Chronic high-sodium diet intake after weaning lead to neurogenic hypertension in adult Wistar rats

Transcription

1 Chronic high-sodium diet intke fter wening led to neurogenic hypertension in dult Wistr rts 1 Pul Mglhães Gomes; 2 Rento Willin Mrtins Sá; 1 Giovn Lopes Aguir; 1 Milede Hnner Sriv Pes; 1 Andréi Crvlho Alzmor; 1 Wnderson Gerldo Lim; 1 Lisndr Brndino de Oliveir; 3 Sen D. Stocker; 2 Vgner Roerto Antunes; 1 Leonrdo M. Crdoso 1 Deprtment of Biologicl Sciences, Institute of Exct nd Biologicl Sciences nd NUPEB, Federl University of Ouro Preto, Ouro Preto (MG), Brzil. 2 Deprtment of Physiology nd Biophysics, Institute of Biomedicl Sciences, University of So Pulo, So Pulo (SP), Brzil. 3 Deprtment of Medicine, Division of Renl-Electrolyte, University of Pittsurgh School of Medicine, Pittsurgh (PA), United Sttes of Americ. Supplementry Dtset

2 Supplementry Methods nd Dt Body weight nd food intke: Food intke nd ody weight of wened Wistr rts were evluted weekly up to the 12 or 15 weeks. On the week 4, 8, 12 nd 15 fter wening, rts were weighed, individully housed in metolic cges (Tecniplst SPA) for single period of 48 hours nd provided with tp wter nd regulr or high-slt powered chow d liitum. At the sme period, food intke ws clculted s the difference etween initil nd the remining mount of powered chow 24 hours lter in the metolic cge. Therefter, rts were gin housed in collective cges nd provided with free ccess to tp wter nd regulr or high-slt powered chow d liitum. 2

3 Supplementry Tle 1S Body weight nd food intke of HS, HS Unlod nd respective control groups. Cont 12W HS Cont 15W HS Unlod Men±SEM n Men±SEM n p Men±SEM n Men±SEM n p Body weight (g) Wening 52±1 (16) 54±1 (16) > ±2 (6) 51±1 (6) > Week 4 204±7 (16) 210±3 (16) > ±5 (6) 216±8 (6) > Week 8 315±6 (16) 314±6 (16) > ±7 (6) 317±11 (6) > Week ±6 (16) 362±5 (16) > ±5 (6) 368±5 (6) > Week ±4 (6) 416±5 (6) > Food intke (g/rt/24h) Week 4 22±1 (10) 24±1 (11) ±2 (4) 24±1 (4) > Week 8 23±1 (10) 24±2 (11) ±2 (4) 23±2 (4) > Week 12 22±2 (10) 24±2 (11) > ±0.3 (4) 17±1 (4) > Week 15 20±1 (4) 18±0.4 (4) > Dt were compred y two wy ANOVA. Cont 12W = control for HS goup; Cont 15W = control for HS Unlod group. 3

4 Supplementry Methods nd Dt Wter intke nd urine output mesurements: On the week 4, 8, 12 nd 15 fter wening, rts were individully housed in metolic cges (Tecniplst SPA) for single period of 48 hours nd provided with tp wter nd regulr or highslt powered chow d liitum. Urine output nd wter intke were precisely mesured grvimetriclly within the lst 24 hours in the metolic cge. Therefter, rts were gin housed in collective cges nd provided with free ccess to tp wter nd regulr or high-slt powered chow d liitum. Urine smples were centrifuged nd stored t -20 C until the iochemicl nlysis. 4

5 Wter Blnce (ml/rt/24h) Urine Output (ml/rt/24h) Wter Intke (ml/rt/24h) A HS (n=11) HS Unlod (n=4) Cont (n=4-10) 0.27 or 0.90% N % N B C W 8W 12W 15W Supplementry Figure 1S Wter intke (A), urine output (B) nd wter lnce (C) of rts from HS, HS Unlod nd respective control groups. Mesurements were performed on the 4 th (4W), 8 th (8W), 12 th (12W) nd 15 th (15W) weeks fter wening for 24 h in metolic cges. Scttered squres (Cont) nd circles (HS/HS Unlod) represent individul vlues nd rs represent verge vlues with its respective SEM for ech group. Differences etween pirs of mens re indicted y different letters. Sme letter mens no sttisticl difference. Two-wy ANOVA followed y Bonferroni s post test; p<

6 Urine N Conc. (mmol/l) Supplementry Methods nd Dt Urine smples were thwed, vortexed nd smple ws tken for sodium concentrtion mesurement using flme photometry technique (MicroNl B462). Sodium in 24 hours were clculted s the product of solute concentrtion y the 24 h urine output. HS (n=11) HS Unlod (n=4) Cont (n=4-10) 0.27 or 0.90% N % N Supplementry Figure 2S Sodium concentrtion in 24-hours urine smples of rts from HS, HS Unlod nd respective control groups. Mesurements were performed on the 4 th (4W), 8 th (8W), 12 th (12W) nd 15 th (15W) weeks fter wening for 24 h in metolic cges. Scttered squres (Cont) nd circles (HS/HS Unlod) represent individul vlues nd rs represent verge vlues with its respective SEM for ech group. Differences etween pirs of mens re indicted y different letters. Sme letter mens no sttisticl difference. Twowy ANOVA followed y Bonferroni s post test; p<

7 Supplementry Methods nd Dt Blood smpling for iochemicl nlysis: In seprte group of rts (10 HS nd 10 Cont), medin incision ws mde in the domen, nd dominl cv vein ws locted nd then injected with 20 µl of heprin (Hemofol 5,000 IU/mL, Cristáli Ltd., SP, Brzil). After thorcotomy, thorcic cv vein ws sectioned right efore right trium nd 2 ml lood smple ws tken. Blood smples were immeditely centrifuged nd plsm set prt. Osmollity in CSF nd plsm smples ws mesured immeditely fter withdrw (fresh smples) The reminder ws frozen in liquid nitrogen nd stored t -80 C until iochemicl nlysis. Biochemicl nlysis: Cretinine nd ure concentrtions were mesured in urine smples from the totl urine volume collected over 24-hour period. Alumin, cretinine nd ure concentrtions were mesured in plsm smples. Colorimetric ssys were performed y commercil kits (Bioclin; Quis Químic Básic Ltd., Belo Horizonte, Brzil). Alumin determintion ws sed on the romocresol green method. Cretinine ws mesured y the modified Jffe method (17) nd ure ws mesured y colorimetric determintion of mmonium deriving from enzymtic degrdtion of ure. Sodium nd potssium concentrtions in urine, plsm nd CSF smples were mesured y flme photometry (MicroNl B462). Sodium, cretinine nd ure excretion in 24 hours were clculted s the product of ech solute concentrtion y the 24 h urine output. Sodium nd wter lnce in 24 h were clculted s the difference etween intke nd urinry excretion of sodium nd wter, respectively. Plsm nd urine osmollity were mesured in freezing point micro osmometer (µosmettetm, Precision Systems). 7

8 Supplementry Tle S2 Biochemicl test results from plsm nd urine smples tken from HS rts nd respective control group. Cont HS Plsm Men±SEM n Men±SEM n p Alumin (mg/ml) 25.3±0.8 (10) 24.5±0.6 (10) Cretinine (mg/ml) ± (10) ± (10) Ure (mg/ml) 0.43±0.019 (10) 0.38±0.020 (10) Sodium conc. (mmol/l) 147±3.7 (10) 146±4.3 (10) Potssium conc. (mmol/l) 4.9±0.24 (10) 4.5±0.23 (10) Osmollity (mosm/kg) 294±1.6 (10) 289±1.7 (10) Urine Cretinine conc. (mg/ml) 0.91±0.052 (10) 0.75±0.075 (11) Excr. cretinine (mg/24h) 15.1±1.31 (10) 19.1±2.60 (11) Ure conc. (mg/ml) 58.1±4.32 (10) 45.2±3.78* (11) Excr. ure (mg/24h) 966±91.7 (10) 1,149±140.6 (11) Osmollity (mosm/kg) 1,553±134 (10) 1,355±87 (8) Cretinine clernce (ml/min) 4.75±0.63 (4) 5.55±0.64 (4) Alumin, cretinine nd ure tests were crried out using commercil kits (BioClin). Plsm sodium nd potssium concentrtions were mesured using flme photometry. Cretinine clernce, excreted cretinine (Excr. cretinine) nd excreted ure (Excr. ure) were clculted s descried in the methods section. *different of control group; unpired t-test. SEM = stndrd error men. Supplementry Methods Left ventricle wll thickness/left ventricle lumen rtio clcultions: To provide further insight on how the rtio ws clculted, we include here the clcultions we crried out to ssess the dt. In the histologicl sections, the 8

9 oundries etween the ventricle wll nd lumen re often irregulr. Therefore, direct mesurement of the lumen dimeter nd the wll thickness my result in imprecise vlues depending on the point where they were mesured. To precisely determine ventricle lumen dimeter nd wll thickness, we performed mthemticl processing of the dt to ssess these prmeters s if the wll/lumen oundries were perfect merged circles with the sme center. The following steps descrie in further detil the process. 1 st step: Using the softwre Leic Qwin Imge Processing nd Anlysis Softwre (Germny), we mesured the totl re (lumen + left ventricle wll) of the left ventricle. The rdius ws then clculted using the eqution 1 s if it ws perfect circle: Eqution 1: R = A π Where, R = rdius A = re mesured y the softwre. π = nd step: Using the softwre, we mesured the lumen re nd clculted the lumen rdius s if it ws perfect circle using the eqution 1. 3 th step: The left ventricle wll thickness ws clculted using the eqution 2: Eqution 2: 9

10 Wt = R Totl R Lumen Where, Wt = left ventricle wll thickness RTotl = rdius of the wll + lumen circle RLumen = rdius of the lumen circle 4 th Step: The left ventricle wll thickness/lumen dimeter rtio ws clculted using the eqution 3: Eqution 3: Rtio = Wt 2 R Lumen The rtios of the sections from ech niml were verged together nd the resulting rtios of ech niml ws verged to produce one men vlue of rtio for ech experimentl group. 10