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作者：Andrew Y. Zhang, Ya-Fei Chen, David X. Zhang, Fu-Xian Yi, Jenson Qi, Patricia Andrade-Gordon, Lawrence de Garavilla, Pin-Lan Li, Ai-Ping Zou作者单位：1 Departments of Physiology and Pharmacology andToxicology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226; and Vascular Research, Johnson and Johnson PharmaceuticalResearch and Development, Spring House, Pennsylvania 19477
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4 E8 `# W* p" H0 q 【摘要】
/ j1 D( F" k2 `% J Recent studies have indicated that urotensin II (UII), a cyclic peptide, isvasoactive and may be involved in cardiovascular dysfunctions. It remainsunknown, however, whether UII plays a role in the control of renal vasculartone and tubular function. In the present study, a continuous infusion ofsynthetic human UII (hUII) into the renal artery (RA) in anesthetized rats was found to increase renal blood flow (RBF) and urinary water and sodiumexcretion (UV and U Na V) in a dose-dependent manner. At a dose of 20ng · kg - 1 ·min - 1, it increased RBF by 20% and UV andU Na V by 94 and 109%, respectively. Nitric oxide (NO) synthaseinhibitor N G -nitro- L -arginine methyl ester( L -NAME) completely abolished hUII-induced increases in RBF andwater/sodium excretion. In isolated, pressurized, andphenylephrine-precontracted small RA with internal diameter of 200 µm,hUII produced a concentration-dependent vasodilation with a maximal responseof 55% at 1.5 µM. L -NAME significantly blocked this hUII-inducedvasodilation by 60%. In denuded RA, hUII had neither vasodilator norvasoconstrictor effect. With the use of 4,5-diaminofluorescein diacetate-basedfluorescence imaging analysis of NO levels, hUII (1 µM) was shown to doublethe NO levels within the endothelium of freshly dissected small RA, and L -NAME blocked this UII-induced production of endothelial NO. Theseresults indicate that UII produces vasodilator and natriuretic effects in thekidney and that UII-induced vasodilation is associated with increasedendothelial NO in the RA. ' S; x+ J, u5 h/ a
【关键词】 natriuretic factor renal circulation sodium reabsorption renal tubule renal hemodynamics
% y% L' T: S( a/ ` UROTENSIN II (UII) is a cyclic peptide with a COOH-terminal hexapeptide sequence, which is conserved across species, including fish, frog,mouse, rat, pig, and human ( 6, 7, 23 ). This cyclic peptide wasoriginally isolated from fish spinal cord and had a structure similar tosomatostatin ( 27 ). Recently,UII was identified as an endogenous ligand for G protein-coupled receptor (GPR14), which is one of these types of orphan receptors and was first clonedfrom rat cDNA library ( 18, 21, 23 ). A human receptor that has75% homology with rat GPR14 was also characterized ( 2 ), and the mRNA for thisreceptor is widely expressed in human heart, brain, pancreas, skeletal muscle,vascular smooth muscle and endothelial cells, spinal cord, and endocrinetissues ( 2, 18, 20 ). This wide distribution ofGPR14 suggested that UII may serve as a circulating hormone to participate inthe regulation of many physiological processes.2 }1 h* B7 j1 }' u0 E
$ V! M5 t% n/ _6 k5 _& gIndeed, early studies reported that human UII (hUII) produced a markedvasoconstriction in many arteries from nonhuman primates, including largecoronary, pulmonary, and carotid arteries ( 2, 8 ). It was found that hUIIinduced vasoconstriction in the isolated large arteries from both rat andhuman with a potency of 6- to 28-fold greater than endothelin-1( 2 ). In anesthetized nonhumanprimates, this cyclic peptide was shown to markedly increase total peripheralresistance ( 2 ). These studiesindicated that UII is one of the most potent vasoconstrictors in mammals,which may play an important role in the regulation of cardiovascularhomeostasis and be importantly involved in circulatory dysfunction. However,recent studies demonstrated that hUII also causes vasodilation in rat smallarteries and human pulmonary arteries( 4, 30 ). It appears that theeffects of UII depend on different vascular beds, vessel sizes, or species. Despite extensive studies on the effects of UII on different vascular beds,little is known regarding the action of UII on renal vascular tone. It remainsunknown whether this cyclic peptide participates in the control of renalhemodynamics and urinary excretion of electrolytes.
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The present study was designed to test whether UII alters renal hemodynamics and influences excretory function of the kidney, therebyparticipating in the regulation of renal function. To address these questions,we first examined the effects of hUII infusion into the renal artery on renalblood flow (RBF), glomerular filtration rate (GFR), and sodium and waterexcretion (UV and U Na V) in anesthetized Sprague-Dawley rats. Withthe use of isolated small renal arterial preparation, we then observed thedirect vasodilator effects of hUII on small renal arteries. To explore themechanism of hUII-induced renal vasodilation, a fluorescent video microscopywas performed to determine the effects of hUII on intracellular nitric oxide(NO) levels in the intact endothelium of these small renal arteries. These experiments provided direct evidence that hUII dilates renal arteries througha NO-dependent mechanism, which may participate in the regulation of renalfunctions in concert with its natriuretic effect.( A0 b" P4 s8 \% I
+ e. j3 J C5 c5 dMATERIALS AND METHODS
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2 v K3 y+ Q- W& \3 ]$ E" l# D5 mDetermination of renal hemodynamics. Male Sprague-Dawley rats (purchased from Harlan Sprague Dawley, Madison, WI) weighing between 250 and300 g were fasted overnight but allowed free access to water. They wereanesthetized with ketamine ( 30 mg/kg body wt im) and Inactin (50 mg/kg body wt ip) and placed on athermostatically controlled warming table to maintain body temperature at37°C. After tracheotomy, cannulas were placed in the right femoral veinand artery for intravenous infusions and measurements of arterial pressure. Anabdominal incision was made, the left kidney was placed in a stainless steelcup to stabilize the organ, and an electromagnetic flow probe (2 mm) wasplaced around the left renal artery to measure RBF as we described previously( 5 ). The two ureters wereisolated and catheterized for collection of urine during experiments. Aftersurgery, the animals received an intravenous infusion of 2% bovine serumalbumin in a 0.9% sodium chloride solution at a rate of 3 ml/h throughout theexperiment to replace fluid losses and maintain a stable hematocrit of 43± 3%.6 f$ n. c; Y6 d5 T0 F
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Analysis of renal function. Sprague-Dawley rats were anesthetized, and the surgery for renal function study was performed as described above.After surgery and equilibration period, continuous measurements of meanarterial pressure (MAP) and RBF were obtained throughout the experiment. Tomeasure GFR, a 0.5-ml bolus of FITC-inulin (8.0 mg/ml) was given, and then asteady intravenous infusion of FITC-inulin (4.0 mg/ml) at 3.0 ml/h continuedthroughout the experiment. After a 1.5-h equilibration period, two 20-min timed collections of urine were made. Blood samples (100 µl) were taken inheparinized hematocrit tubes after each urine collection period. Then, hUII(2.5, 5, 10, or 20ng·kg - 1 ·min - 1 ) was infused into the renal artery for 60 min, and late urine and bloodcollections were repeated. At the end of each experiment, the kidneys wereremoved and weighed, blood samples were centrifuged, and 20-µl plasma and1:50 diluted urine samples were pipetted into a microtiter plate and mixedwith 200 µl HEPES buffer (10 mM) for FITC-inulin measurement withexcitation and emission wavelengths of 480 and 530, respectively, using anautomatic microplate reader (KC4; Bio-Tek Instruments, Winooski, VT). Theurine flow rate was determined gravimetrically, and sodium (Na )and potassium (K ) concentrations of urine samples were measuredusing a flame photometer. GFR was calculated as the product of urine flow andthe ratio of urine-to-plasma FITC-inulin concentrations. GFR, urine flow, andurinary Na and K excretion were factored per gramkidney weight. In an additional group of rats, nitric oxide synthase (NOS)inhibitor N G -nitro- L -arginine methyl ester( L -NAME; 100 µg · kg - 1 · min - 1 ) was infused into the renal arteryfor 30 min, and then the effects of hUII (2.5, 5, 10, or 20 ng ·kg - 1 · min - 1 )on renal hemodynamics and renal function were examined. The hUII (peptide sequence, ETPDCFWKYCV) used in this study was demonstrated to have an HPLCpurity of 95.64%. The identity of this cyclic peptide was confirmed byelectrospray ionization-mass spectrometry analysis with a molecular weight of1387.7, which was consistent with the theoretical molecular weight of 1387.6(data not shown).# Z5 d% b6 `* ^5 x# c9 k& ^2 A1 w
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Preparation of small renal arteries. Male Sprague-Dawley rats weighing between 250 and 300 g were anesthetized with pentobarbital sodium (80mg/kg body wt ip), and the kidneys were rapidly removed and kept in ice-coldHEPES-buffered physiological saline solution (PSS) that consisted of thefollowing composition (in mM): 140 NaCl, 4.7 KCl, 1.6 CaCl 2, 1.17MgSO 4, 1.18 NaH 2 PO 4, 5.5 glucose, 10 HEPES,pH 7.4. The small renal arteries (250- to 300-µm internal diameter) werecarefully dissected on ice and transferred to a 35-mm Sylgard-coateddissecting dish containing ice-cold PSS. In an additional group of rats, theaorta below the left renal artery was isolated and cannulated. After the aortaat a site above the right renal artery was ligated, the kidneys were flushedwith 10 ml of ice-cold PSS following 60 ml of air perfusion to remove theendothelium of the renal arteries. Then, the small renal arteries were dissected to measure agonist-induced NO production. These experiments wereperformed to confirm that NO was derived from the renal arterial endothelium.All these procedures were described in great detail in our previous studies( 16 ).9 C+ \, T$ ]4 {: K# a* [
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Video microscopy of isolated and perfused renal arteries. Dissected segments of small renal arteries were mounted on glass pipettes in awater-jacketed perfusion chamber. The small arteries were perfused and bathedwith PSS that was equilibrated with a 95% O 2 -5% CO 2 mixture and maintained at 37°C. This arterial preparation has been shownto have an intact endothelium( 12, 17, 37 ) in 95% of the arteries asdetermined by a vasodilator response to bradykinin (data not shown). After theartery was mounted, the outflow cannula was clamped, and the artery was pressurized to 80 mmHg and equilibrated for 1.5 h. Internal diameter of theartery was measured using a video system composed of a stereomicroscope (LeicaMZ8, Leica), a CCD camera (KP-MI AU, Hitachi), a video monitor (VM-1221,Hitachi), a video measuring apparatus (VIA-170, Boeckeler Instrument, Tucson,AZ), and a video printer (UP890 MD, Sony). The arterial images were recorded continuously with a video cassette recorder (M-674, Toshiba). The effects ofhUII on arterial diameters were studied by cumulative additions of hUII(0.25-1.5 µM) into the bath solution.5 {, v* j: C& t5 J' ]8 `( _ q0 c
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Measurement of NO levels within the endothelium of small renal arteries. A fluorescent NO indicator, 4,5-diaminofluorescein diacetate(DAF-2DA), which was recently developed by Kojima et al.( 15 ), was used to measure NOlevels within the endothelial cells of freshly isolated small renal arteriesas we described previously( 16 ). Small renal arterieswere dissected as described above. The arterial segment was cut open along itslongitudinal axis and pinned onto the dish with lumen side upward. Care was taken not to disrupt the endothelium. After a 1-h equilibrium period, thearterial segment was incubated with DAF-2DA (10 µM, Calbiochem) in 1 ml ofPSS at room temperature for 30 min. The segment was then rinsed three timeswith PSS, and the dish was mounted on the stage of an epifluorescencemicroscope (Nicon E600) equipped with a x 20 objective and a 490-nmexcitation and a 535-nm emission filter. Digital images were acquired and analyzed using a PC-controlled digital CCD camera (Roper Scientific RTE/CCD-1300-Y/HS) by MetaMorph imaging analysis software (Universal Imaging)as we described previously( 35 ). NO fluorescence wasmeasured every 5 min in the same area of endothelial layer.
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R( e2 g( X/ }. }3 r A$ f A& ~4 nStatistics. Data are presented as means ± SE. Thesignificance of differences within and between groups in multiple groups ofexperiments was evaluated using an analysis of variance for repeated measures,followed by Duncan's multiple range tests (Sigmastat). P significant.
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0 v% Z, Z) ~: V" x: z* mRESULTS
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: ?& }& D9 N: tEffect of hUII on MAP, RBF, and GFR. The effects of hUII on renalhemodynamics in rats are presented in Fig.1. A continuous infusion of hUII (2.5, 5, 10, or 20 ng ·kg - 1 · min - 1 )into the renal artery in anesthetized rats had no significant effect on MAPregardless of the presence or absence of L -NAME ( n = 7; Fig. 1 A ). However,this continuous infusion of hUII produced a concentration-dependent increase in RBF ( Fig. 1 B ). Italso increased GFR with a maximum increase of 23% at 2.5 ng ·kg - 1 · min - 1. At higher concentrations of hUII over 10 ng ·kg - 1 · min - 1,GRF returned to baseline ( Fig.1 C ). After treatment of the rats with L -NAME,hUII-induced alterations of RBF and GFR were completely blocked. Fractionalfiltration (FF) rate was calculated as a ratio of GFR and renal plasma flow,and no significant change was found on this ratio by hUII administration.1 q6 S/ ]/ W% i/ _& _
6 z* C- p7 O# l _2 S# w3 L7 rFig. 1. Effects of intrarenal infusion of human urotensin II (hUII) on meanarterial pressure (MAP), renal blood flow (RBF), and glomerular filtrationrate (GFR) in anesthetized rats. A - C : effects of hUII onMAP, RBF, and GFR under control conditions and after treatment of nitric oxidesynthase (NOS) inhibitor N G -nitro- L -argininemethyl ester ( L -NAME), respectively. * P P L -NAME infusion (control; n = 7).) s, L- L' K8 B& m0 x
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Effects of hUII on renal function. The results of theseexperiments are presented in Fig.2. hUII markedly increased UV( Fig. 2 A ) andU Na V ( Fig.2 B ) in a dose-dependent manner, but it had no effect onpotassium excretion ( Fig.2 C ). At the highest dose of hUII infusion, UV andU Na V were doubled. These hUII-induced increases in UV andU Na V were also completely blocked by L -NAME pretreatment. Consistently, the fractional excretion of sodium (FE Na ) was increased and doubled at the highest dose of hUII administration.7 T: B3 U- D# W- @
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Fig. 2. Effects of intrarenal infusion of hUII on urine flow rate (UV) and urinarysodium excretion (U Na V). A - C : effects of hUII onUV, U Na V, and potassium excretion (U K V) under controlconditions and after treatment of NOS inhibitor L -NAME,respectively. * P n = 6). gkwt, Grams kidney weight.
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) \+ j' }. x L9 }2 ahUII-induced endothelium-dependent vasodilation in small renal arteries. hUII was found to induce an endothelium-dependent vasodilationin a dose-dependent manner. The isolated small renal arteries (with a basalinternal diameter of 258 ± 15 µm) were precontracted withphenylephrine, then concentration-response curves of hUII were determinedusing these vessels. Figure 3 A shows representative microscopic images showing thechanges in the internal diameters of the small renal arteries treated withdifferent compounds. Addition of hUII into the bath solution producedvasodilation in phenylnephrine-contracted arteries. In the presence of L -NAME, hUII-induced vasodilation was significantly blocked. Figure 3 B summarizesthe effects of hUII on vascular diameter of the small renal arteries with andwithout the intact endothelium. Addition of hUII into the bath solutionproduced a concentration-dependent vasodilation with maximal relaxation of55%. Pretreatment with L -NAME (100 µM) for 30 min markedlyinhibited the hUII-induced vasodilation in these phenylephrineprecontracted arteries with the maximal inhibition by 60%. To confirm that hUII-inducedvasodilation is endothelium dependent, the denuded arteries were used toexamine the effect of hUII. In these arteries, no significant vasorelaxationwas observed.' K2 I# o3 m$ W6 v% j3 s% Y, S: {4 {
, S; ?; d3 f% A. z$ _3 aFig. 3. hUII-induced vasodilation in isolated small renal arteries. A :representative photo prints showing the changes in internal diameters of smallrenal arteries treated with hUII or L -NAME hUII. B :summarized data showing the effects of hUII on control (Ctrl), L -NAME-treated, or endothelium-denuded arteries. * P n = 5). PE, precontraction with phenylephrine; EC,endothelial cells.
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1 j; A! }& b: n' r* q( xUII-induced NO production in the intact endothelium of renal arteries. As shown in Fig.4 A, a 30-min infusion of hUII (1 µM) with the renalarteries (internal diameter = 340 ± 35 µm) produced a markedincrease in green NO fluorescence in the endothelium. In the presence of L -NAME, hUII-induced increase in NO green fluorescence wassignificantly attenuated, suggesting that L -NAME is capable ofblocking the hUII-induced increase in NO within the intact endothelium of these freshly isolated renal arteries. Figure 4 B summarizes hUII-induced alterations of NO levels measured by DAF-2T fluorescence intensity in the absence or presence of L -NAME ( n = 7).hUII time dependently produced a significant increase in NO levels within theendothelium of the renal arteries. In the denuded renal arteries, there was nodetectable hUII-induced increase in DAF-2T fluorescence.
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Fig. 4. hUII-induced NO increase in the endothelium of small renal arteries. A : typical fluorescent microscopic images showing NO-induced4,5-diaminofluorescein diacetate (DAF-2) green fluorescence within endothelialcells. B : time course for hUII-induced change in NO levels in therenal arterial endothelium with different treatments. * P n = 7).
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DISCUSSION8 _* I' u& A( L) b1 n- g$ v
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In contrast to the vasoconstrictor effect in large vessels from differentspecies, the present study demonstrated that hUII produced a markedNO-dependent vasodilator response in isolated small renal arteries of rats. Wealso found that hUII induced a strong natriuretic response when directlyadministrated into the renal artery. These results indicate that hUII is aNO-dependent vasodilator and natriuretic peptide in the rat kidney.7 @, ]# z9 U7 @, E2 v3 M
4 i) {( A, }, M$ F" ]In anesthetized rats, we first examined the effects of infusion of hUIIinto the renal arteries on renal hemodynamics and renal functions. It wasdemonstrated that hUII markedly increased RBF in a dose-dependent manner,indicating that hUII may produce renal vasodilation. After pretreatment of therat kidney with NOS inhibitor L -NAME, hUII-induced actions on renalhemodynamics were substantially blocked. It appears that the effect of hUII inthis preparation is associated with NO-dependent mechanism. This is consistentwith previous observations in cerebral and other vascular beds, showing thatthe vascular effects of hUII could be blocked by L -NAME( 4 ). It is well known that NOplays a critical role in the regulation of renal vascular tone and RBF( 36 ). Renal vascularresistance is increased following inhibition of NOS, whereas stimulation ofendogenous NO leads to a decrease in renal vascular resistance and increase in RBF. Many stimuli or agonists such as bradykinin, acetylcholine, ANG II,norepinephrine, endothelin, or shear stress have been reported to activate NOSand produce NO in the kidney( 26, 33 ). The results from thepresent in vivo animal experiments suggest that hUII may be another possibleagonist to stimulate NO production in renal vascular bed.
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& l; ]' _' h2 L( DConsistent with the increase in RBF, hUII also produced a significant increase in GFR, especially at a low dose. By calculating FF rate, we foundthat hUII only increased FF at low-dose range, indicating that lowconcentrations of hUII may produce greater vasodilation in preglomerularvessels compared with postglomerular vessels. When the dose of intrarenallyinfused hUII was increased, however, GFR and FF increase did not occur. Thissuggests that high concentrations of hUII may dilate both pre- andpostglomerular vessels. The present study also demonstrated that hUIIincreased urinary water and sodium excretion, which was blockable by L -NAME. This hUII-induced increase in urinary sodium excretion maynot simply be attributed to the increase in RBF or GFR; it may be associatedwith the direct inhibition of tubular ion transport activity. Indeed,FE Na was found increased by administration of hUII. Given that hUIIincreased FE Na but had no effect on potassium excretion, it ispossible that the natriuretic action of this peptide is attributed toinhibition of the ion transport activity in the collecting duct. When the ratkidney was pretreated with L -NAME, the effect of hUII on sodiumexcretion was completely blocked. Therefore, NO may be involved in UII-inducedchanges in urinary sodium excretion. Numerous studies have demonstrated thatNO can directly act on renal tubules to inhibit tubular ion transportactivity. It has been indicated that the effects of NO on sodium reabsorption are associated with its direct inhibitory action on theNa /H exchange, Na -K -ATPase,and amiloride-sensitive Na channels in different tubules( 36 ). It seems that hUIIstimulates NO production in the kidney and thereby inhibits tubular sodium reabsorption, resulting in diuretic and natriuretic response in concert withits hemodynamic effects.
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- { ]) Q/ Y+ _/ H L3 G/ y0 hDespite intensive studies on the vasomotor response of hUII in othervascular beds, there is no direct evidence showing the effect of hUII on renalvascular tone or renal endothelial function. To provide direct evidence thathUII produced renal vasodilation and to explore the mechanism responsible forhUII-induced vasodilator response, we used isolated, perfused, and pressurized renal artery preparation to examine the effects of hUII on the diameter ofthese small arteries using video microscopy. It was found that addition ofhUII into the lumen of the perfused arteries produced aconcentration-dependent vasodilation in endotheliumintact renal arteries. Whenthese arteries were denuded, hUII-induced vasodilation was completely blocked. Similarly, when these arteries were pretreated with L -NAME, hUII-induced vasodilation was substantially attenuated. These results indicatethat hUII stimulates NO and produces endothelium-dependent vasodilation in therenal arteries, which is consistent with the results obtained from our in vivoanimal experiments. Therefore, we conclude that hUII is a potent NO-dependentvasodilator in renal circulation.- D) {7 b' ~' N+ c) L( @( J% i
# I9 p+ A4 i- A! @& b3 mIn previous studies, however, hUII has been shown to be a potent vasoconstrictor in various arteries, including large coronary, pulmonary, andcarotid arteries isolated from different species, such as rats, dogs, pigs,and monkeys ( 2, 9, 10, 19 ). In some studies, incontrast, this cyclic peptide was not found to have any vasomotor action inhuman arteries and veins of different sizes( 13 ). This led to anassumption that a vasoconstrictor action of hUII may be masked by its potentvasodilator effects in these human vessels. Indeed, hUII has been reported tocause vasodilation in isolated human pulmonary and abdominal resistant arteries ( 30 ). Recent studieshave demonstrated that hUII also dilates the small arteries from differentvascular beds in rats ( 4, 11, 14, 30 ). The results from thepresent study support the view that hUII is a potent vasodilator in small resistance arteries. Taken together, hUII seems to cause vasoconstriction primarily in large conduit vessels but vasodilation predominantly in smallresistance arteries.) y1 H) i h3 c, ~- R# b
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To further test the hypothesis that hUII stimulates NO production in renalarterial endothelium, we directly examined the intracellular NO response tohUII. In these experiments, DAF-2DA, a novel cell-permeable fluorescentindicator of NO, was loaded into endothelial cells, and then NO responses inthese cells were monitored. We found that hUII (1 µM) stimulated theproduction of a strong green fluorescence in the endothelial layer of the renal arteries, which represented the increases in NO levels within renalarterial endothelial cells. The NOS inhibitor L -NAME or the removalof the endothelium completely blocked the hUII-induced increase in NO levelsin this preparation, suggesting that detected NO increases in response to hUIIare derived from the endothelium of these arteries. To our knowledge, theseresults provide the first direct evidence that hUII increases NO levels in therenal arterial endothelium. It should be noted that the arteries used in thisprotocol were relatively large renal arteries. Therefore, the results may notnecessarily suggest that this NO production in these large renal arteries contributes to UII-induced reduction of renal vascular resistance or increasein RBF, because these large vessels are not renal resistance arteries.! S0 H- a! B( I$ n1 r! }
5 }2 {9 I% ^! B. W) D9 T0 ?The mechanism by which hUII stimulates NO production remains unknown.Previous studies showed that hUII is an endogenous ligand for the orphanreceptor GPR14 ( 18 ). Thiscyclic peptide caused concentration-dependent increases in intracellular[Ca 2 ] in HEK-293 cells expressing human GPR14( 2 ). In vascular smooth musclecells, the action of hUII is mediated by an increase in[Ca 2 ] i through the IP 3 signalingpathway ( 25 ). Therefore, it ispossible that hUII activates its receptors on vascular endothelial cells andsubsequently causes intracellular Ca 2 mobilization,resulting in the stimulation of endothelial NOS activity through acalmodulin-dependent mechanism. This Ca 2 -depenent activation of NOS in endothelial cells has been well documented ( 28 ). In a recent study, weprovided direct evidence that NOS activation in the intact arterialendothelium is dependent on cytosolic [Ca 2 ]( 34 ). f- s; C; l3 U: _. M2 f- @
: D$ Y4 a( f1 f* N% `, xAlthough the present study did not determine the role of endogenous UII inthe regulation of renal hemodynamics and renal excretory function due to lackof specific potent antagonists of UII, the finding that this peptide increasesRBF and sodium and water excretion at least indicates that increases in plasma concentration of UII change renal vascular and tubular activities, which mayrepresent the effects of activation of UII system on renal function. However,it remains unknown whether these actions of UII are physiological orpathological, because the plasma concentrations in human or different animalsmeasured in many laboratories have shown the diversity even underphysiological conditions, ranging from 1.9 pM to 2.5 nM depending on the methods used for its measurements. The disparity of the assay results may berelated to the assay formats, reagents, and/or extraction( 8 ). In the present study, thecalculated concentrations of hUII in renal blood were 75-600 pM,which seem to be at physiological range of UII plasma levels. However, we performed preliminary experiments to quantify rat plasma UII concentrationusing RIA and found that plasma concentrations of UII were 4.78 ± 1.2pM in anesthetized rats ( n = 6), which was much lower than calculatedrenal plasma concentrations. If the assay results are true, the highconcentrations of UII during intrarenal infusion may represent a pathologicalcondition related to activation of UII activation as seen in patients withchronic heart failure or chronic renal failure( 8, 29, 31 ). As discussed above,nevertheless, the diversity of the assay results plagues an appropriateevaluation of physiological range of UII plasma levels. Interestingly,previous studies showed that pre-pro-urotensin II and GPR14 mRNAs and UIIproteins exhibited a high abundance in human kidney( 22, 24 ). Recent studies also foundthat hUII mRNAs were abundantly expressed in renal carcinoma cells and thatplasma hUII concentrations were higher in patients with chronic renal failurecompared with normal people( 29, 31 ). By immunohistochemicalexaminations, hUII was found high in endothelial cell and in the distalconvoluted tubules ( 29 ). Allthese results and our findings suggest that UII may be an important targetmolecule in studying renal physiology and pathophysiology.
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+ u$ e9 k" e7 ]5 S5 y1 j7 KIn summary, the present study demonstrated that 1 ) acuteelevations of hUII in the kidney produced an increase in RBF, GFR, and urinarywater/sodium excretion, which was blocked by L -NAME; 2 )hUII stimulated endothelium-dependent vasodilator responses in the small renalarteries when added into isolated, perfused, and pressurized renal arterialpreparation; and 3 ) hUII increased NO levels in the intactendothelium of the renal arteries. These results suggest that UII is aNO-dependent vasodilator and natriuretic peptide in the kidney, which mayparticipate in the control of renal function.8 @8 c) L/ |8 F! B4 y% |
4 \! a, J0 G( VDISCLOSURES
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& T" c: Y; ~# \This study was supported by National Institutes of Health Grants DK-54927,HL-70206, and HL-57244.
- C9 I* K' s" y* \5 z) b- c2 C 【参考文献】9 A1 T! N1 u# ~' X% u
Alderton WK,Cooper CE, and Knowles RG. Nitric oxide synthases: structure, function,and inhibition. Biochem J 357:593-615, 2001./ T$ q: z7 y. l) z0 E. N& W, n4 M+ {
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Ames RS, SarauHM, Chambers JK, Willette RN, Aiyar NV, Romanic AM, Louden CS, Foley JJ,Sauermelch CF, Coatney RW, Ao Z, Disa J, Holmes SD, Stadel JM, Martin JD, LiuWS, Glover GI, Wilson S, Mcnulty DE, Ellis CE, Elshourbagy NA, Shabon U, TrillJJ, Hay DWP, Ohlstein EH, Bergsman DJ, and Douglas SA. Human urotensin-IIis a potent vasoconstrictor and agonist for the orphan receptor GPR14. Nature 401:282-286, 1999.
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