Male gender increases sensitivity to renal injury in responseto cholesterol load - 《American Journal of Physiology》美国生理学杂志干细胞之家



免疫细胞治疗专区	欢迎关注干细胞微信公众号

12 3 4 5 6 7 8 9 10 ... 22 下一页

返回列表

查看: 342511\|回复: 213	go Male gender increases sensitivity to renal injury in responseto cholesterol load [复制链接]

轻羽

新手上路

Rank: 1

积分: 0
威望: 0
包包: 0

楼主

发表于 2009-4-21 13:35 |只看该作者 |倒序浏览 |打印

作者：Diana M.Attia, RoelGoldschmeding, Mahmoud A.Attia, PeterBoer, Hein A.Koomans,  Jaap A.Joles作者单位：1 Departments of Nephrology and Hypertension and Pathology, University Medical Center, 3508 GAUtrecht, The Netherlands; and Department of Pathology, Centre International deToxicologie, 27005 Evreux Cedex, France


      7 k# m/ b0 Y6 F: p* @; L


         ; G  Y; v4 y2 H$ J

         $ x; e7 V% m3 [5 B



      ) Q& s/ l/ Z* K
      【摘要】
   Males are at greater risk for renalinjury than females. This may relate to nitric oxide (NO) availability,because female rats have higher renal endothelial NO synthase (NOS)levels. Previously, our laboratory found susceptibility to proteinuriainduced by NOS inhibition in male compared with female rats.Dyslipidemia and hypercholesterolemia dose dependently decreased renalNOS activity and caused renal injury in female rats. We hypothesized that exposure of male rats to hypercholesterolemia would lead to morerenal injury in male than in female rats due to an a priori lower renalNO system. Female and male rats were fed no, low-dose, or high-dosecholesterol for 24 wk. Cholesterol feeding dose dependently increasedproteinuria in both female and male rats, but male rats developed moreproteinuria at similar plasma cholesterol ( P 0.001).Control males had lower renal NOS activity than control females(4.44 ± 0.18 vs. 7.46 ± 0.37 pmol · min 1 · mgprotein 1; P in males and in females( P significantly more vascular, glomerular, and tubulointerstitial monocyte/macrophage influx and injury than females. Thus under baselineconditions, male rats have lower renal NOS activity than female rats.This may explain why male rats are more sensitive to renal injury byfactors that decrease NO availability, such as hypercholesterolemia. 0 ?% v0 o5 p( h5 ?/ K
      【关键词】 hypercholesterolemia proteinuria renal nitric oxide synthaseactivity9 \1 P! Z! {% t! {* C8 M
   INTRODUCTION5 C0 O' p3 Y$ T! F, B/ V+ p% j/ X, [
7 R' K( R% ^: L# B8 I0 p) z7 T
THE OCCURRENCE OF CARDIOVASCULAR injury is related to gender. Males are at greaterrisk for cardiovascular disease. In females, the rate of cardiovasculardisease increases in early middle age ( 37 ). Whether thisis due to decreased estrogen levels is debated because of variableeffects of estrogen replacement on cardiovascular outcome( 40 ). Similar effects of gender on renal injury have beenrecognized ( 10 ). Aging men have a more rapid rate ofprogression to end-stage renal failure than women ( 21 ).Similarly, aging male rats develop spontaneous proteinuria andglomerulosclerosis, whereas females seem to be resistant to renalinjury ( 6 ). Furthermore, male rats developed more renalinjury in response to mild chronic nitric oxide (NO) synthase (NOS)inhibition ( 42 ). Females might be protected by an enhancedendothelial NO availability. Indeed, whole-body NO synthesis was higherin women compared with men ( 14 ). Furthermore, estrogensupplementation increased circulating levels of nitrate and nitrite inpostmenopausal women ( 33 ). Gender differences in the renalNO system have also been observed. Renal endothelial NOS (eNOS) mRNAand protein levels were higher in female rats compared with male rats( 30 ). However, little is known about the difference inrenal NOS activity between males and females. Thus the question aroseof whether male rats have lower renal NOS activity than female rats.
) s7 O+ M/ ^  Z. i
In a previous study, our laboratory found that cholesterol loadingdecreased renal NOS activity in female rats ( 4 ). Low- andhigh-dose cholesterol loading caused dyslipidemia andhypercholesterolemia, respectively. Dyslipidemia was defined by nosignificant increase in total cholesterol but marked increases in VLDLcholesterol and intermediate density lipoprotein (IDL) cholesterol.Dyslipidemia decreased renal NOS activity in female rats in the absenceof proteinuria, whereas hypercholesterolemia caused proteinuria and renal injury. We hypothesized that exposure of male rats tohypercholesterolemia would lead to more renal injury in male than infemale rats due to an a priori lower renal NO system.0 W6 \' k3 |3 J% o! F5 e' k7 T3 K
1 z7 U% ~3 s% ]
METHODS

Animals. Female and male Sprague-Dawley rats (150-175 g; Harlan-Olac,Blackthorn, United Kingdom) were exposed to a 12:12-h light-dark cycle,ambient temperature of 22°C, and humidity of 60%. Sentinel animals,which were monitored regularly for infection by nematodes andpathogenic bacteria, as well as for antibodies for a large number ofrodent viral pathogens (International Council for Laboratory AnimalScience, Nijmegen, The Netherlands), consistently tested negative forinfection throughout the experiment. The Utrecht University Board forstudies in experimental animals approved the studies.# e& Z% Z9 j) z: n

Experimental protocol. Six groups of rats ( n = 5-8 rats/group) werestudied. Groups 1 and 2 were control females andcontrol males, respectively. Groups 3 and 4 werefemales and males, respectively, on low-dose cholesterol: group3 was fed 0.5% cholesterol 0.125% cholate and group 4 was fed 0.25% cholesterol 0.0625% cholate. Groups 5 and 6 were females and males, respectively, on high-dosecholesterol: group 5 was fed 1% cholesterol 0.25% cholateand group 6 was fed 0.5% cholesterol 0.125% cholate. Thesedifferent regimens were applied because it has been reported that onthe same diet male rats developed higher plasma cholesterol levels thanfemale rats, even though food intake corrected for body weight wasidentical ( 38 ). Our purpose was that male rats wouldachieve comparable cholesterol levels at lower dietary cholesterolconcentrations than females. Cholesterol cholate were mixed throughchow (RMH-TM, Hope Farms, Woerden, The Netherlands). Rats were treatedfor 24 wk, starting at the age of 6 wk.$ l/ ?4 e! P% m
* b. j! ~1 ^4 ?' z( _" I
At the end of the experimental protocol, the kidneys were removed andcut transversely into three slices. The poles were frozen in liquidnitrogen and stored at 80°C until being processed for NOS activityand NOS immunolocalization. The middle slice was immersion-fixed in PBSformaldehyde (4%, pH 7.35) and embedded in paraffin for morphological studies.3 z1 U% s& c+ Z3 u) ]& K' z/ \* ?! m+ ]

Food intake, plasma lipids, renal function, blood pressure, bodyweight, and proteinuria. Food intake was determined every 6 wk. At weeks 0, 6, and 18, blood samples were taken from the tailvein for determination of plasma creatinine, cholesterol, andtriglycerides. At the end of the experiment ( week 24 ), theanimals were anesthetized with 60 mg/kg pentobarbital sodium ip tocollect blood from the vena cava for determination of plasma lipids,lipoproteins, and creatinine. Plasma cholesterol and triglycerides weredetermined enzymatically (Roche Diagnostics, Mannheim, Germany). Plasmacreatinine levels were determined colorimetrically (Sigma, St. Louis,MO). Systolic blood pressure was measured every 6 wk in the consciousrats, starting 1 wk before the start of treatment ( week 0 )by the tail-cuff method (IITC, San Diego, CA). Urine was collectedevery 6 wk starting at week 0 for determination of urinaryprotein and creatinine excretion. The rats were weighed and placed inmetabolic cages for 24 h, with free access to food and water.Urinary protein levels were determined with Coomassie blue.  m" V( ]. z. F
4 J; {4 f. w: L5 l& }! ?; h
Lipoprotein isolation by density-gradient ultracentrifugation. Lipoproteins were separated in terminal plasma samples bydensity-gradient ultracentrifugation ( 41 ) into fivefractions (chylomicrons and VLDLs, D g/ml; LDL, D = 1.019-1.063 g/ml; HDL, D = 1.063-1.21 g/ml). Lipoprotein cholesterol was measured asdescribed above.& q. I1 ^9 \/ P0 h. Z

Urinary thiobarbituric acid reactive substances. Lipid peroxidaton was determined by measurement of thiobarbituric acidreactive substances (TBARS). Urine samples were stored at 80°Cbefore determination of urinary TBARS. Aliquots of 500 µl of urine ormalondialdehyde standards were mixed with 500 µl thiobarbituric acid(1%, pH 1.5) and boiled for 30 min. After the mixture was boiled, itwas left to cool to room temperature. After it cooled, absorbance wasmeasured at 540 nm with a microplate reader ( 11 ). Theresults were expressed as micromoles per day.) @- ]9 |( V6 |7 O9 P
* m6 `" O/ p' I3 n" `6 n7 c
Renal NOS activity. NOS activity was measured by determining the formation of L -[ 3 H]citrulline from L -[ 3 H]arginine. Using an Ultraturrax, analiquot of ~300 mg kidney tissue was homogenized in 1.5 ml ofice-cooled homogenization buffer, pH 7.4, consisting of 50 mmol/l Trisbuffer, 320 mmol/l sucrose, 1 mmol/l EDTA, 1 mmol/l dithiotreitol, 2 mg/l aprotinin, and 100 mg/l phenylmethylsulfonyl fluoride. An aliquotof 50 µl of homogenate was incubated in a final volume of 100 µl at37°C for 30 min in the presence of 1 mmol/l L -citrulline,0.3 mmol/l tetrahydrobiopterin, 300 U/ml calmodulin, 0.5 mmol/l NADPH,1 mmol/l CaCl 2, 0.01 mmol/l L -arginine, and 3.7 kBeq L -[2,3,4,5]-arginine (AmershamPharmacia Biotech, Buckinghamshire, UK) in 50 mmol/l KH 2 PO 4 phosphate buffer, pH 7.2. In anadditional tube, the NADPH was substituted by 100 mmol/l L -NAME to determine nonspecific activity. The reaction wasstopped by the placement of the tubes on ice and addition of 20 mmol/lice-cold HEPES buffer, pH 5.5, followed by separation of arginine andcitrulline on Dowex 50X8-200 (Na form).[ 3 H]citulline was detected by scintillation counting. Allmeasurements were performed in duplicate, and the results are expressedas picomoles per minute per milligram of protein.

NOS immunolocalization. Frozen tissue sections (5 µm) of rat kidneys were fixed in acetoneand rinsed twice with PBS containing 0.1% Triton X-100 (PBST; Tween).Endogenous peroxidase reactions were blocked with 30%H 2 O 2 in a phosphate-citrate buffer, pH 5.8. Tissue sections were incubated for 1 h at room temperature witheNOS or inducible NOS (iNOS) antibody (1:5,000 in 10% PBS;Transduction Laboratories) and then rinsed twice with PBST and fixedwith formalin for 10 min. After a rinsing with PBST, sections wereincubated for 30 min at room temperature with goat anti-rabbitPowerVision (polymerized-horseradish peroxidase-goat-anti-rabbit,Immunologic, Duiven, The Netherlands) and rinsed for 10 min with PBS.For eNOS, detection slides were rinsed for 5 min with acetate buffer(100 mmol/l, pH 4.8) followed by color development with3-amino-9-ethylcarbazole substrate (Sigma). For iNOS, detection slideswere rinsed for 5 min with phosphate-citrate buffer (100 mmol/l, pH5.8) followed by color development with diaminobenzidine. Aftercounterstaining with hematoxylin, sections were covered with paragon.The stained area was quantified morphometrically with Optimas softwarein 20 glomeruli/kidney at ×400 magnification and expressed as thepercentage of the total glomerular area./ c! L( u' K  B/ q
  L$ V3 k' e" A7 q% Z) o' Q% [
Monocyte/macrophage localization. Paraffin sections (3 µm) of formaldehyde-fixed kidney weredeparaffinized and rehydrated. Incubation with the ED-1 mousemonoclonal antibody (kindly provided by Ed Dub, Department of CellBiology, Free University, Amsterdam, The Netherlands) demonstratedmonocytes/macrophages. After application of ED-1 (dilution 1:2,500 inPBS containing 5% BSA and 0.4% sodium azide) to the slides at 22°Cfor 1 h, bound antibody was detected by the DAKO EnVision System (prediluted peroxidase-dextran-conjugated goat anti-mouseantibody and diaminobenzidine color reaction). The number ofED-1-antigen-positive monocytes/macrophages was determined with ×400magnification in all arteries, 50 randomly distributed glomeruli, and20 microscopic tubulointerstitial fields, for determination ofmonocytes/macrophages infiltration. An average score per glomerulus orper field was calculated.

ED-1 and iNOS double staining. Tissue sections prepared as described for NOS staining werepreincubated for 15 min with 10% normal goat serum in PBS and thenincubated with rabbit anti-iNOS antibody (dilution 1:1,000 in 10%normal goat serum, kindly provided by Dr. H. van Goor, Groningen,Netherlands) at 4°C overnight. Next, sections were rinsedwith PBST and incubated for 30 min at room temperature with goatanti-rabbit PowerVision followed by color development in3-amino-9-ethylcarbazole substrate and counterstain with hematoxylin. After blocking of endogenous biotin (biotin blocking kit, Vector Laboratories, Burlingame, CA), the iNOS-stained slides were incubated with biotinylated ED-1 antibody (60 min, room temperature), followed bystreptavidin-FITC (dilution 1:100 in 1% normal rat serum in PBS,Vector Laboratories) for 30 min, and then rinsed in PBST, incubatedwith FITC-anti-streptavidin (dilution 1:100 in 1% normal rat serum inPBS, Vector Laboratories) for 30 min, and rinsed in PBS. Slides werecovered with Pertex.

Morphological studies. Light microscopy was done on 3-µm paraffin sections of theformaldehyde-fixed kidney stained by hematoxylin-eosin. The sections were numbered. The investigators (D. M. Attia and M. A. Attia) were blinded to their identity. Glomerular injury (aneurysms and glomerular fibrosis) was assessed in 50 glomeruli semiquantitatively with a 0-4 scale: 0 = absent, 1 = slight, 2 = mild,3 = moderate, and 4 = marked. Glomerular protein dropletswere assessed by calculating the percentage of affected glomeruli.Tubulointerstitial damage (tubular dilatation, casts, flattened tubularepithelium, and tubular epithelial cell degeneration/necrosis)and cytoplasmic protein droplets in tubular epithelium weresemiquantitatively graded in 20 fields in the same way as glomerularinjury. A total glomerular and tubulointerstitial injury score wasdetermined by summing (1 × score 1 ) (2 × score 2 ) (3 × score 3 ) (4 × score 4 )., |  [: G1 W3 n" h! T

Statistical analyses. Results are expressed as mean ± SE. To assess the influence ofgender on cholesterol feeding, data were tested by two-way ANOVAfollowed by the Student-Newman-Keuls test for multiple comparison. Skewed data sets were either log converted (proteinuria) or ranked (morphological data) before statistical analysis. Analysis of covariance was used to analyze whether differences were present betweenregression coefficients of proteinuria against plasma cholesterol inmale and female rats.
" F% z1 f! r. j+ M8 w6 A
RESULTS

Cholesterol intake. Food intake was higher in male than in female rats (Table 1 ). However, when corrected for thegender-related differences in body weight, food intake was actuallylower in the males (4.3 ± 0.2 vs. 5.4 ± 0.3 g/100 g bodywt). Because of the differences in cholesterol content of theexperimental diets administered to the male and female rats (see METHODS ), 0.5% cholesterol in the chow was the onlyconcentration where cholesterol intake could be directly compared. Atthis concentration, cholesterol intake, corrected for body weight, wasslightly lower in male than in female rats. The data from week18 are presented. They are representative of the whole experiment(Table 1 ).

Table 1. Food and cholesterol intake at week 18 in female and male rats fedincreasing concentrations of cholesterol

Plasma lipids, renal function, blood pressure, and body weight. Both male and female rats fed high-dose cholesterol had similarlyincreased plasma cholesterol. Note that we achieved our goal, namely,that different cholesterol levels in chow resulted in comparable plasmacholesterol levels. Total plasma cholesterol levels were not increasedin rats fed low-dose cholesterol (Fig. 1 ). However, these rats weredyslipidemic. Both VLDL cholesterol and IDL cholesterol levelswere increased. The VLDL levels were increased more in dyslipidemicmale rats than in dyslipidemic female rats. LDL cholesterol levels wereunchanged in dyslipidemic male rats but decreased in dyslipidemicfemale rats. HDL levels remained unchanged. In hypercholesterolemicmale and female rats, the changes in VLDL and IDL cholesterol contentwere even more pronounced. In hypercholesterolemic male rats, LDL andHDL cholesterol were decreased, whereas in hypercholesterolemic femalerats LDL and HDL cholesterol levels remained unchanged (Table 2 ). Cholesterol feeding had no effects onbody weight (Table 1 ), plasma triglycerides, plasma creatinine, andblood pressure (data not shown). Irrespective of diet, plasmacreatinine levels and creatinine clearances were both significantlyhigher in males than in females (group means at 24 wk: 50 ± 2 vs.41 ± 2 µmol/l, and 3.2 ± 0.1 vs. 2.3 ± 0.1 ml/ min,respectively).' G5 i! q* @1 b- r& ~' f& Y
; J- h4 C; `$ |4 S& c
Fig. 1. Plasma cholesterol levels in female ( )and male ( ) control rats, in female ( )and male ( ) dyslipidemic rats, and in female( ) and male ( ) hypercholesterolemicrats. * P P # P & P- J2 G( y: J" e- C& K# D4 E- d
5 g* O# n) {8 E9 @+ g2 a! Q) `
Table 2. Cholesterol concentrations in lipoprotein fractions in female and malerats fed increasing concentrations of cholesterol
; D3 s% R; X4 i8 _3 i; i2 A4 h
Proteinuria. Control male rats spontaneously developed proteinuria in contrast tocontrol females. Dyslipidemic rats showed no increase in proteinuriacompared with controls. Hypercholesterolemic male and female ratsdeveloped significantly more proteinuria than controls, and this wasparticularly pronounced in males (Fig. 2 ). Correcting proteinuria for creatinineclearance did not introduce significant changes in this pattern (datanot shown). The increase in proteinuria was significantly correlatedwith the increase in plasma cholesterol levels both in male and infemale rats. However, at weeks 18 and 24 malerats developed significantly more proteinuria at similar plasmacholesterol levels than did female rats ( P 3 ). The data shown in Fig. 3 are from week 24 of the study. Malecholesterol-fed rats developed more proteinuria at identical plasmacholesterol levels during the entire experiment.  G* U3 V( ^9 H0 V3 j  f

Fig. 2. Proteinuria in female ( ) and male( ) control rats, in female ( ) and male( ) dyslipidemic rats, and in female ( )and male ( ) hypercholesterolemic rats.* P P # P % P% g+ c0 _! k: r

Fig. 3. Linear regression between plasma cholesterol levels andproteinuria in female (; r = 0.8124, P; r = 0.9019, P week 24 of the study.0 `$ v' U: A+ H# h# x

Urinary TBARS. Urinary TBARS were similar in control male and female rats.Dyslipidemic male rats had significantly increased urinary TBARS compared with control male rats and dyslipidemic female rats. UrinaryTBARS were significantly increased in both hypercholesterolemic maleand female rats (Fig. 4 ). Correctingurinary TBARS for creatinine clearance did not introduce significantchanges in this pattern (data not shown).

Fig. 4. Urinary thiobarbituric acid reactive substances (TBARS)in female and male control rats, in dyslipidemic female and male rats,and in hypercholesterolemic female and male rats. * P P # P & P
/ _* ~0 O  i- y0 H" K: R; U3 G: S, `9 d
Renal NOS activity. In control male rats, renal NOS activity was significantly lower thanin control female rats (4.44 ± 0.18 in control male rats vs.7.46 ± 0.37 pmol · min 1 · mgprotein 1 in control female rats; P decreased renal NOSactivity in female rats. In dyslipidemic male rats, renal NOS activitywas markedly decreased and even more so than in female dyslipidemicrats, but renal NOS activity was at control levels inhypercholesterolemic male rats (Fig. 5 ). To explore this remarkable finding, we semiquantitatively analyzed NOSisoform expression by immunohistochemistry (see below).

Fig. 5. Renal nitric oxide synthase (NOS) activity in female andmale control rats, in dyslipidemic female and male rats, and inhypercholesterolemic female and male rats. * P P # P % P & P, H3 L2 ^4 y5 }' V+ g9 p3 E7 O
" o1 _* \: t7 W. ]$ N- Z; w
NOS immunolocalization. Glomerular eNOS immunolocalization was unchanged inhypercholesterolemic males and females compared with control males and females. However, hypercholesterolemic male rats had significantly increased glomerular iNOS-positive glomerular surface area compared with control male rats and hypercholesterolemic female rats (Fig. 6 ). An increase in tubulointerstitialiNOS was also observed in some nephrons in this group (Fig. 6 ), butthis focal effect was not readily quantifiable.1 j; q9 i. H$ P7 R$ L# K9 t- \
8 Z$ a& e8 A- Z$ P% J( ?/ l% D
Fig. 6. Representative light microscopic features ( A, C, D ) and quantitative data ( B and E ) depicting glomerular endothelial NOS (eNOS) and inducibleNOS (iNOS) immunolocalization in control and in hypercholesterolemicfemale and male rats. A : glomerular eNOS immunolocalizationin a hypercholesterolemic female rat. Note the intense red staining inthe vascular endothelium. B : eNOS-positive glomerularcross-sectional surface area (%) in control (black bars) andhypercholesterolemic (gray bars) female and male rats. C :glomerular iNOS immunolocalization in a control male. The iNOS isoformis constitutively expressed in both glomeruli and tubules. D : hypercholesterolemic male rats have significantly moreglomerular iNOS. An increase in tubulointerstitial iNOS was alsoobserved in this group. Note that the iNOS staining is especiallyincreased in a dilated tubule, which probably contains protein casts. E : iNOS-positive glomerular cross-sectional surfacearea (%) in control (black bars) and hypercholesterolemic (gray bars)female and male rats. P % P

ED-1 and iNOS double staining. Double staining for ED-1 and iNOS in hypercholesterolemic male rats(Fig. 7 ) revealed hardly any detectableiNOS expression in infiltrating monocytes/macrophages. The very limitedcolocalization of the two antibodies, in combination with glomerularand tubular iNOS staining, suggests that ED-1-positive cells provide anegligible contribution to the observed increase in renal NOS activityin hypercholesterolemic male rats.6 F5 \7 p' u% ?- l* [
5 k) h; A* u; L3 C* I2 q! |
Fig. 7. Representative microscopic image of double staining foriNOS ( A ) and ED-1 ( B ) showing tubulointerstitialarea of the kidney of a hypercholesterolemic male rat. A :immunoperoxidase staining shows high intensity of tubular iNOS, as alsodepicted in Fig. 6, but practically no staining of the infiltratingmacrophages (arrows). B : immunofluorescence image shows 2 interstitial ED-1-positive macrophages clearly separated fromautofluorescent tubular epithelium.
9 W- F0 ~; _8 C# C
Vascular and glomerular morphology. The number of ED-1-positive cells attached to the endothelium andinfiltrated into the intima and media of arteries was increased only inhypercholesterolemic male rats (Table 3 ).Cytoplasmic protein droplets in glomerular epithelium wereincreased in hypercholesterolemic female rats. In cholesterol-fed malerats, glomerular protein droplets were dose dependently increased.However, hypercholesterolemic male rats had more glomerular proteindroplets than hypercholesterolemic female or dyslipidemic male rats.Dyslipidemic male and female rats had increased glomerularmonocyte/macrophage infiltration. Hypercholesterolemic male rats hadsignificantly more glomerular monocyte/macrophage infiltration thandyslipidemic male rats or hypercholesterolemic female rats. Glomerularinjury dose-dependently increased in cholesterol-fed males but not incholesterol-fed females (Table 3 ).( B  i' K8 _+ J+ t# e- O
, V$ N) E2 k3 j9 o/ u0 o; M" g" v$ w) j
Table 3. Vascular and glomerular morphology in female and male rats fedincreasing concentrations of cholesterol, C3 f. G2 `$ R, R

Tubulointerstitial morphology. Cytoplasmic protein droplets in tubular epithelium were only increasedin hypercholesterolemic males. Dyslipidemic male rats had increasedtubulointerstitial monocyte/macrophage influx, whereas dyslipidemic female rats had no tubulointerstitial monocyte/macrophage influx. Hypercholesterolemic male rats had significantly more tubulointerstitial monocyte/macrophage infiltration thandyslipidemic male rats or hypercholesterolemic female rats.Tubulointerstitial injury dose-dependently increased in cholesterol-fedmales but only in the presence of hypercholesterolemia in females(Table 4 ).

Table 4. Tubulointerstitial morphology in female and male rats fed increasingconcentrations of cholesterol9 B+ x" k2 r  o; L

DISCUSSION

The present study shows that male rats have lower renalNOS activity than female rats. Furthermore, dyslipidemia decreased renal NOS activity in both male and female rats and caused renal injuryin males, whereas females were protected. Hypercholesterolemic malerats developed more extensive renal injury than hypercholesterolemic female rats. Although renal NOS activity was decreased inhypercholesterolemic female rats, it remained unchanged inhypercholesterolemic males possibly because of increased expression ofiNOS in proximal tubules.8 e. j, D& o" g- r: t2 u; K, V
" u5 X- f. L) ?3 {% o; G
It has been shown that renal levels of eNOS mRNA and protein are higherin females than in males ( 30 ). However, the present studyis, to our knowledge, the first study to show that male rats have lowerrenal NOS activity. In a previous study, our laboratory showed thatboth estrogen and androgen contribute to the differences in sensitivityin response to mild chronic NOS inhibition between female and male rats( 42 ). Several studies have suggested that NOS might beregulated by sex hormones. Estradiol increased renal NOS activity( 43 ). Furthermore, uterine NOS activity was increased during pregnancy, when concentrations of estrogen and progesterone arehigh ( 43 ). A study on endothelial cells has shown thatestrogen, by binding to its receptor in the caveolae, promotes theassociation between heat shock protein 90 and eNOS, which increaseseNOS activity ( 36 ). Furthermore, estrogen has been shownto downregulate AT 1 receptor expression ( 29 ).Testosterone, on the other hand, upregulates renal ANG II( 12 ), and it has been shown that inhibition of therenin-angiotensin system increased NOS activity in endothelial cells( 18 ). Thus both estrogen and androgen may contribute to the lower renal NOS activity in male rats. Note that the in vitro NOSactivity assay is conducted in the presence of excess cofactors andsubstrate. This may not be the case in vivo.. U5 i: E, Z- V# h, G9 X6 v$ s
& h5 K% Y" M1 k2 g6 v
Previously, we found that dyslipidemia and hypercholesterolemiadecreased renal NOS activity in female rats ( 4 ). In the present study, we have shown that renal NOS activity was also decreasedin dyslipidemic male rats. We also recently found that hypercholesterolemia decreased renal NOS activity by upregulating renalcaveolin-1 protein abundance via an ANG II-sensitive mechanism ( 3 ). Interestingly, renal NOS activity inhypercholesterolemic male rats was at control levels. In these rats,the iNOS isoform was upregulated, which may be related to the fact thatthey had large amounts of cytoplasmic protein droplets in theglomerular and tubular epithelium. In the setting of severeproteinuria, glomerular and tubular epithelial cells are no longer ableto process filtered protein ( 31 ). The question remainswhether the source of increased iNOS is infiltratingmonocytes/macrophages. Hypercholesterolemic male rats that showed thestrongest increase in arterial, glomerular, and tubulointerstitialmonocyte and macrophage influx also exhibited increased iNOS staining.However, hypercholesterolemic female rats also showed significantlyincreased glomerular and tubulointerstitial monocyte and macrophageinflux but showed no increase in iNOS expression. Expression of iNOSalso occurs in tubular epithelial cells ( 39 ). Using doublestaining, we observed a clear separation of tubular iNOS staining frommacrophage iNOS. Hence, we suggest that iNOS expression may beincreased in tubular epithelial cells by protein reabsorption. Ingeneral, the beneficial effects of NO are attributed to NO synthesizedby eNOS ( 7, 22 ), whereas the excessive amounts of NOproduced by iNOS are thought to generate the damage via peroxynitrite( 19 ). Examples include apoE-iNOS double knockout mice feda Western-type diet, in which the atherosclerotic lesions and theplasma levels of lipoperoxides were lower compared with apoE knockoutmice fed the same diet ( 27 ), and cyclosporine nephropathy,in which glomerular and tubular iNOS expression and activity wereincreased and correlated with the extent of renal injury( 32 ). Therefore, normal total renal NOS activity inhypercholesterolemic males may be due to a reduction of eNOS activitycaused by hypercholesterolemia on the one hand and a secondary increaseof iNOS due to protein resorption on the other hand. In control anddyslipidemic male rats, tubular protein droplets were not significantlyincreased, indicating that the tubular epithelial cells were able toadequately process the filtered proteins in the lysosomes. Previously,we found that renal injury could be prevented by exogenous NOadministration, suggesting that renal injury is NO dependent( 4 ). Thus male rats have lower baseline renal NOSactivity, which is further decreased in the initial phase of dietarycholesterol loading. This may explain why, in general, male rats aremore sensitive to induction of renal injury than female rats.
4 a' _/ j' D0 ^0 M8 E9 t
Gender dependence of renal injury varies according to the model used.Spontaneously hypercholesterolemic male Imai rats were shown to be moresusceptible to developing proteinuria and glomerulosclerosis thanfemale rats ( 35 ). Administration of estrogen to these rats improved renal injury. Thus male gender may interact withhypercholesterolemia to accelerate renal injury. However, in rat modelswhere hypertriglyceridemia is the prominent disorder, such as inanalbuminemic ( 25 ) and obese Zucker ( 16 )rats, female gender and estrogen treatment promoted the development ofglomerulosclerosis, whereas ovariectomy retarded it ( 24 ).

Male cholesterol-fed rats appear to be especially sensitive toglomerular injury, whereas cholesterol-fed female rats developed noglomerular injury. Estradiol may limit the progression of glomerular injury by reducing extracellular matrix production and accumulation ( 28 ). In female rats, estrogen may have played alipid-lowering role. In the present study, female rats had to be fedmore cholesterol than male rats to achieve equal plasma cholesterollevels, despite the fact that when factored for body weight, chowintake was even higher in the females (Table 1 ). This suggests thatfemale rats were protected from dietary cholesterol loading. It hasbeen shown that when fed a similar commercial diet enriched withcholesterol, male and female rats had similar food intake whencorrected for body weight. However, at a comparable chow cholesterolcontent, males developed higher plasma cholesterol levels( 38 ). In our study, these gender-related differences incholesterol metabolism were even more striking because, at similarplasma cholesterol concentrations, cholesterol intake, both in absoluteterms and factored for body weight, was higher in female rats.Estrogens may cause more efficient cholesterol metabolism in females.Indeed, it has been shown that estrogen increases the catabolism andclearance of LDL ( 8 ) and VLDL by increasing activities ofhepatic lipase and lipoprotein lipase ( 9 ). Furthermore, incholesterol-fed ovariectomized rabbits, estrogen replacement attenuatedaortic accumulation of cholesterol ( 20 ). In postmenopausalwomen, the levels of LDL increase and those of HDL decrease. Estrogenreplacement therapy reversed postmenopausal alterations in serumlipoproteins ( 26 ). In contrast to LDL, HDL iscardioprotective ( 34 ) and testosterone administrationdecreases HDL cholesterol ( 15 ). In the present study,hypercholesterolemic female rats had unchanged HDL levels, whereas HDLlevels were decreased in hypercholesterolemic male rats, suggestingthat maintenance of HDL levels in female rats in response to dietarycholesterol renders them less prone to develop renal injury.
3 I1 _/ `5 ~7 M: |9 d0 F( v8 ]9 h
Renal protection provided by estrogen is not only due to itslipid-lowering role. As discussed above, estrogen might be responsible for enhancing renal NOS activity in females, and NO is a potent oxygenradical scavenger. Gender-related differences in lipid peroxidationmight thus contribute to differences in the progression of renal injury( 2 ). Indeed, male dyslipidemic rats had increased lipidperoxidation (as measured by urinary TBARS), whereas urinary TBARSlevels in dyslipidemic females remained unchanged. Thus in the presentstudy, lipid peroxidation was only present in association withinterstitial injury and monocyte/ macrophage infiltration.& `$ h( S' |" P* p: F& b) k3 R

In summary, male rats have lower renal NOS activity than female rats.Furthermore, dietary cholesterol loading decreases renal NOS activityin male and female rats but only causes renal injury in male rats. Thissuggests that a priori lower activity of the renal NO system in malerats, in combination with an increased susceptibility to iNOSinduction, determines their sensitivity to renal injury.* G: C( |; C9 c5 @: x, m

ACKNOWLEDGEMENTS

We acknowledge Dionne van der Giezen, Paula Martens, NelWillekes-Koolschijn, and Hennie IJzerman for technical assistance.( i% x: j  E$ C' l) O
      【参考文献】
1. Albrecht, EW,van Goor H,Tiebosch AT,Moshage H,Tegress AM,andStegeman CA. Nitric oxide production and nitric oxide synthase expression in acute human renal allograft rejection. Transplantation 15:1610-1616,2000.
) m. ]8 A, W; H9 U% b- ]
& d6 t# ?" e/ C: r% T/ q

2. Arnal, JF,Clamens S,Pechet C,Negre-Salvayre A,Allera C,Girolami JP,Salvayre R,andBayard F. Ethinylestradiol does not enhance the expression of nitric oxide synthase in bovine endothelial cells but increases the release of bioactive nitric oxide by inhibiting superoxide anion production. Proc Natl Acad Sci USA 93:4108-4113,1996 .$ ]2 H6 d5 e5 z8 u/ _

3. Attia, DM,Goldschmeding R,Koomans HA,andJoles JA. Hypercholesterolemia decreases renal NO synthesis through an angiotensin II-mediated mechanism (Abstract). J Am Soc Nephrol 12:809A,2001.

: m+ Q$ j) L6 X! m& Z1 D1 ]  F
4. Attia, DM,Ni Z,Boer P,Attia MA,Goldschmeding R,Koomans HA,Vaziri ND,andJoles JA. Proteinuria is preceded by decreased NO synthesis and prevented by a NO donor in cholesterol-fed rats. Kidney Int 61:1776-1787,2002  .

  |) o2 h: i" l# U
5. Ayres, S,Abplanalp W,Liu JH,andSubbiah MTR Mechanisms involved in the protective effect of estradiol-17 on lipid peroxidation and DNA damage. Am J Physiol Endocrinol Metab 274:E1002-E1008,1998 .
3 V3 v" `+ ]$ E( a% A# }6 L
& q, N, p2 }4 E6 W* N  X9 M+ O5 w

6. Baylis, C. Age-dependent glomerular damage in the rat. Dissociation between glomerular injury and both glomerular hypertension and hypertrophy male gender as a primary risk factor. J Clin Invest 94:1823-1829,1994  .
, T& I! L! B* G6 k) d

' K  N, f# O5 z% ]0 C7 {+ i, _% A
7. Chen, J,Kuhlencordt PJ,Satern J,Gyurko R,andHuang PL. Hypertension does not account for the accelerated atherosclerosis and development of aneurysms in male apolipoprotein E/endothelial nitric oxide synthase double knockout mice. Circulation 104:2391-2394,2001 .

% z, l8 q) x# X9 m# J, i4 c
8. Colvin, PL,Auerbach BJ,Case LD,Hazzard WR,andApplebaum-Bowden D. A dose-response relationship between sex hormone-induced change in hepatic triglyceride lipase and high-density lipoprotein cholesterol in postmenopausal women. Metabolism 40:1052-1056,1991  .

+ d* r& e/ Q! a# _3 p4 R

9. Demacker, PN,Staels B,Stalenhoef AF,andAuwerx J. Increased removal of beta-very low density lipoproteins after ethinyl estradiol is associated with increased mRNA levels for hepatic lipase, lipoprotein lipase and low density lipoprotein receptor in Watanabe hyperlipidemic rabbits. Arterioscler Thromb Vasc Biol 11:1652-1659,1991 .$ \$ n5 j& \) u1 E/ H- x* o

& M9 \. J( J$ V  W4 Y- \
( ^9 x6 F3 X0 g6 T4 x1 k
10. Dubey, RK,andJackson EK. Estrogen-induced cardiorenal protection: potential cellular, biochemical, and molecular mechanisms. Am J Physiol Renal Physiol 280:F365-F388,2001 .5 y& {/ j" A" f7 A) S: H. z3 n$ g

4 D2 {: h- `% Y

11. Dyer, RG,Stewart MW,Mitcheson J,George K,Alberti MM,andLaker MF. 7-ketosterol, a specific indicator of lipoprotein oxidation, and malondialdehyde in non-insulin dependent diabetes and peripheral vascular disease. Clin Chim Acta 260:1-13,1977.
  o+ E7 w9 O6 w2 A! E
) b! F8 k3 `' ]; \3 i
/ o9 L5 m+ y2 v
12. Ellison, KE,Ingelfinger JR,Pivor M,andDzau VJ. Androgen regulation of rat renal angiotensinogen messenger RNA expression. J Clin Invest 83:1941-1945,1989  .
0 @; C4 G. ?+ z, f1 z
5 N" J7 G9 C. Y/ m/ q

13. Floege, J,Hackmann B,Kliem V,Kriz W,Alpers CE,Johnson RJ,Kühn KW,Koch KM,andBrunkhorst R. Age-related glomerulosclerosis and interstitial fibrosis in Milan normotensive rats: a podocyte disease. Kidney Int 51:230-243,1997  .3 R0 q  {6 V- X) H" K6 i, F

9 e7 V3 [1 [0 ]) f' A6 H
) m+ `  X8 I; s3 I
14. Forte, P,Kneale BJ,Milne E,Chowienczyk PJ,Johnston A,Benjamin N,andRitter JM. Evidence for a difference in nitric oxide biosynthesis between healthy women and men. Hypertension 32:730-734,1998 .8 q7 r7 j3 o9 P2 k9 ?8 ~$ T

) S" W. ^0 Z8 q3 R! w/ P8 v

15. Frisch, F,andSumida KD. Strength training does not alter the effects of testosterone propionate injections on high-density lipoprotein cholesterol concentrations. Metabolism 48:1493-1497,1999  .! W8 H7 U9 {3 p6 v
/ Z$ _2 U0 R* b

16. Gaddes, MD,Stern JS,van Goor H,Nguyen D,Johnson PR,andKaysen GA. Estrogen accelerates the development of renal disease in female obese Zucker rats. Kidney Int 53:130-135,1998  .
5 e9 ?2 o/ w+ J8 L2 ^

' |+ L8 B3 e& Q$ U% |
17. Gafter, U,Ben-Assat M,andLevi J. Castration inhibits glomerular hypertrophy and proteinuria in uninephrectomized male rats. Eur J Clin Invest 20:360-365,1990  .

18. González Bosc, LV,Kurnjek ML,Müller A,Terragno NA,andBasso N. Effect of chronic angiotensin II inhibition on the nitric oxide synthase in the normal rat during aging. J Hypertens 19:1403-1409,2001  .' ]% g$ @0 K0 p

; V  }4 n. X$ S. _) R
19. Groves, JT. Peroxynitrite: reactive, invasive and enigmatic. Curr Opin Chem Biol 3:226-235,1999  .5 D& \- `8 a( ^( G# |& H! j
( q8 P: I- u7 ]4 B, u5 x: ?! o

20. Haarbo, J,Svendsen OL,andChristiansen C. Progesterons do not affect aortic accumulation of cholesterol in ovariectomized cholesterol-fed rabbits. Circ Res 70:1198-1202,1992 ., |# O7 l5 m! D! k
3 X- e" N6 w# |$ V: F0 M+ c
9 o$ X  e6 H2 c+ u, t* ~
! J* ~7 W# b) T
21. Hannedouche, T,Chauveau P,Kalou F,Albouze G,andLacour B. Factors affecting progression in advanced chronic renal failure. Clin Nephrol 39:312-320,1993  .! |3 x" ^5 m. W2 I' [; _: K- V
" B+ ]+ {  |& R
: W+ o% |; \0 K* L, O' h
( T  P, z1 r/ B. H# h* k
22. Heeringa, P,van Goor H,Itoh-Lindstrom Y,Maeda N,Falk RJ,Assmann KJM,Kallenberg CGM,andJennette JC. Lack of endothelial nitric oxide synthase aggravates murine accelerated anti-glomerular basement membrane glomerulonephritis. Am J Pathol 156:879-888,2000 .5 O7 Y7 k$ ?# e' g

6 Z- ?5 s9 f$ K
/ l' m: z3 T# L3 g  E
23. Ishiyama, A,Atarashi K,Minami M,Takagi M,Kimura K,Goto A,andOmata M. Role of free radicals in the pathogenesis of lipid-induced glomerulosclerosis in rats. Kidney Int 55:1348-1358,1999  .
% n, s0 G/ V, f' D% s) g% x

24. Joles, JA,van Goor H,andKoomans HA. Estrogen induces glomerulosclerosis in analbuminemic rats. Kidney Int 53:862-868,1998  .( @* R. }' V+ q" S8 p

' ~9 u7 l# _9 B  b# @% Y
25. Joles, JA,van Goor H,van der Horst MLC,van Tol A,Elema JD,andKoomans HA. High lipid levels in very low density lipoprotein and intermediate density lipoprotein may cause proteinuria and glomerulosclerosis in aging female analbuminemic rats. Lab Invest 73:912-921,1995  .5 D) O3 N" T/ D( \

/ c0 P2 w5 w# J$ @* h* V& J) \6 x
26. Knopp, RH. Cardiovascular effects of endogenous and exogenous sex hormones over a woman's lifetime. Am J Obstet Gynecol 158:1630-1643,1988  .2 `4 T( A' n$ L% w, R+ n# U9 p
8 I- U, \4 ~: u, A3 A( Y

5 P; h# d# ]( d6 b' f% i
27. Kuhlencordt, PJ,Chen J,Han F,Astern J,andHuang PL. Genetic deficiency of inducible nitric oxide synthase reduces atherosclerosis and lowers plasma lipid peroxides in apolipoprotein E-knockout mice. Circulation 103:3099-3104,2001 ./ f* n: {8 z* e9 i; S# W6 n  L& M

3 i, u0 G5 A+ z3 U+ k7 k1 C4 l

28. Kwan, G,Neugarten J,Sherman M,Ding Q,Fotadar U,Lei J,andSilbiger S. Effects of sex hormones on mesangial cell proliferation and collagen synthesis. Kidney Int 50:1173-1179,1996  .5 s2 j( `7 ]. Y& _* {+ c# t
- [6 n7 e/ W% B. s# N/ u6 n
  M' j6 P' W( c) u8 i
) Z& l' Z% u' T% e# l3 h
29. Nickenig, G,Baümer AT,Grohè C,Kahlert S,Strehlow K,Rosenkranz S,Stäblein A,Beckers F,Smits JFM,Daemen MJAP,Vetter H,andBöhm M. Estrogen modulates AT 1 receptor gene expression in vitro and in vivo. Circulation 97:2197-2201,1998 .2 z' u% b4 a0 Z8 }! f& s" C

; X" B: |7 N2 i$ q4 c9 ]

30. Reckelhoff, JF,Hennington BS,Moore AG,Blanchard EJ,andCameron J. Gender differences in the renal nitric oxide (NO) system. Dissociation between expression of endothelial NO synthase and renal hemodynamic response to NO synthase inhibition. Am J Hypertens 11:97-104,1998  .8 N# k, g* q" h6 v2 m9 @! R
7 n# \* Q: X# B2 S+ j; f' z

31. Remuzzi, G,andBertani T. Pathophysiology of progressive nephropathies. N Engl J Med 339:1448-1456,1998 .
& M* j+ w, o+ ?, L
  s3 g6 C" M4 o/ \% B  v: D: Q

32. Rezzani, R,Rodella L,Corsetti G,andBianchi R. Does methylene blue protect the kidney tissues from damage induced by cyclosporine A treatment? Nephron 89:329-336,2001  .* R! A& R' D4 q8 @0 c

# C0 f+ Y( M( G/ o
5 Q7 A8 N, x! v8 g1 f9 ?
33. Rosselli, M,Imthurn B,Keller PJ,Jackson EK,andDubey RK. Circulating nitric oxide (nitrite/nitrate) levels in postmenopausal women substituted with 17 -estradiol and norethisterone acetate: a 2-year follow-up study. Hypertension 25:848-853,1995 .

7 u8 S- y4 u% |0 r# m0 j
/ G* k$ A/ g6 E. ~: X! ?
34. Rubins, HB,Robins SJ,Collins D,Fye CL,Anderson JW,Elam MB,Faas FH,Linares E,Schaefer EJ,Schectman G,Wilt TJ,andWittes J. Gemfibrozil for the secondary prevention of coronary heart disease in men with low levels of high-density lipoprotein cholesterol. N Engl J Med 341:410-448,1999 .

4 F" O$ H) O3 @3 a3 x- C. k% p/ w
# E6 p( S) `) @. m; i
35. Sakemi, T,Toyoshima H,Shouno Y,andMorito F. Estrogen attenuates progressive glomerular injury in hypercholesterolemic male Imai rats. Nephron 69:159-165,1995  .
8 H" O0 j5 {$ e/ r( y, g% B7 N

0 d/ I: B5 d5 l, t" m5 O7 ^" J* I
36. Strong, KS,Haynes MP,Caulin-Glaser T,Rosneck J,Sessa WC,andBender JR. Estrogen stimulates heat shock protein 90 binding to endothelial nitric oxide synthase in human vascular endothelial cells. Effects on calcium sensitivity and NO release. J Biol Chem 275:5026-5030,2000 .

6 X5 F% N2 X4 A6 ^+ z5 Q! e
37. Sullivan, JM,andFowlkes LP. The clinical aspects of estrogen and the cardiovascular system. Obstet Gynecol 87:S36-S43,1996 .
/ i  v: r0 X. j0 d: A, p. q: ]9 S
' Q( t) O* F2 H. K

38. Terpstra, AHM,Van Tintelen G,andWest CE. The effect of semipurified diets containing different proportions of either casein or soybean protein on the concentration of cholesterol in whole serum, serum lipoproteins and liver in male and female rats. Atherosclerosis 42:85-95,1982  .

/ o- U# q" S: z

39. Tojo, A,Welch WJ,Bremer V,Kimoto M,Kimura K,Omata M,Ogawa T,Vallance P,andWilcox CS. Colocalization of demethylating enzymes and NOS and functional effects of methylarginines in rat kidney. Kidney Int 52:1593-1601,1997  .
2 h& C' i) |, M- a5 z

: a- r4 [/ E+ c6 o
40. Tolbert, T,andOparil S. Cardiovascular effects of estrogen. Am J Hypertens 14:186S-193S,2001  ./ ~% \9 ]* y$ l

41. Van Tol, A,Jansen EHJM,Koomans HA,andJoles JA. Hyperlipoproteinemia in Nagase analbuminemic rats. Effects of pravastatin on plasma (apo)lipoproteins and lecithin: cholesterol acyltransferase activity. J Lipid Res 32:1719-1728,1991 .9 {8 @7 S3 E: {
1 Y* T. S9 e5 L6 c% M

! O( ]9 y- f" q7 m
42. Verhagen, AMG,Attia DM,Koomans HA,andJoles JA. Male gender increases sensitivity to proteinuria induced by mild NOS inhibition in rats: role of sex hormones. Am J Physiol Renal Physiol 279:F664-F671,2000 .

43. Weiner, CP,Lizasoain I,Baylis SA,Knowles RG,Charles IG,andMoncado S. Induction of calcium-dependent nitric oxide synthases by sex hormones. Proc Natl Acad Sci USA 91:5212-5216,1994 .

( s5 o, l. d& \: V! [

44. Zatz, R,andFujihara CK. Glomerular hypertrophy and progressive glomerulopathy. Is there a definite pathogenic correlation? Kidney Int 45:S27-S29,1994.% Q3 K( F3 g4 k! Q6 q
# X& J: o2 z  Z9 [+ {: L
& v1 Y6 n% K: ]6 S. p/ s- ]. x" o: r, e* S

45. Zeier, M,Schönherr R,Amann K,andRitz E. Effects of testosterone on glomerular growth after uninephrectomy. Nephrol Dial Transplant 13:2234-2240,1998 .

收藏0 分享0 顶0 踩0