kidney International 2004 65, 1311-1319DOI: (10.1111/j.1523-1755.2004.00508.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 8 Cationic amino acid transporter-1 (CAT-1), CAT-2, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA. (A) Ethidium-stained agarose electrophoresis gel showing polymerase chain reaction (PCR)-amplified CAT-1, CAT-2, and GAPDH of bone marrow–derived transformed endothelial cells preincubated with agmatine, putrecine or sodium nitroprusside (SNP) (0.5mmol/L) for 24hours. Abbreviations are: CTL, control; Ag, agmatine; Putr, putrecine. (B) Relative amount of CAT-1 and CAT-2 mRNA quantitated by densitometry and expressed as CAT/GAPDH ratio of the same experiments shown in Figure 6a. Data are presented as the mean ± SEM of three different experiments. *P < 0.05 vs. control. Kidney International 2004 65, 1311-1319DOI: (10.1111/j.1523-1755.2004.00508.x) Copyright © 2004 International Society of Nephrology Terms and Conditions