Detailed Methods Experiment I enos / mice were purchased from Jackson Laboratory (Bar Harbor, USA). C57BL/6J mice on the same genetic background were purchased from KBT Oriental (Hamamatsu, Japan). Eleven-week-old enos / mice and agematched wild type mice were fed either a normal salt containing 0.5 % NaCl or high salt diet containing 8% NaCl for various periods. Throughout the experiment, blood pressure of the conscious mice was measured weekly with the tailcuff method (BP98A; Softron, Tokyo). Mice were individually housed in metabolic cages (1 mouse per cage) for a period of 3 days under standardized conditions of 12:12-hour light-dark cycle. Mice were acclimatized to the metabolic cages for 48 hours and then (the 3rd day), 24-hour urine samples were collected with metabolic cages to measure various parameters. Urine samples were collected at days 0, 3, 7, or 23 after start of salt loading. Volume of water intake, urine, and food intake per day were measured in each mouse. Twenty-four hours urine samples were used to measure various parameters such as urinary albumin excretion, urinary NOx, and urinary angiotensinogen. Mice were anesthetized and sacrificed at various specified times after start of high salt diet. Arterial blood was immediately collected by cardiac puncture, and plasma was collected by centrifugation and stored at 808C until use. After perfusion with phosphatebuffered saline, the kidneys were immediately excised from each mouse to evaluate various parameters as described below. Experiment II enos / mice, fed a high-salt diet in the same manner as Experiment I, were treated with (1) vehicle, (2) a subpressor dose of irbesartan (2 mg/kg/day), (3) anti-hypertensive dose of irbesartan (20 mg/kg/day), and (4) tempol (1 mmol/l in the drinking water). Irbesartan, generously gifted from Shionogi & Co., Ltd. (Osaka, Japan), was administered by oral gavage. Tempol (Sigma-Aldrich Co.) was orally given to mice as drinking water. Blood pressure was measured every week in the same manner as Experiment I. At various time points after salt loading, 24 hours-urine samples were collected from each mouse with metabolic cages. Mice were anesthetized with at 1 week or 4 weeks after salt loading, and the kidneys were rapidly excised from each mouse to evaluate various parameters as described below. Measurement of urinary albumin, NOx, and angiotesinogen Urinary albumin concentrations were quantified by using commercially available kit (AssayMax Mouse Albumin ELISA Kit; Assaypro LLC), and expressed as ratio of urinary albumin to creatinine. The levels of urinary NOx and angiotensinogen were measured by ELISA kit, NO 2 /NO 3 Assay Kit-Fx (Donjindo Co) and Mouse Total Angiotensinogen Assay Kit (IBL), respectively. Urinary NOx and angiotensinogen were also corrected for urinary creatinine. Histological examination of kidney tissue The kidney tissues removed from each mouse were immediately frozen in Tissue-Tek O.C.T. embedding medium (Sakura Finetek). For detection of macrophage infiltration, frozen kidney sections were blocked with 0.3% H 2 O 2 for 30 minutes, washed in water, treated with 2% skimmilk in PBS, washed with PBS, and incubated with the primary antibodies (rat anti-mouse CD68, Serotec; dilution, 1:500) overnight at 48C. After being washed with PBS, the primary antibody was reacted with horseradish peroxidase-conjugated anti-rat IgG secondary antibodies (BioSource, Camarillo, CA, USA). The reactions were developed with 3,3 -diaminobenzidine (DakoCytomation, Carpinteria, CA, USA), and counter-stained with hematoxylin. Negative controls were prepared by substitution of the primary antibody with an irrelevant antibody. The number of glomerular CD68 positive cells was counted in more than 100 glomeruli per section, and the average of CD68 positive cell number was obtained in individual mice. For the assessment of glomerular sclerosis, the kidney tissues from each mouse were fixed in 4 % paraformaldehyde overnight. Then, they were embedded in paraffin, sectioned into 5-mm slices, and stained with periodic acid-schiff. The glomerulosclerosis index was semiquantitatively calculated by examining more than 100 glomeruli per section, as described previously. [1] The grades were 0, no sclerosis of glomeruli; I, sclerosis of up to 25% of glomerulus; II, sclerosis of 25%-50% of glomerulus; III, sclerosis of 50%-75% of glomerulus; IV, sclerosis of 76%-100% of glomerulus. For the assessment of tubulointerstitial fibrosis, the kidney sections embedded in paraffin were also stained with Sirius Red F3BA (0.5 % in saturated aqueous picric acid, Aldrich Chemical Company). The positive area of Sirius Red F3BA per field was assessed by using Lumina Vision version 2.2 analysis software. Identification of phospho-enos-positive cells in renal cortex For double immunofluorescence staining of phospho-enos and PECAM1 as a marker of vascular endothelium, the cryosections of kidneys were first incubated with anti-rabbit phospho -enos (Abcam; dilution, 1:200) and with anti-rat
PECAM1 (BD Biosciences Pharmingen, dilution, 1:200) overnight at 48C, followed by incubation with goat anti-rabbit Alexa 488 and goat anti-rat Alexa 594 as secondary antibodies for 1 hour at room temperature. The sections were incubated with DAPI solution (Invitrogen; 100 ng/ml) for a few minutes for counterstaining of all nuclei. All the color figures were merged using Lumina Vision version 2.2 analysis software. Measurement of glomerular superoxide production Dihydroethidium (DHE) was used to evaluate renal superoxide levels in situ, as described previously. [2] DHE fluorescence of glomerular sections were quantified using Lumina Vision version 2.2, analysis software. The mean fluorescence was quantified and expressed relative to values obtained from control mice. Measurement of NADPH oxidase activity For measurement of renal NADPH oxidase activity, excised kidney was homogenized with an Ultraturrax T8, and the supernatant was obtained by centrifugation for 5 minutes at 1,000 xg, for measurement of NADPH oxidase activity by lucigenin chemiluminescence in the presence of 10 mm NADPH and 10 mm lucigenin as electron acceptor.[3] Chemiluminescence was recorded every 30 sec. for 15 min. with chemiluminescence reader (BLR-201; ALOKA, Japan). The count of chemiluminescence was corrected for protein concentration. Protein concentrations were measured by the method of Bradford. Detection of angiotensinogen protein in glomerulus and cortical tubules After antigen activation, paraffin-embedded kidney sections were incubated with the primary rabbit anti-mouse/rat angiotensinogen antibody (IBL, Gumma, Japan) in a 1:300 dilution overnight at 48C. Dako Envision plus system-hrp (Dakocytomation, Carpinteria, CA, USA) was used to detect the binding primary antibodies, and the reactions were counter-stained with hematoxylin. Negative controls were prepared by substitution of the primary antibody with an irrelevant antibody. The glomerular angiotensinogen staining score (grades 0 to þ4) was semiquantitatively evaluated in a blind manner by examining more than 100 glomeruli per section. The grades were 0, 0%; I, 1 to 25%; II, 26% to 50%; III, 51% to 75%; IV, 76% to 100% of glomerulus involved. Angiotensinogen protein levels in cortical tubules were assessed by the positive areas per field area using a microscope with 200 magnification, and the intensities of positive areas were averaged by at least 30 fields per mouse. Double immunostaining of angiotensinogen with PECAM1 or a-sma For double immunofluorescence staining of angiotensinogen and PECAM1 as a marker of endothelial cells or a-sma as a marker of mesangial cells, renal cryosections were first incubated with anti-rabbit angiotensinogen (IBL; see above) and with either anti-rat PECAM1 (see above) or anti-mouce a-sma (SIGMA) overnight at 48C. Then, the sections were followed by incubation with goat anti-rabbit Alexa 568 and with either goat anti-rat Alexa 488 or goat anti-mouce Alexa 488 for 1 hour at room temperature, respectively. All the sections were incubated with DAPI solution (Invitrogen; 100 ng/ml) for a few minutes for counterstaining of all nuclei. All the color figures were merged using Lumina Vision version 2.2 analysis software. Identification of angiotensin II in glomerulus For immunofluorescence staining of angiotensin II, the cryosections of kidneys were incubated with anti-goat angiotensin II (Santa Cruz Biotechnology, Inc.; dilution, 1:200) overnight at 48C, followed by incubation with rabbit anti-goat Alexa 488 as secondary antibodies for 1 hour at room temperature. Preparation of renal cortical protein extracts and Western blot analysis Our detailed method has been described previously. [4] Briefly, after protein extracts of renal cortical tissues were subjected to sodium-dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electric transfer to polyvinylidene difluoride membrane, the membranes were probed with specific antibodies. Antibodies used were as follows: anti-phospho enos Ser1177 (BD Transduction Laboratories; 1:2000), anti-enos (BD Transduction Laboratories; 1:2000), anti-phospho nnos Ser 1417 (Abcam; 1:2000), anti-nnos (BD Transduction Laboratories; 1:2000), anti-inos (Cell Signaling Technology Inc.; 1:1000), anti-ace (Abcam; 1:5000), anti-at1 receptor (Santa Cruz Biotechnology, Inc.; 1:2000), and anti-b-actin (Cell Signaling Technology Inc.; 1:2000). The antibody was visualized using an enhanced chemiluminescence method (ECL Plus; Amersham Biosciences). The intensity of the bands was quantified using NIH Image analysis software v1.61. Phospho-eNOS and phospho-nnos values were corrected for total enos and total nnos detected in the same membrane, respectively. The intensity of each value of inos, ACE, and AT1 receptor was normalized for b-actin detected in the same membrane.
Quantitative real time RT-PCR Total RNA was extracted from kidney tissue using TRIzol Regent (Invitrogen). Complementary DNAs were synthesized by reverse transcription of 1 mg of total RNA using QuantiTect 1 Reverse Transcription Kit (QIAGEN Inc., Hilden, Germany) according to the manufacturer s protocol. Real time PCR reactions were performed to evaluate the expression levels of MCP-1, angiotensinogen and renin, by using Thermal Cycler Dice 1 Real Time System (TaKaRa Bio Inc., Shiga, Japan) with SYBR Green I detection and melting temperature analysis as described previously. [5] cdna was amplified using SYBR 1 Premix Ex Taq TM (Perfect Real Time) PCR kit (TaKaRa Bio Inc.) with the following sets of specific primers; MCP-1, angiotensinogen, renin, or gylceraldehyde-3-phosphate dehydrogenase (GAPDH). To confirm amplification specificity the PCR products from each primer pair were subjected to a melting curve analysis. The threshold cycle (Ct) value, which was determined using crossing point method, was normalized to the respective housekeeping GAPDH (Applied Biosystems, California, U.S.A) Ct value and relatively calculated by setting a calibrator sample in each run. Plasma renin activity (PRA) PRA was measured by a radioimmunoassay kit (Wallac, Tokyo, Japan). Statistical analysis All data are presented as mean SEM. The data on time course experiments were analyzed by two-way ANOVA, followed by Fisher s PLSD test, using StatView for Windows (SAS Institute, Inc. Cary, U.S.A.). In all other data, statistical significance was determined with one-way ANOVA, followed by Fisher s PLSD test. In all tests, differences were considered statistically significant at a value of P < 0.05. References 1 Dong YF, Liu L, Lai ZF, Yamamoto E, Kataoka K, Nakamura T, et al. Aliskiren enhances protective effects of valsartan against type 2 diabetic nephropathy in mice. J Hypertens 28:1554-1565. 2 Yamamoto E, Kataoka K, Shintaku H, Yamashita T, Tokutomi Y, Dong YF, et al. Novel mechanism and role of angiotensin II induced vascular endothelial injury in hypertensive diastolic heart failure. Arterioscler Thromb Vasc Biol 2007; 27:2569-2575. 3 Dong YF, Liu L, Kataoka K, Nakamura T, Fukuda M, Tokutomi Y, et al. Aliskiren prevents cardiovascular complications and pancreatic injury in a mouse model of obesity and type 2 diabetes. Diabetologia 2010; 53:180-191. 4 Yamamoto E, Tamamaki N, Nakamura T, Kataoka K, Tokutomi Y, Dong YF, et al. Excess salt causes cerebral neuronal apoptosis and inflammation in stroke-prone hypertensive rats through angiotensin II-induced NADPH oxidase activation. Stroke 2008; 39:3049-3056. 5 Yamamoto E, Kataoka K, Dong YF, Nakamura T, Fukuda M, Tokutomi Y, et al. Aliskiren enhances the protective effects of valsartan against cardiovascular and renal injury in endothelial nitric oxide synthase-deficient mice. Hypertension 2009; 54:633-638.
Online Figure legends Online Figure 1. Effects of high salt diet on daily water intake (A), daily diet intake (B), daily urine volume (C), urinary sodium excretion (D), and urinary sodium/ potassium ratio (E) in wild type and enos / mice at 0, 3, 7, 23 days of salt loading. Twenty four hours-urine samples were collected from each mouse using metabolic cages, as described in Online Methods. Abbreviations used: Wild, wild type mice; enos, enos / mice; (N), normal salt; (H), high salt. Values are means SEM (Wild (N), n ¼ 7; Wild (H), n ¼ 8; enos (N), n ¼ 7; enos (H) n ¼ 8). Online Figure 2. Effects of high salt diet on blood pressure (A), urinary albumin excretion (B), glomerular macrophage infiltration (C), renal MCP-1 mrna (D), glomerulosclerosis (E), and tubulointerstitial fibrosis (F) in wild type and enos / mice. Abbreviations used are the same as in Online Fig. 1. Values are means SEM (n ¼ 6-9).
Online Figure 3. Upper, middle, and lower panels indicate representative photomicrographs of kidney sections from 4 weeks of salt-loaded mice stained with anti-cd68 antibody (arrowheads) (Magnifications, x400), periodic acid-schiff (PAS) (Magnifications, x400), and Sirius Red F3BA (Magnifications, x200), respectively. Scale bar ¼ 100 mm. Abbreviations used are the same as in Online Fig. 1. Online Figure 4. Effect of high salt diet on urinary NOx excretion (A), p-enos (S1177) (B), phospho-enos localization in renal cortices (C) and glomerular superoxide (D) in wild type and enos / mice. Abbreviations used were the same as in Online Fig. 1. ND, not detected. Top panels in (B) indicate representative western blots of phospho (p)- enos and total (t)-enos. The confocal immunofluorescence images in (C) show enos phosphorylation (green), PECAM1 (red), their combined images, and DAPI (blue) in the same area of renal cortices. The data in (B) and (C) were obtained from mice subjected to a high-salt diet for 4 weeks. Values are means SEM (n ¼ 6 9).
Online Figure 5. Effects of high salt diet on renal p-nnos (A) and inos protein levels (B) in wild type and enos / mice on 4 weeks of high salt diet. Top panels indicate representative western blots of phospho (p)- nnos and inos. Abbreviations used were the same as in Online Fig. 1. Values are means SEM (n ¼ 9). Online Figure 6. Representative photomicrographs of kidney sections stained with DHE at 4 weeks after salt loading. Magnifications, x400. Scale bar ¼ 100 mm. Abbreviations used were the same as in Online Fig. 1.
Online Figure 7. Effect of high salt diet on urinary angiotensinogen excretion (A), renal angiotensinogen mrna (B), and glomerular angiotensinogen protein expressions (C) and tubular angiotensinogen (D) in wild type and enos / mice. Abbreviations used were the same as in Online Fig. 1. Renal angiotensinogen mrna levels (B) in individual mice were normalized to GAPDH mrna levels. Values are means SEM (n ¼ 6 9). Online Figure 8. Immunohistochemical analysis of renal cortical angiotensinogen and glomerular angiotensinogen in mice on 1week of high salt diet. The upper panels show representative photomicrographs of renal cortical sections stained with angiotensinogen. Lower panels indicate higher magnified details of upper panels. Positive areas for angiotensinogen are observed as brown dots in the glomerulus and the apical side of proximal tubules. Abbreviations used were the same as in Online Fig. 1.
Online Figure 9. Effects of high salt diet on glomerular superoxide (A) and glomerular angiotensinogen staining (B) in enos / mice on 0, 1, 2, and 3 days of high salt diet. Abbreviations used: N, normal salt diet; High, high salt diet. Values are means SEM (enos (N), n ¼ 3; enos (Day 1), n ¼ 4; enos (Day 2), n ¼ 4; enos (Day 3), n ¼ 5). Online Figure 10. Representative immunofluorescence staining of angiotensin II (green), DAPI (blue), and their combined images in the same area of glomerulus. Scale bar ¼ 100 mm. Abbreviations used are the same as in Online Fig. 1.
Online Figure 11. Effects of high salt diet on plasma renin activity (A), renal renin mrna expression (B), and renal ACE (C) and AT1R protein expressions (D) in wild type and enos / mice on 4 weeks of high salt diet. (B) Renal renin mrna levels in individual mice were normalized to GAPDH mrna levels. Top panels in (C) and (D) indicate representative western blots of renal ACE and AT1 receptor (AT1R) protein, respectively. Abbreviations used were the same as in Online Fig. 1. Values are means SEM (n ¼ 7 9). Online Figure 12. Upper and lower panels indicate representative photomicrographs of glomerular sections stained with anti-cd68 antibody and periodic acid-schiff (PAS), respectively. Magnifications, x400. Scale bar ¼ 100mm. Abbreviations used: N, normal salt-fed enos / mice; V, vehicle-treated enos / mice on a high-salt diet; Irb(2), irbesartan (2 mg/kg/day)-treated enos / mice on a high-salt diet; Irb(20), irbesartan (20 mg/kg/day)-treated enos / mice on a high-salt diet; Tem, tempol-treated enos / mice on a high-salt diet. Online Figure 13. Upper and lower panels indicate representative photomicrographs of glomerular sections stained with anti-angiotensinogen (AGT) antibody and DHE, respectively. Magnifications, x400. Scale bar ¼ 100 mm. Abbreviations used are the same as in Online Fig. 12.
Online Figure 14. Representative immunofluorescence staining of angiotensin II (Ang II) (green) and DAPI (blue) in the same area of glomerulus. Scale bar ¼ 100 mm. Abbreviations used are the same as in Online Fig. 12. Online Figure 15. Effect of irbesartan and tempol on renal renin mrna (A), and renal ACE (B) and AT1 receptor (AT1R) protein expressions (C) in high salt fed enos / mice. (A) Renal renin mrna levels in individual mice were normalized to GAPDH mrna levels. Top panels in (B) and (C) indicate representative western blots of renal ACE and AT1R protein, respectively. Abbreviations used were the same as in Online Fig. 12. Values are means SEM (n ¼ 6 7).