Effect of salt on hypertension and oxidative stress in a rat model of diet-induc - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:47 |只看该作者 |倒序浏览 |打印

作者：Anca D. Dobrian, Suzanne D. Schriver, Terrie Lynch,  Russell L. Prewitt作者单位：Department of Physiological Sciences, Eastern Virginia Medical School,Norfolk, Virginia 23507 - q+ H1 W1 @2 c4 i

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      【摘要】
   High-salt diet is known to induce or aggravate hypertension in animalmodels of hypertension and in humans. When Sprague-Dawley rats ( n =60) are fed a moderately high-fat diet (32% kcal fat, 0.8% NaCl) for 10 wk,about one-half develop obesity [obesity prone (OP)] and mild hypertension,whereas the other half [obesity resistant (OR)] maintain body weightequivalent to a low-fat control (C) and are normotensive. The aim of thisstudy was to test the effect of high-NaCl diets (2 and 4% NaCl) on the development of hypertension and obesity, oxidative stress, and renal function.Both 2 and 4% NaCl induced an early increase in systolic blood pressure of OPbut not OR or C rats. High-salt intake induced an increase in the size andreduction in number of adipocytes, concomitant to a twofold increase incirculating leptin in OP rats. Aortic superoxide generation indicated a 2.8-fold increase in the OP high-salt vs. normal-salt groups, whereas urineisoprostanes were not significantly increased. Also, hydroxynonenal proteinadducts in the kidney were highly increased in OP rats on 2 and 4% NaCl,indicating oxidative stress in the renal tissue. Urine albumin was increasedthreefold in the OP on 2% NaCl and fourfold in the same group on 4% NaCl vs.0.8% NaCl. Kidney histology indicated a higher degree of glomerulosclerosis inOP rats on high-salt diets. In summary, high-salt diet accelerated thedevelopment but did not increase the severity of hypertension; high saltincreased oxidative stress in the vasculature and kidney and induced kidneyglomerulosclerosis and microalbuminuria. Also, the OP rats on high saltdisplayed adipocyte hypertrophy and increased leptin production. : z9 j, s9 z* y, ?
      【关键词】 glomerulosclerosis kidney leptin sodium dietary' {; N* h) a: h
   OBESITY IS A complex, multifactorial disease that is associated with essential hypertension in 78% of men and 65% of women, asindicated by the data from the Framingham Heart Study( 25 ). Another importantcontributor to hypertension in humans is the excessive consumption of dietarysalt, and epidemiological studies have demonstrated a significant but weakrelation between salt intake and hypertension( 32, 33 ). Some, but not all, interventional studies have shown that salt restriction may lower bloodpressure (BP) ( 19, 33 ). Some recent studiesreport correlation among hypertension, salt sensitivity, and insulin resistance in obese humans( 38 ), whereas others fail toobserve a significant relationship( 8 ). Animal models of obesity, hypertension, and insulin resistance display differences with respect to saltsensitivity. In Zucker rats, there is a clear correlation between salt intakeand the severity of hypertension ( 4, 47 ), whereas in chronichyperinsulinemic Sprague-Dawley (SD) rats, hypertension is not salt sensitive,albeit a shift in pressure-natriuresis relationship was reported( 2 ). One important contributorto hypertension in salt-sensitive animal models and humans seems to be theendothelial dysfunction, in particular the altered vascular reactivity due toan impairment in nitric oxide (NO) production( 22, 31, 36 ). High-salt intake is ableto decrease both plasma levels and urinary excretion of nitrates( 3, 16 ). One possible explanationis a reduced availability rather than decreased production of NO. The ability of NO to quickly interact with superoxide anion, forming the potent oxidantperoxynitrite, is well documented( 43 ). Increased superoxideproduction in both vasculature and kidney was extensively reported in variousforms of hypertension in experimental models and humans( 40 - 42, 46 ). Moreover, we reportedthat obese hypertensive rats on high-fat diet also display increased oxidativestress and reduced NO bioavailability( 12 ). Also, salt sensitivitywas associated with increased oxidative stress in rats( 5, 49 ). Apart from the effects onBP regulation, elevated salt intake was associated with cardiovascular and renal changes leading to end-organ damage( 6 ). Moreover, a recent reportconnects salt intake with oxidative stress and nephrosclerosis inDahl-sensitive hypertensive rats( 48 ). Another important factorinvolved in BP regulation in obesity is leptin( 24 ). Leptin was shown to haveboth a vasopressor effect at peripheral level and, infused in high doses, ahypertensive effect acting at central level( 24 ). However, a recent report suggests that leptin may not contribute to arterial pressure sensitivity tosalt in hyperleptinemic obese rats( 7 ). The aim of our study wasto assess the effect of high-fat, high-salt diets on the development ofhypertension and oxidative stress in a rat model of diet-induced obesity.Moreover, the effect on vascular hypertrophy and kidney sclerosis wasassessed. Additionally, the effect of salt on adiposity and leptin production was also measured.6 }2 A9 [- ^# e+ U! G4 `1 }0 [
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METHODS2 N- d& X: g  d7 |

Animals

All procedures involving animals were approved by the Institutional AnimalCare and Use Committee of Eastern Virginia Medical School. Eighteen male SDrats (300-350 g) individually caged were randomly selected to be fed amoderately high-fat diet (MHF) with 0.8% sodium (32% kcal as fat, ResearchDiets, New Brunswick, NJ), whereas six rats (controls) were fed purified low-fat (LF) diet with 0.8% sodium (10.6% kcal as fat, Research Diets) for 10wk. An identical number of rats was placed on MHF and LF diets, eachcontaining either 2 or 4% sodium (high-salt diets). Food and water wereprovided ad libitum throughout the experiment. Body weights (BW) and lengthswere measured initially and then weekly together with food intake. Rats fed the MHF diet on both low and high salt diverged into distinct groups based onBW gains. Assignment of rats into obesityprone (OP) ( n = 8) andobesity-resistant (OR) ( n = 8) groups was performed as describedpreviously ( 12 )./ i# _5 N. p+ i- t6 G, `3 R$ H9 t
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Systolic BP

The onset and development of hypertension were assessed by using thetail-cuff method with a Narco Biosystems Electro-Sphygnomanometer (Houston,TX). BP was measured under conscious conditions at the beginning of theexperiment and at 1, 5, 8, and 10 wk of diet. The average of five pressurereadings was recorded for each measurement.+ W) r$ u8 U/ C" e  t- N
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Assessment of Oxidative Stress" f4 F$ z3 k" c! o; {* O

Superoxide anion production was measured in isolated aortic rings using amethod previously described( 12, 21 ). Briefly, 5-mm aorticrings were preincubated in Krebs-bicarbonate buffer, at 37°C, for 30 minand then transferred to a cocktail containing 5 µmol/l lucigenin andimmediately measured, every 2 min, for 15 min total, using a scintillationcounter set in the out-of-coincidence mode. The readings were plotted and thearea under the curve was integrated. Results were normalized per milligram ofDNA measured using the Hoechst 33258 dye as described( 27 ). The specificity of thereaction was tested by the ability of 50 U/ml of SOD to quench thechemiluminescence at the end of the measurement.0 G! v9 V4 q& W( i) p! G
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Free 8-isoprostane F 2. Isoprostaneswere measured by EIA using a kit from Cayman Chemicals as previously described( 12 ). Urine collected inmetabolic cages over a 24-h period was supplemented with 0.05% butylatedhydroxytoluene and spiked with 8-[ 3 H]isoprostane. The samples (1ml) were passed on an affinity column (Cayman Chemicals) and only the freeisoprostanes were eluted using 95% methanol. The eluate was evaporated todryness under a stream of N 2 and the pellet was resuspended in a1-ml assay buffer. Each sample was assayed in duplicate at two different dilutions and corrected for individual recovery of8-[ 3 H]isoprostane, and the results were averaged. Nitrate/nitritewas assayed both in plasma and urine (diluted 1:50 in PBS) using a LDH colorimetric method with a kit from Cayman Chemicals.4 T1 e/ o. p: E+ H7 z  {( g3 s

Immunohistochemistry for 4-hydroxy-2-nonenal. Kidneys were fixed in 10% buffered formaline for 3 h and paraffin embedded. The sections wereincubated with a polyclonal antibody recognizing 1:1 Cys, His, Lys-4hydroxy-2-nonenal "Michael" adducts (Calbiochem, dilution 1:750).The slides were then reacted with biotinylated secondary goat anti-rabbitantibody (1:500 dilution; Vector Laboratories, Burlin-game, CA), with theABC-Elite avidin reagent (Vector Laboratories), and finally with the ImmunoPure Metal Enhanced DAB Substrate kit (Pierce, Rockford, IL).% S& W# o. y2 b  Q

Vascular Hypertrophy and Kidney Sclerosis

Aortic wall area. Thin sections of the paraffin-embedded tissue were stained for 1 min with toluidine blue and analyzed as described previously ( 10 ).

Kidney histology. Kidneys were fixed in 10% buffered formalin for4 h and embedded in paraffin. Sections were stained using the periodicacid-Schiff (PAS) reagent and counterstained with hematoxylin. To evaluate thedegree of segmental sclerosis, three independent investigators examined theslides in a blind fashion, mixing the slides after covering the protocolnumbers. In each case, 80-100 glomeruli were examined for each slide andindividually graded on a scale of 0 to 2  according to the degree ofglomerular sclerosis. Grade 0 was a normal looking glomerulus; grade 1 was characterized by mild expansion of mesangial matrix, noocclusion in the glomerular capillaries or adhesion to Bowman's capsule; and grade 2 included expansion of the mesangial matrix, usually focalwith adhesion to Bowman's capsule and some degree of capillary occlusion. Ascore representing the sum of grades was obtained for each rat.: H  C% i4 ~( P% k8 u6 L; x& j4 T
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Adipocyte Morphometry0 J* |- p2 w# Q% ^' g; i

Adipose tissues from the same depot and group were pooled and collagenasewas digested according to Rodbell and Krishna ( 39 ). Adipocytes were washedseveral times to remove collagenase and centrifuged to separate adipocytesfrom preadipocytes, stromal cells, and vascular membranes. Cell diameter of 1,200 cells was measured with the Image 1 Analysis System (Universal Image, West Chester, PA). Mean cell diameter was used to estimate mean cellvolume. Cell size (µg lipid/cell) was calculated by multiplying cell volume(pl) by lipid density ( 0.915 g/ml). Cell lipid content was determinedaccording to the method of Dole( 14 ). Cell lipid content andcell size were used to calculate cell number.# I# V7 L, Q, [# v) y+ g, e

Statistics  W( A2 e' V, l" c

Data are means ± SE. To determine the significance between differentgroups, two-way or three-way ANOVA was performed followed by Tukey's post hoctest. P statistically significant.6 W9 Y* f  u& \8 F4 d% k

RESULTS
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Effect of Salt on BW, Adiposity, Adipocyte Morphometry, andLeptin4 s' Q/ n; C0 p4 z* ?9 G9 ?# q4 ]

After 10 wk of diet, BW in the OP groups on both high- and regular-salt diets was significantly higher than those in the corresponding OR and control(C) groups ( Table 1 ). Inaddition, no significant difference in BWs was detected between each of theOP, OR, and C groups on 0.8, 2, and 4% NaCl, respectively( Table 1 ). The result is inaccordance with daily food intake data, indicating that high-salt diets didnot result in increased food consumption in OP, OR, or C rats on therespective diets vs. their counterparts on the low-salt diet( Fig. 1, A and B ). However, from the beginning of the experiment until week 8, the OP rats ate significantly more than OR rats on a similardiet ( Fig. 1, A and B ). Also, the average food intake in all experimentalgroups reaches a peak after 3 wk on the respective diets, followed by adecrease by week 5 and a subsequent relatively stable level until the end of the experiment ( Fig. 1, A and B ), indicating that the highest salt intakes occurredin the first 3-5 wk on the diet. The increased BWs in the OP groupscompared with OR and C groups were also mirrored by the elevated adiposity. Both the epididymal and retroperitoneal fat depots were significantly increased in the OP groups compared with OR and C, but no significant differences were recorded between the high- and low-salt groups ( Table 1 ). Furthermore, theobesity index was higher in the OP groups compared with OR and C and was notinfluenced by the salt intake ( Table1 ). In contradistinction, the adipocyte morphometry and numberwere different among the OP, OR, and C groups placed on low- vs. high-saltdiets. For all OP, OR, and C, the 2% NaCl diet induced an increase by12-20% in cell volume and 12-15% in cell size with the highest effect on OP adipocytes ( Table2 ). Also, a decrease in adipocyte number was measured for OP( 30%) and OR ( 12%) groups on 2 vs. 0.8% NaCl diet, with nodifference for C adipocytes ( Table2 ). This indicates hypertrophy of adipocytes from the OP rats, significantly exacerbated by the high-salt intake. In accordance with previousfindings ( 29 ), our resultsindicate an increase in circulating leptin for OP rats compared with OR and Cafter 10-14 wk of diet ( Table2 ). Interestingly, both the 2 and 4% NaCl diets significantlyincreased, by 40%, plasma leptin in the OP rats, and only the 4% NaCldiet induced a significant increase in the leptin levels in OR and C rats,compared with their counterparts on 0.8% NaCl( Table 2 ). The latter result suggests that obesity and high salt are both important in regulation of leptinlevels. Moreover, the finding that the 4% NaCl, but not 2% NaCl, dietincreased leptin levels in both OR and C lean groups of rats suggests thateven in the absence of obesity, and independently of the amount of fat in thediet, a high enough content of NaCl (in our particular experiment 4 vs. 2%) could modulate the leptin levels.
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Table 1. BW and adiposity in OP, OR, and C rats on 0.8, 2, and 4% NaCldiets
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Fig. 1. Average daily food intake in obesity-prone (OP), obesity-resistant (OR),and control (C) groups on 2% NaCl ( A ) and 4% NaCl ( B )compared with 0.8% NaCl groups. Food consumption was measured weekly andcorrected for spillage for each individual rat. Average amount per day wasplotted for the week when systolic blood pressure had also been measured.*Significant compared with OR in the same salt group ( P

Table 2. Effect of salt on adipocyte parameters and leptin production in OP, OR,and C rats
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Effect of High-Salt Diet on BP and Plasma Renin Activity in OP, OR,and C Rats

Systolic BP measured in the conscious rats at the beginning of the dietindicated an average of 122 ± 3.9 mmHg. Starting with week 5,the OP rats on both 2 and 4% NaCl displayed a significant increase in BP withan average of 160.2 ± 5.2 and 156.5 ± 4.4 mmHg, respectively, asopposed to all the other groups that were either normotensive or borderline hypertensive ( Fig. 2, A and B ). At week 8, the OP rats on 0.8% NaCl dietwere moderately hypertensive with an average BP of 154 ± 3.2 mmHg,whereas the OP rats on both 2 and 4% NaCl did not show a further significantincrease in their systolic BP compared with week 5 ( Fig. 2, A and B ). By the end of the experiment ( week 10 ), allthree OP groups (on 0.8, 2, and 4% salt) had a similar increase in BP thataveraged 158 mmHg. Also, the OR and C groups on high- and normal-saltdiets were normotensive ( Fig. 2, A and B ). In the OP rats on 0.8% NaCl, the increase insystolic BP was paralleled by an approximately twofold increase in plasmarenin activity (PRA), as measured at the end of the experiment( Fig. 2 C ). The 2% NaCldiet induced a 40% reduction in PRA in the OP rats and slightly decreased PRA in the OR and C rats ( Fig.2 C ). In addition, the 4% NaCl diet induced a significantreduction in PRA in OP, OR, and C groups compared with their respectivecounterparts on 0.8 and 2% NaCl diets ( Fig.2 C ). The ability of the OP rat groups to adequatelyrespond to the different increase in dietary salt at week 10 mayexplain the lack of difference in the systolic BP between the three OP groupsat that time point.: h& @' u) L+ t
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Fig. 2. Systolic blood pressure (BP) in OP, OR, and C on 2% NaCl ( A ) and4% NaCl ( B ) compared with 0.8% NaCl diets. Systolic BP was monitoredfrom the beginning of the study using the tail-cuff method. OP rats on highsalt have significantly increased BP starting with week 5, whereas OPrats on regular salt are hypertensive starting with week 8 on thediet. Plasma renin activity ( C ) was measured in terminal plasmasamples from OP, OR, and C rats on 0.8% NaCl, 2% NaCl, and 4% NaCl, using aRIA method. Data represent means ± SE of 6 rats/group. * Significantcompared with OR and C groups; #significant compared with counterpart on 2%salt; *significant compared with counterpart on 4% salt ( P
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Oxidative Stress in Rats Fed Regular- and High-Salt Diets

The systemic oxidative stress measured as the excreted free 8-isoprostaneF 2 in 24-h urine samples indicated an 2.5-fold increase in the OP groups on both normal (0.8% NaCl)- and high (2 and 4% NaCl)-salt diets, compared with the respective OR and C groups( Fig. 3 A ), indicatingthat high salt does not further increase systemic oxidative stress in theobese rats. However, the ability of thoracic aortic rings to generatesuperoxide anions, measured as lucigenin chemiluminescence, is double in OPrats on both 2 and 4% NaCl vs. OP rats on regular salt, indicating an increasein oxidative stress in the large vessels of obese animals( Fig. 3 B ). Also, asignificant increase induced in response to high salt was measured in C ratsand the same trend was present in the OR rats( Fig. 3 B ). The latterresult indicates that high salt increased superoxide formation independent ofobese state and the amount of dietary fat. In addition, the high-salt intakeand obesity, but not dietary fat, seem to have a synergistic effect onsuperoxide generation. Also, the urinary nitrate/nitrite is four- to fivefolddecreased in OP rats on both regular and salt-supplemented diets, compared with the OR and C counterparts ( Fig.3 C ). The result indicates that salt intake does notfurther decrease nitrite/nitrate excretion, despite its significant effect onsuperoxide generation in the vasculature. Therefore, nitrite/nitrite formationseems to be modulated mainly by the obese state per se and not critically bythe high-fat or high-salt content in the diets. Kidney immunohistochemistry using a polyclonal antibody for 2-hydroxy-4-nonenal protein adducts indicatesa similar staining pattern in all groups on both regularand high-salt diets;however, the intensity of the staining is much higher in the OP, OR, and Crats on 4% vs. regular-salt diets ( Fig. 4, G - L ). The most intense staining is noticedin the distal tubules, thick ascending limb, and to a lesser extent in thecortical proximal tubules, whereas it is virtually absent in the glomeruli. Asshown in Fig. 4, G - L, the staining is more prominent in allOP, OR, and C rats on high salt ( Fig. 4, G - I ) vs. regular salt( Fig. 4, J - L ), suggesting an increased local free radical production in the kidney cortex induced by high-sodium intake. Thecontrol in which the primary antibody was replaced with nonimmune serum showsno staining ( Fig.4 M ).

Fig. 3. Free urinary isoprostanes ( A ), aortic superoxide generation( B ), and nitrate/nitrite excretion ( C ) in OP, OR, and C ratson 0.8% NaCl, 2% NaCl, and 4% NaCl diets. Urinary free isoprostanes weremeasured using an EIA method. In both OP groups on high- and regular-Na diet,the isoprostanes are significantly increased compared with the respective ORand C groups. Superoxide anion generation by aortic rings was measured bylucigenin chemiluminescence as described under METHODS. High-Naintake induces an increase in superoxide production in the OP group but not inthe OR and C. Nitrate/nitrite excretion, measured by a colorimetric LDHmethod, indicates an approximately fourfold reduction in OP rats on bothregular- and high-salt diets, compared with OR and C groups. Salt intake doesnot further reduce nitrate/nitrite excretion. *Significant compared with ORand C; #significant vs. 2% NaCl counterpart; *significant vs. 4% NaClcounterpart ( P

Fig. 4. Periodic acid-Schiff (PAS)-hematoxylin staining of the kidney cortex inhigh-salt OP ( A ), OR ( B ), and C ( C ) rats andnormal-salt OP ( D ), OR ( E ), and C ( F ) rats.Immunohistochemistry using a 2-hydroxy-4 nonenal (HNE) antibody in 4% NaCl OP( G ), OR ( H ), and C ( I ) rats and normal (0.8%)-saltOP ( J ), OR ( K ), and C ( L ) rats; method controlusing nonimmune serum instead of primary antibody ( M ). PAS stainingindicates glomerulosclerosis and increased matrix deposition in the cortex ofOP rats on high-salt ( A ) and to a lesser extent in OP rats onregular-salt diet. Also, HNE staining is more intense in the OP, OR, and Crats on high salt ( G - I ) vs. normal salt( J - L ), with the same distribution pattern. Bar = 10µm.

Effect of Salt on Vascular Hypertrophy, Kidney Sclerosis, andExcretory Function
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Vascular hypertrophy was measured as aortic cross-sectional wall area.Results indicated that in all the OP groups (0.8% NaCl, 2% NaCl, and 4% NaCl),there is a significant increase in wall area compared with the respective ORand C groups ( Fig. 5 A ). However, high salt did not induce a further increasein wall area in OP rats, suggesting no additional effect on vascular hypertrophy ( Fig. 5 A ).To address the possible morphological changes in the kidney, we usedPAS-hematoxylin staining followed by morphometric analysis. In accordance withour previous data ( 11 ), in OPrats on regular-salt diet, a mild sclerosis with most of the lesions in arelatively early stage was noticed, as opposed to OR and C rats that displayeda normal kidney histology ( Fig. 4, D - F ). The OP rats on 4% NaCl displayed numerous and more advanced lesions of the glomeruli as well as significantmatrix deposition throughout the cortex( Fig. 4 A ). Theglomerular lesions displayed capillary loop collapse, mesangial matrixexpansion, and sometimes adhesion to Bowman's capsule. In addition,interstitial fibrosis and glomerular membrane thickening were noticed( Fig. 4 A ). The changesnoticed in the 2% NaCl groups were somewhat intermediate between the 4 and0.8% NaCl counterparts (not shown). In OR and C rats, a normal histologicalappearance was observed regardless of the amount of NaCl in the diets.Morphometric analysis indicated a mean ± SE mesangial score of 16.9± 1.4 for the OP rats on 4% NaCl compared with 14.2 ± 0.8 and 10.9 ± 1.2 for the OP rats on 2% NaCl and 0.8% NaCl, respectively. Thescores for the OR rats on 4, 2, and 0.8% NaCl were 9.4 ± 1.1, 8.7± 1.2, and 8.4 ± 1.3, respectively, and the scores for the Crats on 4, 2, and 0.8% NaCl were 8.8 ± 1.4, 8.2 ± 1.2, and 8.4 ± 1.3, respectively. To test the possible changes in the renalfunction, protein, creatinine, and albumin excretion were measured. OP rats onboth high-salt diets did not display overt proteinuria or significantlyincreased protein excretion compared with OP rats on low salt. Also, thecreatinine values were similar among all groups. However, OP rats on 2% NaCl had mild albuminuria (5.6 ± 0.42 mg/24 h) compared with OP on regularsalt (2.12 ± 0.47 mg/24 h) ( Fig.5 B ). In addition, the OP group on 4% NaCl had asignificantly higher albumin excretion (7.8 ± 0.53 mg/24 h) comparedwith both 2% NaCl and 0.8% NaCl counterparts( Fig. 5 B ). The results indicate that the changes in renal morphology, paralleled by albuminuria, aredependent on the salt content in OP rats only, suggesting a synergistic effectfor salt and obesity but not for the dietary fat.

Fig. 5. Vascular hypertrophy ( A ) and albumin excretion ( B ) in OP,OR, and C rats on normal (0.8%)- and high (2 and 4%)-salt diets. Wall area( A ) was measured on aortic sections stained with toluidine blue, withthe use of a video-based image system with edge-tracking software. Albumin( B ) was measured using an ELISA kit in 24-h urine samples, collectedin metabolic cages at the end of the study. Data represent means ± SEof 6 rats/group. *Significant compared with OR and C; #significant comparedwith 2% NaCl counterpart; *significant compared with 4% NaCl counterpart( P( n! S. g! R$ U6 H9 A
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DISCUSSION4 O3 ]) D6 W& I# l4 i

This rat model of diet-induced obesity was shown to develop moderatehypertension subsequent to the accretion of visceral adiposity, suggesting arole for metabolic factors associated with obesity in the development ofhypertension ( 11, 12 ). We also reportedincreased oxidative stress in the vasculature, plasma, and urine of obeseanimals at both early (3 wk) and 10 wk) stages of the diet( 11, 12 ). Oxidative stress wasdocumented in a variety of animal models, such as the spontaneously hypertensive ( 42, 44 ), Dahl-sensitive( 45, 46 ), or ANG II-infused rat( 28 ). In addition, animportant role for free radicals in BP regulation was shown in a model oflead-induced hypertension ( 9, 50 ), in 1K1C  renalhypertension ( 13 ), in chronicrenal failure ( 51 ), and in amodel of glutathione-depleted rats ( 52 ). Several mechanisms wereproposed for explaining the effect of free radicals production on BPregulation. It was recently demonstrated that endogenously produced superoxideanion can decrease NO bioavailability in the thick ascending limb and therefore increase NaCl reabsorption and induce hypertension ( 37 ). Also, chronicallyincreased oxidative stress induced in the medulla of uninephrectomizedSprague-Dawley rats was shown to lower medullary blood flow leading tohypertension ( 34 ).Hypertension in spontaneously hypertensive rats seems to involve reduced NOavailability in macula densa cells( 54 ). Some forms ofhypertension in humans, including essential hypertension associated withobesity, are influenced by an increase in the amount of dietary salt intake( 17, 53 ). The aim of the present study was to assess the effect of increased salt intake on the development ofhypertension and associated mechanisms involving oxidative stress andend-organ damage in obese rats. The data showed that the increase in dietarysodium up to 2 or 4% induces a more rapid elevation in BP, after only 5 wk ofdiet, instead of 8 wk in OP rats on a regular-salt diet. Although we do not have PRA data at week 5, it is reasonable to assume that the latterresult could be explained by the inability of OP rats to adequately respond toan increase in the dietary salt early (at week 5 ) on the diet.Conversely, the lack of difference in the BP between OP rats on the threedifferent diets at the end of the experiment is reflected in the ability of OPrats to reduce their PRA according to the different levels of salt intake.Also, the similar daily food intake for OP rats on 0.8, 2, and 4% NaCl at anytime point throughout the experiment rules out the possibility of a highersalt intake in the early (up to week 5 ) as opposed to late part ofthe diet, which could have accounted for the earlier increase in BP in the 2and 4% NaCl vs. 0.8% NaCl OP group. Another possible explanation for theearlier increase in BP in OP rats on a high-salt diet may be related to thepressor effect of leptin, as recently reported( 23 ). Our own data or datareported by others ( 29 ) indicate that OP rats are hyperleptinemic by the end of the diet. Data showedthat high-salt diets induced a significant 40% increase in plasma leptin in OPrats. Interaction between high-salt intake and obese state significantlyincreased circulating leptin in the OP groups on high- vs. regular-salt diet.Also, there was a lower, although significant, increase in plasma leptin inlean OR and C groups on 4% NaCl, compared with 0.8% NaCl, suggesting thathigh-salt intake could contribute to elevated plasma leptin independently ofobesity and high dietary fat. A recent report by Correia et al.( 7 ) demonstrated that highamounts of circulating leptin can act centrally to increase BP in rats.Increased leptin may act centrally as a pressor agent in the initial stages ofthe diet, before full onset of obesity, but it is unable to have any effectlater, possibly due to the onset of leptin resistance.1 @6 h5 ?. w/ U* ]/ `& [
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Also, high-salt diet induced a significant increase in the adipocyte size,especially in the OP rats. Adipocyte hypertrophy may potentiate the insulinresistance in OP rats, due to the increased fatty acids efflux and increasedcirculating triglycerides. It was shown that salt increases circulating levelsof fatty acids ( 17 ), and ourdata indicating adipocyte hypertrophy suggest a possible increase incirculating fatty acids.

Oxidative stress was reported previously for this animal model in bothprehypertensive ( 11 ) stage andafter the development of moderate hypertension( 12 ). In the present study, wetested whether salt has an effect on free radicals formation in the obeserats. Data indicated that 2 and 4% NaCl diets did not enhance freeisoprostanes excretion in OP, OR, or C rat groups compared with theircounterparts on 0.8% NaCl diet. However, the superoxide production by aorticrings is significantly increased in OP rats on both high-salt diets vs. 0.8%NaCl group. Urine isoprostanes are considered a reliable marker to quantifysystemic oxidative stress( 30 ). However, recent reports indicate that in rats, under certain conditions such as increased oxygentension ( 26 ) orNADPH-stimulated free radical production ( 15 ),F 2 isoprostanes were not increased, althoughother oxidative stress parameters were elevated. Although this study does not provide data to support this hypothesis, it is possible that the increasedvascular superoxide production mainly originates from a vascular NAD(P)Hoxidase ( 18, 35 ) and hence isoprostanes F 2 could not accurately reflect the increasedaortic oxidative stress. Nevertheless, increased superoxide production in the aorta does not seem to affect vascular hypertrophy. The wall area in OP ratgroups on high- and regular-salt diets is increased vs. the OR and C, but nodifferences were measured among the three OP groups on 4, 2, and 0.8% NaCl.The results suggest that vascular remodeling is due to the elevation in BPrather than directly related to free radicals production in rats on high-saltdiet. The presence of increased hydroxynonenal protein adducts in the kidneysof OP rats on high-salt vs. normal-salt diets indicates elevated free radicalsproduction in the renal tissue in the former. One possible source of freeradicals in the kidney may originate from high-leptin production by the localinfiltrates of adipose tissue. Leptin was shown to induce oxidative stress inthe endothelial cells in culture( 1 ). Therefore, it is possiblethat increased local leptin production may contribute to reactive oxygenspecies generation. High dietary fat does not appear to have a direct effect,because both the OR and C groups displayed similar levels of lipid peroxidation. Conversely, high-salt intake (4% NaCl) induced increased renallipid peroxidation in all study groups (OP, OR, and C), suggesting a role forhigh salt independent from obesity and dietary fat. However, the higher lipidperoxidation in the OP group on 4% NaCl vs. 0.8% NaCl and the higherperoxidation in all OP groups compared with their OR and C counterparts on similar diets indicate a possible synergistic effect of obesity and salt onrenal lipid peroxidation./ d4 {, o) U) o: x/ F

A direct or indirect effect of high-salt intake, possibly via free radicalsproduction, could be responsible for the kidney glomerulosclerosis in the OPrats. Salt was shown to induce smooth muscle cells and myoblasts hypertrophyin vitro ( 20 ). Also, oxidativestress seems to be directly involved in the renal dysfunction in Dahlsalt-sensitive rats ( 48 ).Therefore, it is reasonable to assume that the higher degree of renal damage in the OP rats on high salt vs. normal salt is likely to be independent of apressor effect and rather due to the production of local excess leptin and/orfree radicals. In conclusion, our results indicate that 1 ) high-saltdiet induces an earlier increase in systolic BP in OP rats (5 wk on 4 and 2%NaCl vs. 8 wk on 0.8% NaCl), possibly due to the inability of OP rats toreduce their renin production in response to increased NaCl intake early inthe diet; 2 ) salt does not affect fat accretion, but it inducesadipocyte hypertrophy and increased leptin production, independently fromdietary fat and in synergy with obese state; 3 ) high-NaCl intakeinduces increased vascular and renal oxidative stress, independently fromdietary fat and synergistically to obesity; and 4 ) high-salt dietaccelerates kidney sclerosis, which correlates with renal oxidative stress,but it is, at least in part, independent of a direct pressor effect and does not affect vascular hypertrophy, which is probably the direct result of higharterial pressure. In this model, the concurrent effect of metabolic factorsrelated to obese state and high-salt intake seems to induce kidney sclerosisand moderate hypertension. The finding could be relevant for human pathology,indicating that increased salt intake in obese individuals with moderate hypertension may lead to accelerated end-organ damage.! F) e8 W( J+ x0 s  b% p9 d" z/ m

DISCLOSURES% @; k$ n9 k1 ]8 E
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This study was supported by National Institutes of Health Grant HL-54810, aGrant-in-Aid from the American Heart Association (AHA), and a postdoctoralfellowship from the MidAtlantic Affiliate of the AHA to Dr. A. D. Dobrian.) S& T; t( ~2 ^! P) \+ |8 ~
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