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作者：KazuakiMoridaira, JeremiahMorrissey, MelanieFitzgerald, GuangjieGuo, RuthMcCracken, TimothyTolley,  SauloKlahr作者单位：Departments of Internal Medicine and Cell Biologyand Physiology, Washington University School of Medicine atBarnes-Jewish Hospital, St. Louis, Missouri 63110-1092
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      【摘要】0 V7 y$ \- k( W1 L' A* w8 e
   Unilateral ureteral obstruction(UUO) is a well-established model for the study of interstitialfibrosis in the kidney. It has been shown that the renin-angiotensinsystem plays a central role in the progression of interstitialfibrosis. Recent studies indicate that endothelin, a powerfulvasoconstrictive peptide, may play an important role in some types ofrenal disease. To investigate the effects of angiotensin II onendothelin and its receptors in the kidney, mice were subjected to UUOand treated with or without enalapril, an orally activeangiotensin-converting enzyme inhibitor, in their drinking water (100 mg/l). The animals were killed 5 days later. Using RT coupled with PCR,we measured the levels of endothelin-1, endothelin A, and endothelin B(ET B ) along with transforming growth factor-, TNF-,and collagen type IV mRNA expression in the kidney with UUO and thecontralateral kidney along with interstitial expansion in the kidneycortex by a standard point counting method. We found that enalapriladministration ameliorated the increased expression of ET-1 mRNA in theobstructed kidney by 44% ( P expression was significantly increased inthe obstructed kidney, it was not affected by enalapril. We found thatenalapril treatment increased ET B mRNA expression by 115%( P (measured by Westernblot) in the kidney with an obstructed ureter. Enalapril treatmentalone inhibited the expansion of interstitial volume due to UUO by52%. Cotreatment with enalapril and the ET B receptorantagonist BQ-788 inhibited the expression of interstitial volume byonly 19%. This study confirms that enalapril inhibits the interstitialfibrosis in UUO kidneys. It also suggests a beneficial and unforeseeneffect of enalapril on the obstructed kidney by potentially stimulatingthe production of nitric oxide through an increased expression of theET B receptor. # l9 b+ x2 M( L
      【关键词】 nitric oxide formation fibrosis enalapril angiotensinconvertingenzyme endothelin B receptor
   INTRODUCTION) B0 ?9 l4 T( i; L' ?' |

TUBULOINTERSTITIAL FIBROSIS develops in a variety of kidney diseases ( 29, 30, 33 ). Available data indicate that angiotensin II plays a centralrole in the initiation and progression of renal disease by autocrine,paracrine, intercrine, and endocrine pathways. Experimental ureteralobstruction is a well-established model for the study of interstitialfibrosis ( 20, 25, 26 ). Intrarenal concentrations ofangiotensin II increase rapidly after the onset of ureteral obstructionin the ligated obstructed kidney ( 10 ). Angiotensin II, inturn, upregulates the expression of transforming growth factor- (TGF- ), TNF-, and other growth factors and cytokines that lead tothe accumulation of ECM proteins and to renal damage ( 20-23, 28 ). We previously reported the beneficial effects of angiotensin-converting enzyme (ACE) inhibitor on the progression oftubulointerstitial fibrosis in the UUO model ( 19-21, 23, 28 ). ACE inhibitors were found to blunt TGF- and TNF- expression concomitant with amelioration of histological changes thatoccur in the kidney during disease. A more precise identification of these other factors that contribute to the initiation of and/or progression of kidney fibrosis is the subject of the study.
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In the last decade, a number of studies have suggested that endothelin,a powerful vasoconstricting peptide ( 48 ), is also involvedin the progression of chronic renal disease ( 2, 15, 31 ).Endothelin has at least three isopeptides: endothelin-1 (ET-1),endothelin-2 (ET-2), and endothelin-3 (ET-3) ( 18 ). Their effects are mediated through two different receptors: endothelin A(ET A ), selective for ET-1 and ET-2, and endothelin B(ET B ), nonselective to the three ET isopeptides ( 16, 46, 47 ). Among the three ETs, ET-1 appears to be the mostimportant in pathophysiological conditions in the kidney. Indeed, ithas been shown that renal tissue can synthesize and release ET-1 andalso expresses both ET A and ET B receptors( 3 ).
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It has been shown in the last few years that angiotensin II canstimulate the synthesis and release of ET-1 in endothelial cells orvascular smooth muscle cells ( 7, 9, 12, 34, 44 ). However,the role and interactions between angiotensin II and ET-1 in theprogression of renal fibrosis due to ureteral obstruction are stillunclear. Therefore, we used the ACE inhibitor enalapril to explore theeffects of angiotensin II on the mRNA expression of ET-1,ET A, and ET B in mice with UUO. RT-PCRwas utilized for the semiquantitative analysis of mRNA. To confirm theeffects of enalapril, we also measured other cytokines, such asTGF-, TNF-, and collagen type IV mRNA, as a marker of ECM proteins and evaluated histological changes by using a standard pointcounting method. This would validate any observed change(s) inthe pattern of endothelin gene expression. This would help to integratethe contribution of the endothelin in the progression of renal diseasethat probably was initiated by the increase in angiotensin II.$ l& O( h+ H9 `2 }4 w  r% [7 C
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MATERIALS AND METHODS

Animals and reagents. Female C57BL/6 mice (~25-30 g) were purchased from Harlan(Indianapolis, IN). Enalapril, BQ-788, and Tri Reagent, a reagent forRNA isolation, were supplied by Sigma (St. Louis, MO). Avian myeloblastosis virus reverse transcription kits and Taq polymerase were obtained from Promega (Madison, WI).

Experimental design. Initially, mice were divided into two groups: those receiving enalaprilin the drinking water (100 mg/l; n = 9) and those receiving water alone as a control ( n = 9). Each groupwas given water with or without enalapril from 1 day before obstruction of the ureter through 5 days of obstruction. Unilateral ureteral obstruction (UUO) was performed as described previously( 19-23 ). In brief, a midline abdominal incision wasmade while the mice were under ketamine HCl and xylazine HCl (20:3;87.0:13.4 mg/kg ip) anesthesia, and both ureters were exposed. The leftureter was ligated with 4-0 silk at one-third the distance fromthe bladder to the kidney. Animals were allowed to drink or eat normalrodent chow ad libitum after surgery. Subsequently, additional groups of mice were prepared to receive enalapril in the drinking water withor without daily injections (ip) of the ET B receptorantagonist BQ-788 at a dose of 1 mg/kg ( 48 ).. R% _! Q3 u( ~, `6 a9 S1 B1 d
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After 5 days of UUO, the mice were anesthetized with an overdose ofketamine HCl-xylazine HCl, and the kidneys were immediately harvested,decapsulated, and washed in ice-cold PBS. The kidneys were prepared fortotal RNA isolation and histological examination as describedpreviously ( 19-23 ).# R. N3 R& V3 n" {) W
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Preparation of RNA. Total RNA was isolated from each kidney by the guanidinium-thiocyanatemethod ( 5 ). In brief, portions of the kidneys were homogenized in 1 ml of Tri Reagent. Total RNA was precipitated withisopropanol. The RNA pellets were washed in 75% ethanol, air dried,and dissolved in RNase-free distilled water. The quantitative analysisof total RNA was performed at 260 and 280 nm.
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RT-PCR. Total RNA extracted from the kidneys was reverse transcribed intofirst-strand cDNA in RT buffer (10 mM Tris · HCl, 50 mM KCl, 5 mM MgCl 2, 1 mM deoxynucleotide triphosphate mixture, and 1 U/µl RNase inhibitor), 0.5 U/µl avian myeloblastosis virus RT, andoligo(dT) primers. The incubation conditions (42°C for 1 h followed by 95°C for 5 min) were established by using a DNA Thermal Cycler (Perkin-Elmer).5 a" Q# _) T( o: e0 }
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PCR amplification was performed for ET-1, ET A,ET B, TGF-, TNF-, collagen type IV, and GAPDH with theprimers shown in Table 1. Successively,each of the cDNAs were amplified in PCR amplification buffer (10 mMTris · HCl, 50 mM KCl, 1.5 mM MgCl 2, and 200 µM deoxynucleotide triphosphate mixture) containing 1.25 U/µl Promega Ampli Taq DNA polymerase and 25 pM primers for eachamplification under the following conditions: denaturing at 94°C for1 min, annealing at different temperatures (Table 1 ) for 1 min, and extension at 72°C for 1 min up to their optimal cycles (Table 1 ).Preliminary studies were performed to determine the appropriate numberof cycles needed for linear amplification of the target DNA. Inaddition, each pair of primers was confirmed to amplify the objectivecDNA by digestion of the product with at least two restriction enzymesand dideoxy chain-termination DNA sequencing.1 h4 h$ B( G9 j% x+ x

Table 1. Primers for polymerase chain reaction
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Relative quantitative analysis of mRNA. Products amplified by PCR were separated by 2.0% agarose gelcontaining ethidium bromide. The gels were visualized with UV light andwere photographed with Polaroid Type 665 positive-negative films. Theintensity of bands was measured by densitometry for quantification. Therelative level of each mRNA was determined by normalizing the quantityof specific cDNA to the amount of GAPDH cDNA./ E) ?. f) K* g9 U2 x. j
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Relative quantitative analysis of ET B protein. Portions of kidney cortex were solubilized with Laemmli sample bufferby heating to 95°C for 5 min followed by brief centrifugation aftercooling. The protein content of the supernatant was determined byBradford assay and diluted to 2 mg/ml of sample. Twenty-five microgramsof total protein were separated by means of 10% acrylamide gelscontaining sodium dodecyl sulfate. Gel lanes containing kidney extractswere flanked by lanes containing prestained molecular massmarkers. Proteins in the acrylamide gel wereelectrophoretically transferred to nitrocellulose membranes inTris-glycine buffer containing 20% methanol with the prestainedmarkers used as a guide for transfer efficiency. The location andrelative amount of ET B antibody (1:100) were prepared inrabbit catalog no. AER-002 (Alomone Labs, Jerusalem, Israel) followedby a goat anti-rabbit IgG-horseradish peroxidase conjugate (Santa CruzBiotechnology, Santa Cruz, CA) and Luminol reagent SC-2048. Similarresults were obtained with an anti-rat ET B antibody 1:100made in sheep (no. 324755, Calbiochem-Nova Biochem, San Diego, CA) anda donkey anti-sheep IgG-horseradish peroxidase conjugate (A3415, Sigma).0 {( c5 \5 p$ r3 U
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Immunohistochemical analysis. Coronal sections of kidney were placed in the fixative Histochoice(Amresco, Solon, OH). The sections were dehydrated, embedded inparaffin, cut into 4-µm sections, dewaxed, and rehydrated as described previously ( 41 ). The rehydrated sections weretreated overnight at 4°C with 1:100 dilations of rabbit anti-ratET B antibody (Alomone) followed by an alkaline phosphataseconjugated goat anti-rabbit IgG (A9919, Sigma) and evaluated with SigmaFAST-Fast Red/Naphthol (F4648).
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Morphometric analysis. A standard point counting method was used to quantitate the volume ofthe renal interstitium ( 39 ). The relative volume of therenal cortical interstitium (V v int ) was determined onsections by using Mason-Trichrome stain. Ten separate nonoverlappingmicroscopic fields of each kidney section were averaged to yield thescore of each kidney. Each microscopic field contained one glomerular cross section to maintain consistency. The score for 9-11 separate animals for each treatment modality was then averaged.% @& n- W. X3 d. ^& {7 _
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Statistical analysis. All data represent means ± SD. Intergroup comparisons wereperformed by ANOVA and Student's t -test. P the criterion of significance.

RESULTS
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RT-PCR. We measured the expression of TGF-, TNF-, and collagen IV mRNA inmice with UUO with or without enalapril in the drinking water. Thisserved as a measure of the effectiveness of the enalapril treatment togauge any subsequent effects on endothelin gene family members.

Figure 1 shows the mRNA expression ofTGF- in UUO mice treated with or without enalapril. TGF- mRNA was significantly increased in the obstructed kidney compared withthe contralateral kidney ( P the expression of TGF- inthe obstructed kidney by 74% ( P whereas ithad no effect on the contralateral kidney.: p% S( U0 q$ n4 x

Fig. 1. Relative levels of transforming growth factor- (TGF- ) mRNA expression in the contralateral and obstructed kidneyswith or without enalapril treatment at 5 days of unilateral ureteralobstruction (UUO). The ratio of vertical axis refers to TGF- /GAPDH.CK, contralateral kidney; OBK, obstructed kidney. Values are means ± SD; n = 5. * P P8 |; }9 ^8 Z5 I, D

There was also a significant increase of TNF- mRNA in the obstructedkidney ( P in untreated animals. Enalapril blunted the increased expression of TNF- mRNA in the obstructed kidney (28%), which wasstatistically significant ( P of TNF- mRNA in the contralateral kidney(Fig. 2 ).

Fig. 2. Relative levels of TNF- mRNA expression in thecontralateral and obstructed kidneys with or without enalapriltreatment at 5 days of UUO. The ratio of vertical axis refers toTNF- /GAPDH. Values are means ± SD; n = 5. * P P

The expression of collagen IV mRNA in the obstructed kidney wasthreefold greater than in the contralateral kidney ( P This increment in the obstructed kidney was reduced (39%) byenalapril treatment. This was statistically significant( P had no effect on the collagentype IV mRNA content of the contralateral kidney (Fig. 3 ).
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Fig. 3. Relative levels of collagen type IV (Col IV) mRNAexpression in the contralateral and obstructed kidneys with or withoutenalapril treatment at 5 days of UUO. The ratio of vertical axis refersto collagen type IV/GAPDH. Values are means ± SD; n = 5. * P P

ET-1 mRNA expression was fivefold greater in the obstructed kidney thanin the contralateral kidney ( P mice. Enalapril decreased the expression of ET-1 mRNA in the obstructed kidney by 44% ( P the expression of ET-mRNA in the contralateralkidneys (Fig. 4 ).
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Fig. 4. Relative levels of endothelin-1 (ET-1) mRNA expression inthe contralateral and obstructed kidneys with or without enalapriltreatment at 5 days of UUO. The ratio of vertical axis refers toET-1/GAPDH. Values are means ± SD; n = 9. * P P: u. ]$ [: Z5 Y' @0 d
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ET A mRNA in the obstructed kidney was 70-85% higherthan that of the contralateral kidney ( P significant effect on the expression ofET A mRNA in the obstructed or the contralateral kidney(Fig. 5 ).4 k# P( _1 |+ {$ U8 g% B
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Fig. 5. Relative levels of endothelin A (ET A ) mRNAexpression in the contralateral and obstructed kidneys with or withoutenalapril treatment at 5 days of UUO. The ratio of vertical axis refersto ET A /GAPDH. Values are means ± SD; n = 9. * P
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No significant changes were observed in the expression ofET B mRNA between the obstructed and contralateral kidneysof animals receiving water. Enalapril treatment increased theexpression of ET B mRNA in the obstructed kidney by 115%( P treatment had no effect onET B mRNA in the contralateral kidney (Fig. 6 ), although there was a 49% increasedue to the presence of ACE inhibition. Another group of animals( n = 3 each) was subjected to a sham operation in whichthe ureter was manipulated but not ligated or to UUO for 5 days. TheET B mRNA content was not significantly different (0.20 ± 0.13, 0.24 ± 0.06, and 0.26 ± 0.10) for the kidneys ofsham-operated mice, the contralateral kidneys, or the kidneys with anobstructed ureter, respectively. At the same time, the amount of ET-1mRNA was significantly increased greater than threefold in the kidneywith an obstructed ureter but was not different in the contralateralkidneys from that of the kidneys from sham-operated mice.# ~1 y8 }! N' b# T% [, H: G; f8 z
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Fig. 6. Relative levels of endothelin B (ET B ) mRNAexpression in the contralateral and obstructed kidneys with or withoutenalapril treatment at 5 days of UUO. The ratio of vertical axis refersto ET B /GAPDH. Values are means ± SD; n = 9. * P

Western blot analysis of total protein extracts from the kidneys withan obstructed ureter revealed a threefold increase ( P = 0 B protein at ~53 kDabetween untreated animals (0.37 ± 0.10 relative densitometerunits) and enalapril-treated animals (1.12 ± 0.10 relativedensitometer units) (Fig. 7 ). This bandat ~53 kDa is consistent with previous reports of the approximate size of the ET B receptor ( 31 ). This increasein ET B protein appears to be uniform throughout tubules ofthe renal cortex (Fig. 7 ). Only a few cells of glomeruli appear tocontain ET B. This pattern of low-level protein expressionof ET B in cortical tubules is similar to that found byLehrke et al. ( 35 ) in human renal biopsy specimens.
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Fig. 7. Western blot for ET B protein. Resultsrepresent 25 µg of total protein from kidneys with an obstructedureter from 3 separate untreated and 3 separate enalapril-treated mice.The migration of prestained molecular mass markers is indicated on the right.

Morphometric analysis. The relative volume of the cortical interstitium was expressed asV v int (Fig. 8 ). UUO of 5 days duration showed a significant increase ( P n = 9) of V v int in theobstructed kidney (37.0 ± 1.1%) compared with the contralateralkidney (6.0 ± 0.1%). This is consistent with our previousstudies ( 23, 41 ) and reflects what is observed in humanureteral obstruction ( 39 ). Enalapril treatmentsignificantly ameliorated ( P increment by52% in the kidneys with an obstructed ureter (23.7 ± 1.4%, n = 11). No significant difference was observed between the contralateral kidneys of untreated mice and mice treated with enalapril. Other mice ( n = 10) were treated with acombination of oral enalapril and an intraperitoneal injection of theET B -specific receptor antagonist BQ-788 ( 48 ).The decrease in interstitial volume due to enalapril treatment wassignificantly ( P blunted by theET B antagonism (Fig. 9 ). There remained a significant decrease ( P untreated mice with UUO withrespect to the change in V v int.

Fig. 8. Immunohistochemical localization of ET B inthe renal cortex. Results depict sections of kidney with an obstructedureter of untreated mice (A) or mice treated with enalapril(B).
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Fig. 9. Effect of enalapril administration on interstitial volumeof the kidney with an obstructed ureter. The relative volume of theinterstitium (V v int ) of the kidney cortex wasdetermined as described in MATERIALS AND METHODS. Valuesare means ± SD; n = 9 for the untreated group, n = 11 for the enalapril-treated group, and n = 10 for the enalapril-BQ788-treated group.* P P P
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DISCUSSION

In this study, we found that enalapril, an ACE inhibitor, bluntedthe increased expression of ET-1 mRNA by 44% ( P in the obstructed kidney. In the setting of UUO, therenin-angiotensin system is upregulated ( 10 ). AngiotensinII, in turn, stimulates the expression of TGF-, TNF-, collagenIV, and various cytokines or vasoactive compounds that play roles inthe progression of tubulointerstitial fibrosis ( 20-23, 28 ). We have previously shown that enalapril administrationblunted the increased expression of TGF- and collagen IV mRNA in UUOrats ( 19-21, 23 ). In this study, enalaprilsignificantly suppressed the increase of TGF- mRNA in the kidney ofmice with an obstructed ureter. These data suggest that angiotensin IIeither directly or indirectly upregulates ET-1 expression in the kidneywith an obstructed ureter. However, the fact that ET-1 mRNA expressionin the kidney with an obstructed ureter did not return to the normallevel because of ACE inhibition indicates the existence of otherfactor(s) involved in the upregulation of ET-1 in the obstructed kidney.$ T8 v0 l/ A3 @0 ~+ y

Recently, Feldman et al. ( 11 ) reported on the levels ofET A and ET B mRNA expression in UUO rats at 5 days after the onset of obstruction. They showed that ET A mRNA expression was significantly elevated in the obstructed kidneycompared with the contralateral kidney. The level of ET B mRNA expression was not affected by ureteral ligation. We obtainedsimilar results in UUO mice with ureteral obstruction of the sameduration. It seems there is not a difference between rats and miceconcerning the expression or change of ET A andET B mRNA in the model of UUO.* s% y/ U( o6 t7 K

Enalapril administration had no effect on the expression ofET A mRNA in both kidneys when compared with those notreceiving enalapril. Unexpectedly, enalapril administration increasedthe level of ET B mRNA expression by 115%( P of protein threefold( P suggeststhe existence of a feedback system between ET-1 and its receptorET B. Lehrke et al. ( 35 ) found that ACEinhibition decreases ET B expression in biopsy specimensobtained from patients with chronic renal disease. Our study ismeasuring more acute effects.* y# ~# i/ n! x3 o
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In addition to lowering angiotensin levels, ACE inhibitors affectkininase II and increase the levels of bradykinin ( 10 ), which in turn modulate nitric oxide production by the endothelial cells( 36, 40, 52 ). Nitric oxide may have two opposite effects on renal disease. It is known that a moderate amount of nitric oxide isbeneficial in the prevention of experimental renal disease ( 41 ). It has been reported that nitric oxide has a directeffect on matrix protein synthesis ( 32, 51 ). Ourlaboratory has previously shown an effect of nitric oxide on theprevention of interstitial fibrosis caused by UUO by the administrationof L -arginine, a nitric oxide donor ( 41 ). Onthe basis of those data, we suggested that one of the reasons for thebeneficial effects of enalapril was the increased production of nitricoxide modulated by the upregulated bradykinin in the kidney with anobstructed ureter. On the other hand, ET-1 has various biologicaleffects and acts quite differently depending on its binding to twodifferent types of receptors ( 4, 31 ). ET-1 can alsoincrease the production of nitric oxide by the endothelial cellsthrough the ET B receptor ( 8 ). In the kidney,ET B is expressed at ~10 times the level ofET A throughout the tubule epithelium ( 6, 31, 49, 50 ). Our data suggest that enalapril may ameliorate thepathology of the obstructed kidney by increasing the level of nitricoxide not only through the kinin-bradykinin system but also byupregulation of the ET B receptor. Interestingly, Wong etal. ( 53 ) reported the downregulation of ET B incardiomyopathic hamsters and showed that enalapril therapy restoredET B receptor density in these animals. In another study,renal injury was exacerbated in rats genetically deficient in theET B receptor compared with normal rats in adeoxycorticosterone-salt hypertension model ( 38 ). Furthermore, Forbes et al. ( 13 ) have shown that a dualET A -ET B antagonist exacerbated long-termabnormalities in renal function consequent to an ischemicepisode. This suggests that the ET B has an overallbeneficial effect on renal pathophysiological processes. In someexperimental models of renal disease, ET B expression is increased ( 35, 42, 55 ). In a previous study of a rabbit model of partial bladder outlet obstruction, there was a downregulation of ET B receptors in the medulla ( 24 ). This wasmeasured 6 wk after partial bladder obstruction, whereas our studyfocuses on the renal cortex and more acute effects of obstructivenephropathy. Our study points to a beneficial effect of theET B receptor on the expansion of interstitium as blockadewith BQ-788 partially but significantly reversed the effects ofenalapril. Additional studies measuring nitric oxide production andblockade of the bradykinin B2 receptor would help to fully elucidatethe beneficial mechanisms of ACE inhibition.7 z2 ?5 C) B# G4 ^

In the present study in the kidney with an obstructed ureter, enalaprilameliorated the mRNA expression of TGF- and TNF- major cytokinesin the regulation of ECM proteins. Morphometric analysis also showedthat enalapril treatment significantly suppressed the expansion of theinterstitium in the obstructed kidney. These results are consistentwith our previous data in rats and mice ( 19-21 ). Thedose of enalapril might need to be tailored for the model of renaldisease, because Ikoma et al. ( 17 ) showed the greatereffect with low dose (50 mg/l) than high dose (200 mg/l). Ikoma et al.focused on glomerular fibrosis, whereas our laboratory has focused ontubulointerstitial fibrosis ( 19-23 ).
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Administration of enalapril did not decrease TNF- mRNA levels on apercent basis in the kidney with an obstructed ureter as much as itdecreased TGF- mRNA. This is consistent with previous observationsof the rats ( 10 ) and mice ( 29 ) in whichenalapril treatment succeeded in blunting TNF- mRNA expression butnot to the extent seen for TGF-. Angiotensin II appears tocontribute to the early phase of increase in TNF- mRNA expression inthe obstructed kidney ( 10 ). The source of TNF- synthesis may be different according to the time after the obstructionand level of ACE inhibition./ O) {" P+ A2 R2 F
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Again, using mice with UUO, we have shown the beneficial effects ofenalapril on the progression of interstitial fibrosis and expansion dueto the unilateral ureteral ligation. This is a well-establishedobservation. Enalapril administration blunted the increased mRNAexpression TGF- and to a great degree ameliorated the histologicalappearance of tubulointerstitial fibrosis in the obstructed kidney ofmice with UUO. However, the level of TGF- mRNA and the histologicalchange in the obstructed kidney did not return completely to thecontrol level even when treated with enalapril. These results stronglysuggest the existence of other factors besides angiotensin II that areinvolved in the progression of interstitial fibrosis in this setting orthe escape of some pathophysiological factor to ACE inhibition. Thepresent results point to endothelin expression as being a factor, inpart, in renal fibrosis." T  h$ k6 |% i4 p4 n

In summary, we have shown that enalapril significantly but incompletelyblunts the increased expression of ET-1 mRNA in the obstructed kidneyfrom UUO mice at 5 days after the onset of obstruction. To evaluate thedirect interaction between ET-1 and tubulointerstitial fibrosis in theUUO model, however, experiments using selective ET A andET B receptor blockers would be necessary. On the contrary, enalapril increased the mRNA expression of ET B receptor inthe obstructed kidney. These data may be very important because ET-1 can promote nitric oxide production by endothelial cells through ET B and implicates another potential mechanism by which ACEinhibitors exert a beneficial effect on renal disease apart fromlowering angiotensin II formation. A moderate amount of nitric oxide is beneficial to prevent and alleviate tubulointerstitial fibrosis. It maybe worthwhile exploring the role of ET B in the kidney with an obstructed ureter for more details and its interaction among therenin-angiotensin, kinin-bradykinin, and nitric oxide systems.

ACKNOWLEDGEMENTS
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The assistance of Monica Waller in the preparation of thismanuscript is acknowledged.
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