Journal of the American Society of Nephrology 621

Beneficial Influence of Ketanserin on Autoregulation ofBlood Flow in Post-lschemic Kidneys1Marleen Verbeke, Bernd Sm#{244}llich,Johan Van De Voorde, Leo de Ridder, and Norbert Lameire2

M. Verbeke, L. de Ridder, Laboratory for Histology,University of Gent, Gent, Belgium

B. Sm#{244}llich, Department of Medicine, Wi Pieck Univer-sity, Rostock, Germany

J, Van De Voorde, Department of Physiology andPhysiopathology, University of Gent, Gent, Belgium

N. Lameire, Nephrology Department, University of

Gent, Gent, Belgium

(J. Am. Soc. Nephrol. 1996; 7:621-#{243}27)

ABSTRACTThe influence of ketanserin, a S2-serotonergic recep-tor blocker, on the impaired renal hemodynamics ina clamp model of renal ischemia in rats was investi-gated in this study. Serotonin-induced vasoconstric-tion of the renal vascular bed was augmented after

ischemia. This constriction is blocked by ketanserin

(0.05 mg/kg iv bolus, followed by 0.1 mg/kg per hinfusion). The influence of the same ketanserin treat-

ment on the response of RBF versus a stepwise lower-ing of the renal perfusion pressure was studied inpost-ischemic kidneys with an established loss of au-toregulation of RBF. An almost perfect restoration of

the autoregulatory response was apparent after theS2-serotonergic antagonism. Despite this beneficialeffect on renal hemodynamics, renal function,judged by measurement of GFR and urinary sodium

excretion rate, was not influenced by an acute infu-sion of ketanserin in post-ischemic kidneys. It is sug-

gested that serotonin plays a pivotal role in the sup-pression of autoregulation of RBF by a S2-serotonergicreceptor-mediated vasoconstrictor effect in the post-ischemic kidney. It most likely masks the potentialmyogenic dilatory response of the smooth musclecells in renal preglomerular microvasculature. Resto-ration of the renal autoregulatory capacity by S2-

serotonergic receptor antagonism could be of clini-cal relevance in human post-ischemic acute renal

failure.

Key Words: 5-Hydroxytryptamine, serotonin, vasoconstriction.

arcuate arteries, myogenic response

, Received May 9, 1995. Accepted November 16, 1995.2 correspondence to Dr. N. Lameire, Nephrology Department, university of Gent,

De Plntelaan 185, B-9000 Gent, Belgium.

104&6673/07040621 103.00/0Journal of the American Society of Nephrologycopyright C 1996 by the American Society of Nephrology

R enal biopsies obtained from patients during the

maintenance phase of ischemic acute tubular

necrosis show histological evidence of fresh ischemic

lesions, suggesting recurrent intermittent insults of

renal hypoperfusion ( 1) . Moreover, in the norepineph-

line and renal-artery clamp animal model of post-

ischemic acute tubular necrosis, subsequent hemor-

rhagic blood pressure reduction is associated with

functional and histological evidence of recurrent renal

damage (2,3). Such ongoing ischemic injury has been

explained by a loss of autoregulation of RBF so that

the kidneys are unable to protect themselves against

subsequent falls in renal perfusion pressure (RPP). A

loss of autoregulation of RBF has been reported in a

number of post-ischemic (4-7) and in at least one

model of toxic acute renal failure, induced by cyclo-

sporine (8).

A number of factors playing a possible role in this

loss of autoregulation after acute tubular necrosis

have been defined. In both the clamp and the norepi-

nephrine model, structural alterations in the renal

vasculature were observed (6,9). Moreover, in the

norepinephrine model, increased activity of non-a-

adrenergic mechanisms (10), endothelial cell injury,

and increased renovascular smooth muscle calcium

overload ( 1 1) have been described.

In a previous study, we showed that ketanserin, a

selective serotonin �2 antagonist, not only increased

RBF and GFR in the normal rat kidney, but also

preserved the autoregulation of RBF to perfusion

pressures of 70 to 75 mm Hg. In the normal rat

kidney, autoregulation of RBF is lost below a perfu-

sion pressure of 90 to 95 mm Hg. These data indicate

that a basal serotonin activity influences the hemody-

namics in normal kidneys ( 1 2). In the same study, we

found that serotonin infusion induced a pronounced

vasoconstriction, dependent on S2-receptor stimula-

tion, followed by a less pronounced dose-independent

vasodilation, the latter probably mediated by S1-re-

ceptor activation. Furthermore, in a recent study,

Endlich et aL (1 3) visualized the intrarenal vascular

effects of serotonin. It was found that basal serotonin

S2 activity predominantly constricts the arcuate arter-

ies, whereas serotonin S� activity elicits a vasodilation

in interlobular and afferent arterioles. Moreover, ad-

ministration of serotonin impaired the autoregulation

of GFR.

These data led us to investigate whether an S2-

receptor blocker, ketanserin, is able to influence the

autoregulatory capacity in post-ischemic kidneys. A

possible amelioration of the autoregulation after isch-

emia by serotonin �2 blockade could be of obvious

clinical importance. It was found that the renal vaso-

constrictive response to intrarenal serotonin was aug-

Ketanserin and Repair of Autoregulation

622 Volume 7 . Number 4 . 1996

mented after ischemia. This vasoconstriction could be

blocked by serotonin �2 antagonism. The most impor-

tant observation was, however, that serotonin �2 an-tagonism led to the reappearance of the autoregula-

tion of RBF in post-ischemic kidneys.

METHODS

Laboratory Animals

The studies were performed In male Wistar rats (Iffa Credo,Brussels, Belgium) of 285 to 320 g body weight. All animalexperimentation was conducted in accord with the National

Institutes of Health Guide for the Care and Use of Laboratory

Animals. Twenty-four h before each surgical Intervention,the animals were deprived of food, but received tap water cuiUbitum. They were anesthetized with an ip injection of so-

dium pentobarbital (60 mg/kg: Nembutal: Ceva, Brussels,

Belgium) and placed on a heating table that maintained bodytemperature at 37#{176}C.

Study of the Autoregulation of Renal BloodFlow in Control Conditions

The femoral artery and vein were cannulated with polyeth-ylene PE5O tubings. Subsequently. an isotonic saline solu-tion was infused continuously Into the femoral vein at a rateof 0.0425 mL/min. The femoral arterial pressure was moni-tored with a pressure transducer (Harvard Apparatus, Millis,MA) attached to a recorder (Harvard Apparatus). Only ani-mals with a basal mean arterial blood pressure ofat least 115mm Hg were studied.

The aorta and right renal artery were exposed via a smallabdominal incision. A string was placed around the aortaabove both renal arteries. Both ends of the string werepassed through a fixed metallic tube. By gradual compres-sbon of the aorta, it was possible to lower the RPP by steps of5 mm Hg. A small blood-flow sensor with a inner diameterranging between 0.6 to 0.8 mm (Skalar Medical, Deift, The

Netherlands) was placed on the right renal artery, allowingRBF monitoring by an electromagnetic square wave flowmeter (Skalar Medical) and recorded on a electronic record-ing module (Harvard Apparatus). Calibration of the flowprobe was conducted according to Arendshorst et aL ( 1 4). ForIntrarenal administration of drugs, a catheter was insertedInto the right suprarenal artery, according to a techniquedescribed by Smits et aL (1 5) and as used in our previous

experiments (12). In the autoregulation studies, RPP, consid-ered to be equal to the femoral artery blood pressure, wasgradually reduced to 70 mm Hg by steps of 5 mm Hg, andboth RBF and RPP were continuously recorded. At every levelof RPP, RBF was allowed to equilibrate for at least 2 to 3 min.

Clamping of the Renal Artery

After the first autoregulation curve was obtained, unilat-eral renal ischemia was provoked by clamping the right renalartery for 40 mm. Immediately after clamping, and at regularIntervals, the macroscopic aspect of the clamped kidney wasverified to assure the completeness of the ischemia. Afterrelease of the vascular clamp, and after the completeness ofthe renal reperfusion was visually observed, the femoralartery and vein were closed and the Incisions sutured. Therats recovered with free access to food and water.

Study of the Autoregulation of Renal BloodFlow in Post-lschemic Conditions

The animals were restudied 48 h or 7 days after renalreperfusion. They were reanesthetized, a tracheotomy wasperformed, and catheters were placed in the intact femoralvein and artery. The vein was used for systemic administra-tion of either isotonic saline or ketanserin (Janssen Pharma-ceutica, Beerse, Belgium). The arterial blood pressure wasmeasured in the femoral artery. and again only animals witha systemic blood pressure of at least 1 1 5 mm Hg werestudied. The abdominal incision was reopened, and the aortaand right renal artery were freed from surrounding tissueand prepared for the study of autoregulation of RBF as

described above. After confirmation of the loss of autoregu-lation of RBF in this second curve, different study protocols

followed.

Study Protocols for Post-lschemic Kidneys withan Established Loss of Autoregulation of RenalBlood Flow

1. Dose-response studies. Bolus injections of serotonin(5-hydroxytryptamine: Sigma Chemical Company, St. Louis,MO) were delivered into the right renal artery while RBF wascontinuously measured. Increasing doses of serotonin (0.3,0.5, 0.75, and 1 jtg) were injected before and during ketan-scm administration (bolus of 0.05 mg/kg, followed by asustained infusion of 0. 1 mg/kg per h. dissolved in Isotonicsaline at a rate of0.0425 mL/mln: N = 6) or the same amountof saline (N = 6) as described for the autoregulation studiesin the normal kidneys (12). Dose-response curves were stud-

ied 48 h after reperfusion.To exclude eventual enhanced effects ofintrarenai doses of

serotonin because of the decreased basal RBF in the post-ischemic kidneys, the dose-response study was repeated insix additional animals, using doses lowered to a similarextent as the RBF (80% of the former serotonin doses).

To be sure that the dose of ketanserin used in the presentexperiments on post-ischemic kidneys was devoid of a-ad-renergic blocking activity, as demonstrated in our formerstudy on normal kidneys (12), dose-response curves to intra-renal phenylephrine, a pure a-agonist (0. 1 , 0.3, 0.5 and 0.7�kg: Sigma Chemical Company, St. Louis, MO), were exam-

med before and during ketanserin administration.2. Clearance studies. Standard clearances were per-

formed, as described previously (12), at 48 h (N = 15) and 7days (N = 12) of reperfusion, before and during ketanserinadministration (see Protocol 1). In brief, the anesthetizedanimals were iv-infused with saline at a rate of 0.0425mL/min. A pressure transducer was connected to the carotidartery for continuous measurement of mean arterial pres-sure. Inuline (3.3%) was then added to the Infusion andadministered during 1 h before further urine and bloodsampling. In the meantime, both urethers were catheterized(PE- 10). Urine of the left and right kidney was collected

separately during three consecutive periods of 10 rain while

the iv infusion with Inuline continued. In the middle of thefirst and third urine collection, a blood sample was taken.

Three clearance collections were made before and during theketanserin administration. Inulin was measered in urine andserum samples with the anthrone method ( 1 6): sodium was

determined by means of a Klina flame photometer (BeckmanInstruments, Fullerton, CA).

3. Autoregulation. Ketanserin: This protocol involved twosubgroups of animals. A first group of 15 rats was studied 48

Verbeke et al

Journal of the American Society of Nephrology 623

h after renal reperfusion. Ketanserin was applied as de-

scribed in Protocol 1 . In these circumstances, renal vasocon-

trictive response to increasing doses of serotonin is blocked,although the vasodilatory response is well preserved (12:present results). After 45 to 60 mm of the ketanserin infu-sion, a third autoregulation curve was obtained while theinfusion was continued. In the second subgroup of six ant-mals, the same protocol was performed 7 days after kidneyreperfusion.

Saline: A group of animals (N = 10) was studied as

described for the ketanserin protocol, except that an isotonicsaline solution at a rate of 0.0425 mL/min was administeredbefore and during the third autoregulation analysis. Sixanimals were studied after 48 h and four animals after 7 days

of kidney reperfusion.

Morphological Study

After the last autoregulation curve, the kidneys of six ratsat 48 h of reperfusion were perfusion-fixed at a constant

pressure of 120 mm Hg by retrograde aortic perfusion with

1.25% glutaraldehyde in 0. 1 M cacodylate buffer (17). Thekidneys were perfused until a complete washout of the

vascular tree was observed, and processed for light micros-copy and electronmicroscopy.

Kidney slices were further immersion-fixed in buffered

formalin and embedded in paraffin. Five-jzm sections were

stained with hemalum-eosin and studied with a Jenavallichtmicroscope (Jena, Germany).

Thin cortical fragments were further immersion-fixed in2% glutaraldehyde in the same buffer, postfixed in OsO4, andembedded In ERL resin (Serva, Heidelberg, Germany). Afterstaining with uranyl and lead, ultrathin sections were exam-med by means of a Jeol 1 200 EX2 electronmicroscope(Tokyo, Japan).

Statistical Analysis

The results are given as the means ± SE. Comparison

between absolute RBF data were analyzed by the paired orunpaired Wilcoxon’s test. Analysis of the autoregulatloncurves was performed by linear regression analysis of theRPP to RBF data. Differences in results in each protocol werecompared by analysis of variance of the mean slope of theregression lines. The results of the dose-response studieswere compared by means of a t test for unpaired observa-tions. A P value of < 0.05 was considered to be significant.

RESULTS

Dose-Response Studies

Table 1 depicts the percentage changes in RBF in

response to increasing doses of intrarenal serotonin in

both control and ischemic kidneys, 48 h after renal

reperfusion. The mean absolute RBF of the clamped

kidneys after 48 h of reperfusion, as measured in the

six rats subjected to a dose-response analysis, was

3.97 ± 0.43 mL/min. As in previous experiments incontrol rats (12), intrarenal serotonin elicited a bipha-

sic response of RBF: a pronounced dose-dependent

vasoconstriction was followed by a dose-independent

vasodilation. The dose-response curve of RBF in isch-

emic kidneys was significantly different from the con-

trol curve at each dose ofserotonin. Both the vasocon-

strictive and vasodilating responses to serotonin were

clearly enhanced after ischemia. As in control kidneys

(12), ketanserin completely blocked the serotonin-

induced vasoconstriction, but did not influence the

vasodilatory component.

Table 2 represents the responses to serotonin at

doses lowered to 80%, to compensate for the decrease

ofthe basal RBF (4.92 ± 0.31 mL/min in control rats;

see Reference 1 2). The serotonin-induced vascular

responses to these lowered doses were still signifi-

cantly enhanced in post-ischemic kidneys versus con-

trol kidneys.

The responses to intrarenaily applied, augmenting

doses of phenylephrine were not altered by ketan-

serin, demonstrating that the ketanserin treatment

used is devoid of a-adrenolytic activity (Table 3).

Autoregulation Studies

Figure 1 represents the influence of ketanserin on

the autoregulation curves in rats, 48 h after reperfu-

sion. Before ischemia, normal autoregulation of RBF

was observed in 12 rats. The linear regression line

between 1 15 and 95 mm Hg in RPP is y = 0.005x +

4.32 (r� - 0.95). The difference in RBF at RPP of 115

mm Hg and 95 mm Hg, respectively, was only 2%,

(4.86 ± 0.44 mL/min versus 4.77 ± 0.45 mL/min). A

complete loss of autoregulation was observed in all

animals 48 h after reperfusion. The slope of the re-

gression line after 48 h between 1 1 5 and 75 mm Hg

was significantly different from the slope of the control

curve between 1 1 5 and 95 mm Hg. During ketanserin

infusion, the RBF at 1 15 mm Hg had a tendency to

increase (from 3.73 ± 0.75 mL/min before to 4.31 ±

0.80 mL/min during ketanserin infusion; not signifi-

TABLE 1 . Dose response to usual doses of intrarenal serotonin in control and post-ischemic kidneys, 48h afterreperfusion#{176}

Bolus Serotonin (hg)vC

0.3

VD

0.5

VC VD

0.

VC

75

VD

1.0

VC VD

Controlkidneys 18±4 7±2 21±5 10±3 33±5 12±4 40±4 12±4Post-ischemic kidneys 23 ± 5 21 ± 4b 40 ± 6b 25 ± 2b 51 ± 6b 32 ± 3b

59 � 7b 35 �

Post-ischemic kidneys (during ketanserin) 0b 19 ± 3b0b 25 ± 5b

0b 28 ± 5b 5 � 4b34 � 5b

a The biphasic response is represented as a mean percentage of RBF decrease and a mean percentage of RBF increase versus the basal RBF

(± SE). VC. vasoconstriction; VD, vasodilatation.b p < 0.05 versus control kidneys (N = #{243}).

5.6

C

�4.4

E�3.2

0,

5.61-

5.0

4.4

3.8

3.2

C

E

ELA-

..1...� 4�.4� �

Ketanserin and Repair of Autoregulation

624 Volume 7 ‘ Number 4 ‘ 1996

TABLE 2. Dose response to usual doses of intrarenal serotonin in control and post-ischemic kidneys, 48 h afterreperfusiona

Bolus Serotonin 20%Lowered (�Lg)

0.25 0.4 0.6 0.8

VC VDVC VD VC VD VC VD

Controlkidneys 5±2 3± 1 17±3 6±2 26 ±4 8±3 34±5 9±2Post-ischemic kidneys 17 ± 3b 14 ± 4b 30 ± 5b

17 ± 4b 41 ± 5b 22 ± 3b 48 ± 7 25 ± 5b

a The biphasic response is represented as a mean percentage of RBF decrease and a mean percentage of PBF increase versus the basal PBF

(:t: SE). VC, vasoconstriction; VD, vasodilatation.b p < 0.05 versus control kidneys (N = 6).

TABLE 3. Response of intrarenal bolus administration of phenylephrine on RBF (as a mean percentage ofdecrease of the basal RBF), before and during ketanserin administration in post-ischemic kidneys after 48 hand 7 days of reperfusion

Bolus Phenylephrlne (��g) 0.1 0.3 0.5 0.7

48 h reperfusion (N = 4)Before ketanserin 24.6 ± 5.5 50.7 ± 7.0 66.5 ± 7.8 76.7 ± 6.3During ketanserin 21.2 ± 2.4 45.3 ± 4.3 70.9 ± 3.9 79.8 ± 4.7

7 days reperfusion (N = 6)Before ketanserin 22.6 ± 2.8 43.3 ± 5.3 61.5 ± 6.3 69.4 ± 6.7During ketanserin 24.9 ± 2.8 47.2 ± 4.7 58.7 ± 4.5 68.1 ± 5.0

. I 1 1 1 1 1 1 111

75 85 95 105 115

R PPmmHg

Figure 1 . El: autoregulation of RBF in pre-ischemic kidneys; �:RBF versus RPP in posf-ischemic kidneys, 48 h of reperfuslon;0: reappearance of autoregulation of RBF in post-ischemickidneys, 48 h of repertusion, effect of ketanserin infusion (N =

15).

cant INS]). At a pressure of95 mm Hg, RBF decreased

only with 4.5% from the control value at 1 15 mm Hg.

The slope of the regression line between 1 1 5 and 95

mm Hg during ketanserin (y = 0.OlOx + 3. 176; r� =

0.981) is not significantly different from the slope of

the pre-ischemic control regression line.

In another six animals, studied before ischemia and

after 48 h of renal reperfusion, it is shown that the

infusion of isotonic saline, instead of ketanserin, does

not alter the linear regression line obtained before the

saline infusion. The regression line is linear between

1 15 and 75 mm Hg: y = 0.028x + 0.502, (r� = 0.99).The slope of this regression line is significantly differ-

ent from the slope before ischemia (P < 0.00 1 ; results

not shown).

Figure 2 depicts the results after ketanserin addi-

tion, at 7 days of reperfusion. All six animals showed

a complete loss of autoregulation. Infusion of ketan-serin completely restored autoregulation of RBF in a

similar way as in the experiments performed after 48 h

of reperfusion. The regression line of RBF at RPP

between 1 1 5 and 75 mm Hg after reperfusion was y =

0.022x + 1 .505 (r� = 0.99). During ketanserin, the

regression line between RPP 1 1 5 to 95 mm Hg was y =

0.009x + 3.068 (r� = 0.92). The differences in slopes

between these regression lines were highly significant

(P< 0.001).

Infusion of saline in these animals had no influence

on the autoregulation capacity. The slope of the re-

gression line during saline infusion is not different

from the slope before infusion. The results obtained in

I1 � � � � I I I I I I

75 85 95 105 115RPP (mmHg)

Figure 2. L�: RBF versus RPP in post-ischemic kidneys, 7 days ofreperfusion; 0: reappearance of autoregulation of RBF inpost-ischemic kidneys, 7 days of reperfusion, effect of ketan-serin infusion (N = 6).

Verbeke et al

Journal of the American Society of Nephrology 625

the four rats studied after 7 days of reflow were verysimilar to those studied after 48 h of reperfusion(results not shown).

Clearance Studies

At 48 h of reperfusion, 1 1 of the 1 5 post-ischemic

kidneys were anuric. The GFR, diuresis, and FEi�,�a in

the four non-anuric kidneys were 0. 15 ± 0.04 mL/

min, 1.52 ± 0. 15 pL/mln, and 0. 14 ± 0.02% respec-

tively. At 7 days of reperfusion, no post-ischemic

kidneys were anuric. The ketanserin infusion lowered

the MAP, but did not alter renal function significantly.

The clearance results before and during ketanserin

administration are summarized in Table 4.

Morphological Study

In lightmicroscopic sections, necrotic lesions are

visible in the tunica media of arcuate and interlobular

arteries of post-ischemic kidneys. These findings are

confirmed electronmicroscopicaily: condensed nuclei

are observed in small cortical arteries as an indication

of myonecrosis (Figure 3).

DISCUSSIONThe most interesting finding of this study is the

beneficial effect of the S2-serotonin antagonist ketan-

scm on the autoregulation of RBF in the post-isch-

emic rat kidney: after ketanserin administration, this

autoregulation reappeared almost perfectly at 48 h or

at 7 days of renal reperfusion.

The normal renal autoregulatory response to a re-

duction in perfusion pressure is independent of renal

innervation and accomplished by an intrinsic myo-

genic vasorelaxation (18) of predominantly the affer-

ent arterioles, and, to a lesser extent, of the interlob-

ular arterioles (19) and the arcuate arteries (20). Only

in the later phase of autoregulation is tubuloglomeru-

lar feed-back activated (21). The study presented here

confirms previously published data that showed that

the normal autoregulatory response of RBF to a re-

Figure 3. Electronmicroscopic picture of a pycnotic nucleus(arrow) in the tunica media of an arcuate artery from apost-lschemic kidney, 2 days of reperfusion (bar = 1 �m; 1:tunica intima; 2: tunica media; 3 : tunica adventitia).

duction in RPP is lost after an unilateral renal isch-

ernic insult (4-7,9, 1 1). Several mechanisms can be

offered to explain the loss of autoregulation of RBF.

The most obvious explanation is a direct ischemic

TABLE 4. Mean values (± SE) of the parameters, obtained during clearances of post-ischemic right kidneysafter 48 h or 7 days of reperfusion, and left non-lschemlc kidneys before and during ketanserin administration

ParameterBefore Ketanserin During Ketanserin

Right Left Right Left

48 h of reperfusion (N = 15)GFR (mL/min) 0.04 ± 0.02 1.61 ± 0.09 0.04 ± 0.02 1.59 ± 0.09Dluresls (�L/min) 0.40 ± 0.18 7.09 ± 0.71 0.31 ± 0.14 6.21 ± 0.57FENa (“#{176}) 0.04 ± 0.01 0.08 ± 0.03 0.03 ± 0.01 0.07 ± 0.02MAP (mm Hg) 113 ± 4 100 ± 6 (N = 7)#{176}

7 days of reperfuslon (N = 12)

GFR (mi/mm) 0.27 ± 0.04 1.76 ± 0.16 0.24 ± 0.05 1.53 ± 0.14Diuresis (�L/min) 2.01 ± 0.16 5.18 ± 0.48 1.78 ± 0.1 7 4.96 ± 0.41FENO(%) 2.9±2.55( N=4) 0.04±0.03(N=6) 2.34± 1.7( N=4) 0.09±0.04(N=6)MAP (mm Hg) 108 ± 2 94 ± 3�

a p < 0.05 before versus during ketansenn.

Ketanserin and Repair of Autoregulation

626 Volume 7 . Number 4 ‘ 1996

insult to the wall of the renal microvasculature. Vas-

cular smooth muscle necrosis has indeed been de-

scribed in previous papers studying the renal artery

clamp model (6,9) and this finding is confirmed in the

study presented here. In this model, besides the mor-

phological evidence, an impaired vasodilatory re-

sponse to both endothelium-dependent and -indepen-

dent agents was found, also suggesting a predominant

smooth muscle cell damage (9).

Serotonin is a known renal vasoconstrictor in the

rat (12, 13,22,23) and the contraction is the result of

the activation of �2 serotonergic receptors on the

plasma membrane of the vascular smooth muscle cell

(24). Besides its vasoconstrictor action, serotonin also

elicits a vasodilatory response mediated by the sero-

tonin � receptors, as was shown in the canine (25)

and the rat kidney (12,23). Ketanserin is a S2-seroton-

ergic receptor blocking agent (26). It is relevant to note

that ketanserin also has a-adrenergic blocking char-

acteristics, besides its anti-serotonergic action. The

dose applied in this study, however, is devoid of

a-blocking actions: this dose does not reduce the RBF

response to increasing doses of intrarenally adminis-

tered phenylephrine, either in normal kidneys (12), or

in post-ischemic kidneys (present study).

There is evidence that normal renal hemodynamics

are influenced by serotonin. This is indicated by the

fact that antagonism of serotonin S2-activlty, by ap-

plying the same ketanserin-treatment as used in this

study, increases RBF and GFR in rats. Ketanserin also

maintains the autoregulation of RBF and GFR of the

normal rat kidney in pressure ranges below the nor-

mal autoregulation area ( 1 2). The role of serotonin in

normal renal hemodynamics can also be deduced

from the work of Endlich et al. (13). They visualized

serotonin effects on the preglomerular vessels of hy-

dronephrotic kidneys and showed that autoregulation

of glomerular blood flow disappears after the addition

of serotonin.

In the post-ischemic kidneys of this study, RBF

shows a tendency to rise during the infusion with

ketanserin, although not to a significant degree. On

the other hand, GFR, FENa and diuresis are not al-

tered during this acute administration of ketanserin.

The majority of the clamped kidneys remained anuric,

48 h after reperfusion. GFR was restored to approxi-

mately 25 to 30% ofnormal at 1 wk ofreperfusion, but

renal function was not influenced by the acute admin-

istration of the �2 antagonist.

The fact that the lost renal autoregulation response

in post-ischemic rats can be reversed by a 45 to

60-mm infusion of an �2 antagonist might imply that

the intrinsic myogenic response is not damaged, but

only overwhelmed by a stronger vasoconstrictive in-

fluence. This contractile action might be the conse-

quence of the enhanced serotonin �2 activity in the

post-clamp kidneys. An argument in favor of this

hypothesis is the increased vasoconstrictory response

to serotonin in the post-ischemic kidneys, compared

with the response in control kidneys. This increased

vasoconstriction persisted even when serotonin doses

were corrected for the RBF decrease after ischemia.

According to Endlich et al. (1 3), the constriction of

the arcuate arteries in the presence of serotonin is the

limiting factor for the adaptation of renal vascular

resistance to a lowering of RPP in the non-ischemic

kidney. The restrictive influence of serotonin on the

autoregulation might thus be a result of its contractile

effect on arcuate arteries. Yet, the fact that serotonin

�2 antagonism prolongs the autoregulation reactiondown to 70 to 75 mm Hg in a normal rat kidney (12)

might be a result of the blockade of the predominant

constriction ofthe arcuate arteries because ofbasal �2

receptor stimulation, inhibiting autoregulation at a

RPP lower than the normal threshold of 90 to 95 mm

Hg. The autoregulation is not prolonged down to 70 to

75 mm Hg by �2 antagonism in the post-ischemic

kidneys, as observed before in control rat kidneys ( 12).

It can be hypothesized that this is because of vascular

smooth muscle cell damage. Damage was shown mor-

phologically under the form of focal necrosis of the

vascular smooth muscle cells in the arcuate and

interlobular arteries.

It is interesting to note that besides the smooth

muscle cell damage, endothelial injury can also play a

role in the clamp model of renal ischemia (9, 1 1). This

could make the renal vessels more vulnerable to the

constrictive influence of serotonin. Almost all of the

serotonin in the blood is contained in the platelets and

is released when platelets aggregate (27). Vascular

sensitivity to serotonin, which is released from aggre-

gating platelets, is enhanced when the endothelium is

damaged (28). HypoxIa-induced endothelial dysfunc-

tion is believed to augment the contractile effect of

platelet products, further reducing the luminal area

and increasing the obstruction to blood flow (24).

Morever, after ischemia (e.g., in patients with periph-

eral arterial obstructive diseases or after an acute

myocardial infarction), a serotonin-induced platelet

aggregation could be observed, suggesting a potential

vicious circle of subsequent vasoconstrictions (29).

However, in the kidney, the platelets might not be the

only source of serotonin, as it is shown that non-blood-perfused kidneys (30) and proximal tubules (31)

are able to synthetize this agent. From all of this, it is

thus tempting to speculate on a serotonergic influence

as a mechanism not only determining the pressure

range of autoregulation of RBF in normal kidneys, but

also contributing to loss of autoregulation in post-

ischemic rat kidneys.

The serotonergic influence in post-ischemic kidneys

could be more pronounced because ofan upregulation

of S2-receptor number and/or sensitivity, or to an

increased concentration of serotonin in the post-isch-

emic kidney. Furthermore, it is known that threshold

amounts of serotonin can enhance the vasoconstric-

tor response to other substances, e.g. , norepineph-

rine, epinephrine, thromboxane A�, prostaglandin

F2a, angiotensin II (32), and of endothelin (33), all

agents that exert important effects on renal hemody-

Verbeke et al

Journal of the American Society of Nephrology 627

namics. Also, these indirect effects are antagonized by

ketanserin (32).

In conclusion, the reappearance of the autoregula-tory response in a post-ischemic kidney of a ketan-

serin-treated rat, together with an increase in seroto-

mn-induced renal vascular resistance, suggests an

enhanced serotonin S2-receptor-mediated effect in in-

trarenal arteries after an ischemlc insult. This sugges-

tion implies that serotonin could play a key role in

determining the effective renal autoregulatory range.

In normal conditions, this might limit the expression

of autoregulation at a RPP lower than 95 mm Hg; in

the post-ischemic conditions of the present experi-

ments, it could completely eliminate the autoregula-

tion. The finding of a pivotal role played by serotonin

in the expression of autoregulation in post-ischemic

conditions could be of important clinical relevance.

Serotonin �2 antagonism might lead to an interrup-

tion of the chain of recurrent injuries because of the

loss of autoregulation after an initial insult and/or to

a quicker repair process.

ACKNOWLEDGMENTSThis study was supported by the University of Gent Research Fund.Grant No 01 1 16492. Special acknowledgement is given to D. Kes-teloot, M. -A. Waterloos. T. Dheuvaert. M. Dhont, and A. Vermaerke for

the excellent technical assistance.

REFERENCES1 . Solez L, Morel-Maroger L, Sraer J: The morphology of

acute tubular necrosis in man: Analysis of 57 renalbiopsies and comparison with glycerol model. Medicine(Baltimore) 1979:58:362-376.

2. Kelleher SP, Robinette JB, Miller F, Conger JD: Effect ofhemorrhagic reduction in blood pressure on recoveryfrom acute renal failure. Kidney mt 1987;3l:725-730.

3. Conger JD, Schultz MF, Miller F, Robinette JB: Re-sponses to hemorrhagic arterial pressure reduction indifferent ischemlc failure models. Kidney Int 1994:46:318-323.

4. Adams PL, Adams P, Bell PD, Navar LG: Impaired renalblood flow autoregulation In ischemic acute renal failure.Kidney Int 1980:18:68-76.

5. Williams RH, Thomas CE, Navar LG, Evans AP: Hemo-dynamic and single nephron function during the main-tenance phase of ischemic acute renal failure In the dog.Kidney Int 1981:19:503-515.

6. Matthys E, Patton M, Osgood R, Venkatachalam M,Stein J: Alterations In vascular function and morphologyin lschemic autoregulation. Kidney Int 1983:23:7 17-724.

7. Robinette JB, Conger JD: Angiotensin and thromboxanein the enhanced renal adrenergic nerve sensitivity ofacute renal failure. J Chin Invest 1990:86:1532-1539.

8. Kaskel FJ, Devarajan P, Arbeit LA, Moore LC: Effects ofcyclosporine on renal hemodynamics and autoregulationin rats. Transplant Proc 1988:20:603-609.

9. Conger JD, Robinette JB, Hammond WS: Differences invascular reactivity in models of ischemic acute renalfailure. Kidney mt 1991:39:1087-1097.

10. Kelleher SP, Robinette JB, Conger JD: Sympatheticnervous system in the loss of autoregulation of ischemicacute renal failure. Am J Physiol 1984:246:F379-F386.

1 1 . Conger JD, Robinette JB, Schrier RW: Smooth musclecalcium and endothelium-derived relaxing factor in theabnormal vascular responses of acute renal failure. JChin Invest 1988:82:532-537.

12. L,ameire N, Matthys E, Kesteloot D, Waterloos MA:Effect of a serotonin blocking agent on renal hemody-

namics in the normal rat. Kidney Int 1990:38:823-839.13. Endlich K, Kuhn R, Steinhausen M: Visualization of

serotonin effects on renal vessels of rats. Kidney Int1993:43:314-323.

14. Arendshorst WJ, Finn WE, Gotschalck CW: Autoregu-lation of blood flow in the rat kidney. Am J Physbol1975:228:F127-F133.

15. SmitS JFM, Kesbergen CM, Van Essen H, Klelnjans JC,Struycker-Boudier HAJ: Chronic local infusion into therenal artery of unrestrained rats. Am J Physiol 1983:244:H304-H307.

16. Fflhr J, Kaczmarczyk J, Kruttgen CD: Eine einfachecolorimetrische Methode zum Inulinbestimmung f#{252}rNierenclearance-Untersuchungen bei Stoffwechselge-sunden und Diabetikern. KlIn Wochenschr 1955:33:729-730.

17. Donohoe JD, Venkatachalam MA, Bernard DB, Levin-sky GN: Tubular leakage and obstruction after renalischemla: Structural functional correlation. Kidney Int1978:43:314-323.

18. Hura C, Stein JH. Renal blood flow. In: Windhager EE,Ed. Handbook of Physiology. Section 8: Renal physiol-ogy. New York: Oxford University Press: 1992:1129-1184.

19. Casellas D, Moore LC: Autoregulation of intravascularpressure in preglomerularjuxtamedullary vessels. Am JPhysiol 1993: 264:F315-F321.

20. Stelnhausen M, Blum M, Fleming JT, Holtz FG, ParekhN, Wiegman DL: Visualization of renal autoregulation inthe split hydronephrotic kidney ofrats. Kidney mt 1989:35:1151-1160.

2 1 . Holstein-Rathlou NH, Wagner AJ, Marsch DJ: Tubulo-glomerular feedback dynamics and renal blood flowautoregulation in rats. Am J Physiol 199 1 :260:F53-F68.

22. Collis MG, Vanhoutte PM: Vascular reactivity of isolatedperfused kidneys from male and female spontaneouslyhypertensive rats. Circ Res 1977:41:757-767.

23. Verbeuren TJ, Mennecier P, Laubie M: 5-Hydroxytryp-tamine-induced vasodilatation in the isolated perfusedrat kidney: Are endothelial 5-HT1A receptors involved?Eur J Pharmacol 1991:201:17-27.

24. Vanhoutte PM, Houston DS: Platelets, endothelium, andvasospasm. Circulation 1985:72:728-734.

25. Blackshear JL, Orlandi C, Hollenberg NK: Serotoninand the renal blood supply: Role of prostaglandins andthe 5HT-2 receptor. Kidney Int 1986:30:304-3 10.

26. Van Neuten JM, Janssen PAH, Van Beck J, XhonneuxR, Verbeuren TJ, Vanhoutte PM: Vascular effects ofketanserin (R 41468), a novel antagonist of 5-HT2 sero-tonergic receptors. J Pharmacol Exp Ther 1981:218:217-230.

27. Crawford NV: Systemic venous platelet-bound andplasma free serotonin levels in non-carcinoid malig-nancy. Clin Chim Acta 1965: 12:274-278.

28. Cohen RA, Shepherd JT, Vanhoutte PM: Inhibitory roleof the endothelium in the response of isolated coronaryarteries to platelets. Science (Washington DC) 1983:221:273-274.

29. Dc Cree J, Leempoels J, Demoen B, Rods V. VerhaegenH: Effect of ketanserin on the hyperreactivity of plateletsto 5-hydroxytryptamine in patients with cardiovasculardiseases. J Cardbovasc Pharmacol 1985:7[Suppl7I:S26-528.

30. Stier CT, McKendall G, Itskovitz HD: Serotonin forma-tion in non blood-perfused rat kidneys. J Pharmacol ExpTher 1984:228:53-56.

3 1 . Sole MJ, Madapaui Matam M, Baines AD: An activepathway for serotonin synthesis by renal proximal tu-bules. Kidney mt 1986:29:689-694.

32. Janssens WJ, Van Neuten JM. Amplifying effect of sero-tonin in the control of vascular tone. In Paolettl R,Vanhoutte PM, Brunello N, Maggi FM, Eds. Serotonin:From Cell Biology to Pharmacology and Therapeutics.Dordrecht: Kluwer Academic Publishers: 1990:69-76.

33. Yang BC, Nichols WW, Lawson DL, Mehta JL: 5-Hy-droxytryptamine potentiates vasoconstrictor effect of en-dothelin- 1 . Am J Physiol 1992:262:H93 1-H936.