General inhibition of renocortical cyclooxygenase-2expression by the renin-angio - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:50 |只看该作者 |倒序浏览 |打印

作者：HayoCastrop, JürgenKlar, CharlotteWagner, KlausHöcherl,  ArminKurtz作者单位：1 Institut für Physiologie und Pharmakologieder Universität Regensburg, D-93040 Regensburg,Germany % L' L! _7 O' ~" q



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      【摘要】' x9 a. s( ?: j. L: Q+ p
   Because across-the-board data indicatethat renin and cyclooxygenase-2 (COX-2) expression in the kidney cortexare regulated in parallel and because ANG II can inhibit COX-2expression, the purpose of our study was to characterize a potentialgeneral inhibitory feedback of the renin-angiotensin system onrenocortical COX-2 expression in vivo. Rats were fed a high-,normal-, or low-salt diet or were chronically infused with furosemide(60 mg · kg 1 · day 1 ) orthe left renal artery was clipped, and the animals were treated inaddition to or without the angiotensin-converting enzyme inhibitor ramipril (10 mg · kg 1 · day 1 ). Ahigh-salt diet reduced expression of COX-2, whereas a low-salt diet,furosemide infusion, and renal artery stenosis stimulated COX-2expression. Additional angiotensin-converting enzyme inhibition led tofurther increases in renocortical COX-2 expression by 62, 136, 300, 50, and 70% for a high-, normal-, and low-salt diet, furosemide infusion,and renal artery stenosis, respectively. Thus our data suggest ageneral inhibitory effect of the renin-angiotensin system onrenocortical COX-2 expression.
      【关键词】 renin angiotensinconverting enzyme inhibition renal cortex prostaglandins
   INTRODUCTION
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CYCLOOXYGENASE-1 AND -2 (COX-1 and -2) are the two known isoforms ofcyclooxygenases that convert arachidonic acid in a two-step reactioninto PGH 2, which is then further processed to a variety ofprostanoids by specific enzymes ( 14 ). COX-1 is designated as the housekeeping isoform of cyclooxygenase, whereas COX-2 is considered the inducible isoform that is induced, for example, underinflammatory conditions ( 5, 15 )./ c9 R1 b, m; G( f2 @& b& u
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In the renal cortex of the rat, COX-2 is not only constitutivelyexpressed in the macula densa and neighboring cells of the thickascending limb (TAL) of Henle [cortical TAL (cTAL)] ( 8, 20 ) but also its expression is influenced by a variety ofparameters that are not related to inflammation. For example, COX-2expression in the renal cortex is inversely related to the oral saltintake in such a way that high-salt intake leads to a reducedexpression of COX-2 whereas low-salt intake stimulates the expressionof COX-2 ( 8, 11, 22, 25 ).! ~( L* X. U( H" y
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Besides different salt diets, a reduced salt transport activity of themacula densa cells, as caused by loop diuretics, was recently shown toincrease the expression of COX-2 in the renal cortex ( 13, 24 ). In this context, additional experiments with cultured cTALcells have shown that both pharmacological inhibition of the Na-K-2Clcotransport and reduction of the chloride concentration in the culturemedium cause an upregulation of COX-2 ( 1, 24 ). It has beensuggested that reduced salt transport activity of the cTAL cells underthese conditions stimulates the expression of COX-2 via the p38 MAPkinase pathway ( 1, 24 ).; t7 V2 _3 j0 t& q$ A

Not only salt intake and salt transport of the macula densa but alsothe renal perfusion pressure influences the expression of COX-2 in therenal cortex ( 9, 12, 21 ). In this context, it has beendemonstrated that renal artery stenosis, which reduces the renalperfusion pressure, leads to an upregulation of the expression ofCOX-2, whereas an increased systemic blood pressure decreases theexpression of COX-2 ( 9, 12, 21 ).' e& b6 L% C% i, j/ T

Under all of these conditions that influence the expression of COX-2 inthe renal cortex, a striking parallel regulation of the synthesis andsecretion of renin has been observed, suggesting a possibleinterdependency of the expression of both enzymes. A number of studieshave already investigated the influence of COX-2 expression on theexpression of renin ( 2, 3, 6, 7, 10-12, 16-19, 23 ), whereas less is known about the role of renin and ANG IIfor the control of renocortical COX-2 expression ( 2, 22 ).
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During a normal- and low-salt diet, an inhibitory effect of ANGII on the expression of COX-2 was suggested in the rat in such a waythat both angiotensin-converting enzyme (ACE) inhibitors andangiotensin AT 1 receptor antagonists were found tomoderately increase basal COX-2 expression and to enhance theexpression of COX-2 stimulated by a low-salt diet ( 2, 22 ).6 M% g1 q$ y+ B; M" I1 R
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Whether these stimulatory effects of ANG II antagonists on COX-2expression really reflect direct involvement of ANG II in the controlof COX-2 expression or are more secondary to the aggravation of saltdeficiency is less clear. We have previously found that ANG IIantagonists also increase renocortical COX-2 expression in rats on anormal- and low-salt diet during a mineralocorticoid clamp, whichprevents changes of systolic blood pressure and of glomerularfiltration ( 11 ). It appears conceivable from these datathat COX-2 expression in the renal cortex is, at least in part,negatively controlled by ANG II in vivo. If so, any alteration in theactivity of the renin system and in consequence of ANG II formationwould be expected to modulate COX-2 expression. Because there exists astriking parallel modulation of renocortical COX-2 expression and theactivity of the renin system by salt intake, by renal perfusionpressure, and by loop diuretics, it is possible that changes of COX-2expression are limited by concomitant changes of ANG II formation. Inconsequence, the apparent stimulation of COX-2 expression under a givencondition would lead to an underestimation of the strength of thestimulus driving COX-2 expression. In turn, one would expect a generalpotentiation of COX-2 expression by ANG II antagonists, depending onthe renin activity.0 e$ ]% ?8 S% Z8 Y4 ?3 M

To address the question of whether such a general negative-feedbackmechanism of the renin-angiotensin system on the expression ofrenocortical COX-2 exists, we have systematically investigated theinfluence of ACE inhibition on the expression of COX-2 under theaforementioned different conditions that lead to a parallel regulationof the expression of both COX-2 and renin.
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We found an increased expression of renocortical COX-2 during ACEinhibition under all conditions examined that was most pronounced during a low-salt diet.* E) N- E$ u  V" F9 M1 y' E

MATERIALS AND METHODS

Animal Experiments# p+ i/ ]8 I# C  y6 j" W
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Male Sprague-Dawley rats (180-200 g) were used in theexperiments. All animal experiments were conducted in accordance with Guide for the Care and Use of Laboratory Animals (Washington, DC: National Academy Press, 1996) and German laws onthe protection of animals.4 }* q& h1 Z8 s  P3 Y5 ^3 c
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Ten groups of rats ( n = 7 each) were treated as follows.' C; q+ g% d! V( H" ^
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Group I. Rats were fed a normal-salt diet [0.6% NaCl (wt/wt), Altromin, Lage,Germany] over a period of 7 days.

Group II. Rats were fed a low-salt diet [0.02% NaCl (wt/wt), ssniff specialdiets, Soest] over a period of 7 days.
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Group III. Rats were fed a high-salt diet [8% NaCl (wt/wt)] over a period of 7 days.

Group IV. Rats were infused with furosemide (60 mg · kg 1 · day 1 ) viasubcutaneously implanted osmotic pumps (model 2ML1, Alza, Palo Alto, CA) for 7 days. These animals had access to tap water and a salt solution (0.9% NaCl, 0.1% KCl). Rats with empty osmotic pumps servedas controls.
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Group V. In rats with kidney artery stenosis, the left renal arteries wereclipped (0.2-mm-inner-diameter silver clips, Degussa) under pentobarbital sodium anesthesia. In controls, the left renalartery was only touched with forceps (sham clipped).

Animals in groups VI-X were treated similarly to groups I-VI but also received the ACE inhibitorramipril (10 mg · kg 1 · day 1 ) viadrinking water during the last 3 days of the experiment.

Organ Sampling, o& s6 F& ?2 u4 Y9 F; \' M

Animals were killed by decapitation and the kidneys wereremoved. Blood samples were taken for determination of plasma renin activity (PRA). The kidneys were cut in longitudinal halves. From oneof these halves, the cortex was dissected under a stereomicroscope. Pieces of kidney cortex were frozen in liquid nitrogen and stored at 80°C until isolation of total RNA or protein. The second half wasused for immunohistochemistry., u! E$ S/ p7 S/ E) h

Extraction of RNA& V5 W" K  p! s  ^6 J
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Total RNA was extracted from the cortex basically according tothe acid-guanidinium-phenol-chloroform protocol of Chomczynski andSacchi ( 4 ). RNA pellets were dissolved indiethylpyrocarbonate-treated water, the yield of RNA was quantified byspectroscopy at 260 nm, and samples were placed in aliquots and storedat 80°C until further processing. The quality of extracted RNA wasconfirmed by the observation of intact 18S and 28S rRNA bands after gel electrophoresis in an ethidium bromide-stained agarose gel.. \% c' i4 I0 c. h

RNase Protection Assays for COX-2, Renin, and Cytoplasmic -Actin mRNA

COX-2, renin, and -actin mRNA levels were measured by RNaseprotection assays. In brief, after linearization and phenol/chloroform purification, the plasmids yielded radiolabeled antisense cRNA transcripts by incubation with SP6 polymerase (Promega) and[ - 32 P]GTP (Amersham-Pharmacia) according to thePromega riboprobe in vitro transcription protocol. The lengths of thecRNA transcripts were 394, 347, and 351 bp for COX-2, renin, and -actin, respectively. Five times 10 5 counts/min of thecRNA probes were hybridized with 20 µg of total RNA (COX-2, renin), 1 µg of total RNA ( -actin), or 20 µg of tRNA (negative control) at60°C overnight and were then digested with RNase A/T1 (RT/30 min) andproteinase K (37°C/30 min). After phenol/chloroform extraction andethanol precipitation, protected fragments (370, 323, and 303 bp inlength for COX-2, renin, and -actin, respectively) were separated onan 8% polyacrylamide gel. The gel was dried for 2 h, bands werequantitated in a PhosphorImager (Instant Imager 2024, Packard), andautoradiography was performed at 80°C for 1-3 days.

Determination of PRA7 a# `& L7 u% G

PRA was determined utilizing a commercially availableradioimmunoassay kit for ANG I (Sorin Biomedica, Düsseldorf, Germany).5 b; a8 f% e' U2 s# {

COX-2 Immunoreactivity
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After fixation in methyl-Carnoy's solution (60% methanol, 30%chloroform, and 10% acetic acid), tissues were dehydrated by bathingin increasing concentrations of methanol, followed by 100%isopropanol. The tissue was embedded in paraffin and 4-µm sectionswere cut with a Leitz SM 2000R microtome (Leica Instruments, Oberkochel, Germany). After deparaffinization, endogenous peroxidase activity was blocked with 3% H 2 O 2 in methanolfor 20 min at room temperature. Sections were layered with the primaryantibody (dilution 1:1,000; COX-2, Santa Cruz) and incubated at 4°Covernight. After addition of the second antibody (dilution 1:1,000;biotin-conjugated, rabbit anti-goat immunoglobulin G), the sectionswere incubated with avidin D horseradish peroxidase complex (VectastainDAB kit, Vector Laboratory) and exposed to 0.1% diaminobenzidinetetrahydrochloride and 0.02% H 2 O 2 as a sourceof peroxidase substrate. Each slide was counterstained withhematoxylin. As a negative control, we used the secondary antibodyonly, without incubation with the primary antibody.$ v. y+ r0 {6 s% `" I" }9 {6 t
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Statistics
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Data are presented as means ± SE. Levels of significancewere calculated by ANOVA followed by Bonferroni's test for multiple comparisons. P significant.6 `3 B9 F$ A$ \' c$ j
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RESULTS

Influence of ACE Inhibition on the Expression of COX-2 and Renin inthe Renal Cortex During Different Salt Diets1 ]" Q2 j" X. N" t% F0 B( U

To obtain information about the efficacy of the differentmaneuvers to modulate the intrarenal renin system, we measured renin mRNA in addition to COX-2 mRNA.; Q0 w% H  N, S3 S2 k( _9 Y  m
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ACE inhibition by ramipril (10 mg · kg 1 · day 1 ) during anormal-salt diet increased the renocortical mRNA abundance of COX-2 andrenin by 125 ± 27 and 300 ± 30% compared with untreatedcontrols (Figs. 1 and 2 ). In parallel with the mRNA, COX-2protein immunoreactivity, semiquantitated as a percentage ofimmunopositive macula densa, also increased, from 5 ± 0.7%positive glomeruli in controls to 18 ± 2.1% positive glomeruliin ramipril-treated rats (Fig. 2 ).
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Fig. 1. Renin mRNA abundance in the renal cortex of rats after ahigh [8% NaCl (wt/wt)]-, normal [0.6% NaCl (wt/wt)]-, andlow-salt diet [0.02% NaCl (wt/wt)], chronic furosemide infusion (60 mg · kg 1 · day 1 ), and renalartery stenosis (clipped and contralateral kidney), respectively, withand without angiotensin-converting enzyme (ACE) inhibition by ramipril(10 mg · kg 1 · day 1 ). cpm,Counts/min. * P n = 7 each.
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Fig. 2. Cyclooxygenase-2 (COX-2) mRNA abundance ( top )and immunoreactivity ( bottom ), as percentage ofimmunopositive glomeruli, in the renal cortex of rats after a high-,normal-, and low-salt diet, chronic furosemide infusion, and renalartery stenosis (clipped and contralateral kidney), respectively, withand without ACE inhibition by ramipril. * P n = 7 each.* y1 h% k0 w& V+ i0 W: c

A low-salt diet [0.02% NaCl (wt/wt)] led to a parallel stimulationof the mRNA expression of both COX-2 and renin, which increased by75 ± 22 and 100 ± 25%, respectively, compared with rats on a normal-salt diet [0.6% NaCl (wt/wt)]. Additional ACE inhibition byramipril further increased the mRNA levels of both COX-2 and renin by325 ± 35 and 400 ± 17% compared with a low-salt diet without ramipril administration (Figs. 1 and 2 ).; z9 ]6 |, I5 O% @$ k- l4 ~( b: q* x
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In parallel with mRNA, COX-2 protein also increased during a low-saltdiet to 17 ± 3 compared with 5 ± 1% immunopositiveglomeruli in controls and was further increased after ramipriltreatment (50 ± 4% of immunopositive glomeruli), as shown inFigs. 2 and 3.
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Fig. 3. Immunohistochemistry for COX-2 in the renal cortex of a rat after anormal-salt diet ( A ) and a low-salt diet in combination withramipril ( B ) in low (×100, left ) and highmagnification (×400, right ). A low-salt diet in combinationwith ramipril led to the highest COX-2 immunoreactivity (arrows) of allexperimental conditions.' W7 s9 D1 w# e2 g' ?/ H4 s/ C

Contrary to a low-salt diet, a high-salt diet [8% NaCl (wt/wt)]decreased the mRNA abundance of both COX-2 and renin to 50 ± 5 and 70 ± 9% of the control levels, respectively. Ramipril incombination with a high-salt diet elevated the mRNA abundance of COX-2and renin to values found during a normal-salt diet (Figs. 1 and 2 ).
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Influence of ACE Inhibition on the Expression of COX-2 and Renin inthe Renal Cortex During Chronic Furosemide Infusion with SaltSubstitution; o3 v1 i; i9 {* U% i# v
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Loop diuretics were recently shown to increase the expression ofCOX-2 and renin in parallel in the renal cortex ( 13, 24 ). We therefore investigated the influence of ACE inhibition on the expression of COX-2 during chronic furosemide infusion with salt substitution. Furosemide infusion (60 mg · kg 1 · day 1 ) increasedthe mRNA abundance of COX-2 and renin by 240 ± 50 and 182 ± 22% compared with control levels, respectively. We also found higherCOX-2 protein levels of 34 ± 5% immunopositive glomeruli compared with 5 ± 1% in untreated animals. Both COX-2 mRNA and protein levels were further increased by ramipril (45 ± 19 and 50 ± 11%, respectively) compared with furosemide treatmentwithout ACE inhibition. In parallel, renin mRNA levels were enhanced by 208 ± 27% compared with furosemide infusion without ramipriltreatment (Figs. 1 and 2 ).
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Influence of ACE Inhibition on the Expression of COX-2 and Renin inthe Renal Cortex During Unilateral Renal Artery Stenosis
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Because not only salt intake and salt transport of the maculadensa but also renal perfusion pressure is known to influence theexpression of COX-2 in the renal cortex ( 9, 12, 21 ), weinvestigated the influence of renal perfusion pressure on the expression of COX-2 in the renal cortex during ACE inhibition.
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By left renal artery stenosis (0.2-mm-inner-diameter silver clip), bothCOX-2 and renin mRNA were increased by 60 ± 20 and 100 ± 25% compared with the level in sham-operated rats, respectively. During ACE inhibition, the mRNA expression of both enzymes was furtherupregulated by 82 ± 20 and 180 ± 31%, respectively. COX-2 immunohistochemistry changed similarly to the mRNA. Thus COX-2 proteinwas also increased in the clipped kidney (28 ± 4% ofimmunopositive glomeruli compared with 7 ± 2% in sham-operatedrats, not shown), and ramipril treatment further increased the COX-2protein abundance to 35 ± 4% immunopositive glomeruli (Figs. 1 and 2 ).

In the contralateral kidney, COX-2 and renin mRNA abundances werereduced to 70 ± 10 and 45 ± 6% of the level insham-operated animals, respectively. COX-2 immunoreactivity in thecontralateral kidney was only slightly reduced (Figs. 1 and 2 ).
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Ramipril treatment during renal artery stenosis increased both corticalCOX-2 mRNA expression and COX-2 immunoreactivity in the contralateralkidney (Fig. 2 )." X# x. m5 Y6 j, E) x! o8 @
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PRA and Relative Enhancement of COX-2 Expression by ACE Inhibition. w- ]) L, J% Q
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In view of the different potencies of ACE inhibition to enhanceCOX-2 expression after ACE inhibition during the different experimentalmaneuvers, we considered the plasma renin activities (PRAs) under thesedifferent conditions to determine a possible interrelationship betweenthe degree of activated renin system as reflected by PRA and therelative enhancement of COX-2 expression in response to ACE inhibition.
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Average PRA was 5 ± 1 ng ANGI · h 1 · ml 1 during anormal-salt diet. A high-salt diet reduced PRA to 35 ± 5%,whereas a low-salt diet increased PRA to 300 ± 20% of thecontrol level. Furosemide infusion elevated PRA sevenfold, which wasthe highest increase in PRA of all maneuvers examined. Renal arterystenosis increased PRA by 300 ± 25% compared with control high-salt diet (Fig. 4 ).
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Fig. 4. Plasma renin activity (PRA; ng ANGI · h 1 · ml plasma 1 ) ofrats after a high-, normal-, and low-salt diet, chronic furosemideinfusion, and renal artery stenosis. * P n = 7 each.

For the relative enhancement of COX-2 mRNA expression by ACEinhibition, we found an order of increases as follows: low-salt renal artery stenosis, clippedkidney renal arterystenosis, contralateral kidney (Fig. 5 ).

Fig. 5. Relative enhancement of COX-2 mRNA expression by ACEinhibition (compared with the respective treatment without ACEinhibition) in relation to PRA and respective pretreatment of a high-,normal-, and low-salt diet, furosemide infusion, and renal arterystenosis (clipped and contralateral kidney). A good correlation( y = 18 18 x, r 2 = 0.99) between the relative COX-2 mRNAenhancement by ACE inhibition and the respective PRA during thepretreatment was observed for experiments in which dietary salt intakewas modulated. No clear correlation between PRA and enhancement ofCOX-2 by ACE inhibition was found for the other conditions( y = 126 2.4 x, r 2 = 0.5). Values are means ± SE.
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Thus despite the only moderately elevated PRA during a low-salt dietcompared with chronic furosemide infusion, ACE inhibition during alow-salt diet led to a six times higher enhancement of COX-2 mRNAlevels than ACE inhibition during chronic furosemide infusion. Theenhancement of the COX-2 expression by ACE inhibition was by farhighest during a low-salt diet.( m; [! f5 K# U( o
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As a consequence, we found a good positive correlation between PRA andthe relative enhancement of COX-2 expression by ACE inhibition for thedifferent salt diets only but not for furosemide treatment or forunilateral renal artery stenosis.9 G- w, G0 `; H" u( c6 V

DISCUSSION+ x- |3 K& T' r7 I1 Q

The purpose of our study was to investigate whether there exists ageneral negative effect of the renin system on the expression of COX-2in the renal cortex of rats. For this purpose, we investigated theinfluence of ACE inhibition under a number of conditions that lead to aparallel regulation of the expression of both COX-2 and renin.

In accordance with previous reports, also in this study a low-salt diet( 8, 11, 22, 25 ), chronic furosemide infusion ( 13 ), and renal hypoperfusion induced by renal arterystenosis ( 9, 12, 21 ) led to increases of renocorticalCOX-2 and renin expression, whereas a high-salt diet and contralateralrenal artery stenosis reduced the expression of both enzymes ( 9, 12, 25 ).+ @" q. q, f3 J+ N% ]  U" S, W

Under all of these conditions, ACE inhibition increased the expressionof renocortical COX-2, regardless of whether the expression wassuppressed, normal, or stimulated by the pretreatment maneuver. Thesedata are in accordance with reports that COX-2 expression is increasedin response to ACE inhibition in rats on a normal- or low-salt diet( 2, 22 ). Our findings fit also with an in vitro studydescribing a direct inhibitory effect of ANG II on the COX-2 expressionof cultured cells of the cTAL of the rabbit ( 2 ). Thus ourdata suggest a general negative effect of the renin-angiotensin-systemon renocortical COX-2 expression.
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Despite the general enhancement of COX-2 expression by ACE inhibition,both the relative and the absolute increases of COX-2 expression inresponse to ACE inhibition were different for the examined pretreatmentconditions. Thus the relative enhancement of COX-2 expression duringACE inhibition was not related to the level of COX-2 expression duringthe pretreatment without ACE inhibition.# k7 b0 _2 w3 v& r# C. {3 U8 ?0 r

Further analysis revealed that the relative enhancement of COX-2expression during ACE inhibition was correlated with PRA only duringmodulations of salt intake but not during treatment with loop diureticsnor during unilateral renal artery stenosis. Such a stimulus-dependentbut PRA-unrelated efficacy of ACE inhibition renders the concept lesslikely that the general negative effect of renin activity on COX-2expression is only mediated by a direct inhibitory effect ofcirculating ANG II on the COX-2-expressing cells. It appears as ifCOX-2 expression becomes more sensitive toward negative regulation byANG II during modulations of salt intake than during changes of therenal perfusion pressure or during changes of salt transport in theloop of Henle. How salt intake, in particular low-salt intake,sensitizes cTAL and macula densa cells toward the actions of ANG IIremains to be clarified. In any case, this mechanism is not specificfor rats, because we also found a strong potentiation of COX-2 geneexpression in the mouse kidney cortex by the combination of a low-saltdiet with ACE inhibition (Wagner, unpublished observations).

For all experimental conditions, we found a striking, almost linear,correlation for the expression of both COX-2 and renin (Fig. 6 ). This correlation continued to existduring ACE inhibition; thus ACE inhibition enhanced the expression ofboth enzymes to the same extent. These data are in good accordance witha number of reports describing a parallel regulation of the expression of COX-2 and renin under a variety of experimental conditions ( 2, 9-13, 16, 19, 22, 25 ). It has therefore been proposed thatCOX-2-derived prostanoids may mediate a stimulation of renin expressionand secretion. This concept is controversially discussed, becauseinhibition of COX-2 activity led in some studies to reduced reninexpression ( 2, 3, 6, 7, 19, 21, 23 ) whereas in otherstudies COX-2 inhibitors did not influence renin expression ( 10, 12, 16-18 ). A second possible explanation for the finding that COX-2 and renin are regulated in parallel under various conditions would be the existence of a common, yet unknown, coregulator of COX-2and renin expression, which is affected by the different treatments andcontrolled by a negative feedback of the renin system.* a1 Y4 r  K* l, J9 _. B2 V# C
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Fig. 6. Correlation between COX-2 mRNA and renin mRNA abundance(values are means ± SE of the respective treatments) for allexperimental conditions examined. (Regression curve: y = 5 0.074 x, r 2 = 0.79).$ ~* s4 e/ {1 l  \2 z

In summary, our findings suggest a general inhibitory effect of ANG IIon the expression of COX-2 in the renal cortex, which becomes mostapparent during modulations of salt intake.2 z2 g2 }) k! {9 B7 O0 K( |. m/ s
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A general enhancement of COX-2 expression by ANG II antagonists leadingto an enhanced formation of prostanoids in the kidney cortex could beof clinical relevance, because prostanoids are relevant for renalperfusion (vasodilation) and for tubular salt and water transport (diuresis).
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ACKNOWLEDGEMENTS

We thank Anna M'Bangui for technical assistance.
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