Beneficial Effect of Serotonin 5-HT2 -Receptor Antagonism on Renal

J Am Soc Nephrol 10: 28–34, 1999

Beneficial Effect of Serotonin 5-HT2-Receptor Antagonism on Renal Blood Flow Autoregulation in Cyclosporin-Treated Rats

MARLEEN VERBEKE,* JOHAN VAN DE VOORDE,† LEO DE RIDDER,* and NORBERT LAMEIRE‡ *Laboratory for Histology, †Department of Physiology and Physiopathology, and ‡Renal Division, Department of Internal Medicine, University of Gent, Gent, Belgium.

Abstract. Renal blood flow (RBF) autoregulation reappears in tion acutely reappeared. Intrarenal bolus injections of a selec- postischemic rat kidneys during serotonin (5-HT2) antagonism. tive 5-HT2-agonist, 2,5 dimethoxy-4-iodoamphetamine hydro- The aim of the present study was to analyze whether 5-HT2 chloride, elicited a significantly stronger renal vasocontraction antagonism can ameliorate impaired RBF autoregulation in rats in cyclosporin A-treated rats than in control rats. This finding treated with 20 mg/kg per d cyclosporin A during 10 d. was also observed with serotonin after nitric oxide-synthase

Autoregulation of RBF was assessed during stepwise lowering blockade. These results (1) show the importance of 5-HT2- of renal perfusion pressure from 110 to 70 mmHg by gradual receptor-mediated vasoconstriction in the suppression of vaso- compression of the aorta. Autoregulation was lost in the cy- dilatory autoregulation of RBF in experimental cyclosporin closporin A-treated rats. During administration of the 5-HT2 A-induced renal dysfunction and (2) demonstrate that the com- antagonist ritanserin (0.6 mg/kg intravenous bolus, followed by plete loss of RBF autoregulation is not due to damage of the 1.2 mg/kg per h intravenous infusion during 1 h), autoregula- vascular smooth muscle cells.

Serotonin (or 5-hydroxytryptamine, 5-HT) in the circulation is (14–18). Therefore, platelet aggregation and thrombus forma- contained within the platelets. In addition, de novo synthesis of tion may occur, followed by a pronounced 5-HT release. This 5-HT in the kidney has also been described (1,2). 5-HT is finding is supported by observations of both glomerular and known to exert a dual influence on vascular tone. Stimulation nonglomerular vascular thrombosis in cyclosporin A-treated of 5-HT2 receptors on smooth muscle cells elicits vasoconstric- patients (19,20) and by the demonstration of increased aggre- tion. This response is either direct or is provoked by amplifi- gation and 5-HT release from human platelets, preincubated cation of vasoconstrictive effects of other agents (3). On the with cyclosporin A (21). other hand, activation of endothelial 5-HT1-receptors leads to There is evidence that 5-HT is important for the effective vasodilation, a nitric oxide (NO)-mediated indirect effect (4). expression of autoregulation of RBF in normal rats (22,23). A

Endothelial NO prevents platelet adhesion and aggregation beneficial effect of the 5-HT2-antagonist ketanserin on the loss and, hence, thrombus formation and further 5-HT-release (4). of RBF autoregulation was found in a renal artery clamp model Immunosuppressive treatment with cyclosporin A is associ- of ischemic acute renal failure in rats, i.e., administration of ated in both humans and animals with a number of complica- ketanserin resulted in an acute reappearance of the autoregu- tions, including vasculotoxicity and nephrotoxicity. Chronic lation while lowering renal perfusion pressure (RPP) (24). administration of cyclosporin A to rats leads to a decrease of In rats, a positive correlation between whole blood cyclo- renal blood flow (RBF) and GFR (5–8). Moreover, loss of sporin A and 5-HT levels has been found, as well as an inverse autoregulation of RBF after cyclosporin A treatment has been correlation between whole blood cyclosporin A and renal cor- observed in rats (9,10). Absence of the vasodilatory autoregu- tical blood flow (25). More recently, functional and morpho- lation makes the kidney vulnerable to hypoperfusion damage logic renal protection against cyclosporin A-induced nephro- and renders it more susceptible to ischemia. A contribution of toxicity by ketanserin was reported in rats (26). The potential ischemia to cyclosporin A-induced renal damage has often role of 5-HT2-receptor activation in the suppression of auto- been suggested (5,11–13). regulation after cyclosporin A treatment was never studied. Cyclosporin induces endothelial damage in vivo and in vitro The present study was designed to explore this hypothesis in a rat model of cyclosporin A-induced nephrotoxicity.

Received December 31, 1997. Accepted July 10, 1998. This work was presented in part at the spring meeting of the Dutch Society for Materials and Methods Nephrology (1996). Correspondence to Dr. Norbert Lameire, Nephrology Department, University Laboratory Animals Hospital, De Pintelaan 185, B-9000 Gent, Belgium. Phone: 32 9 240 4526; The studies were performed in male Wistar rats (Iffa Credo, Brus- Fax: 32 9 240 4599; E-mail: [email protected] sels, Belgium) of 285 to 320 g body wt. All animal experiments were 1046-6673/1001-0028$03.00/0 conducted in accord with National Institutes of Health Guide for the Journal of the American Society of Nephrology Care and Use of Laboratory Animals. Rats received 20 mg/kg per d Copyright © 1999 by the American Society of Nephrology cyclosporin A (Sandimmun, Sandoz, Basel, Switzerland, oral suspen- J Am Soc Nephrol 10: 28–34, 1999 Cyclosporin, Serotonin, and Autoregulation 29

sion) by oral gavage during 10 consecutive d. Animals of the control arginine (L-NNA, bolus of 0.1 mg/kg; Sigma) in control (n ϭ 4) or group were treated for an identical period with an equivalent amount cyclosporin A-treated rats (n ϭ 8).

(0.05 to 0.06 ml) of the solvent, olive oil. At day 10, the rats were Dose–response curves to a selective 5-HT2-agonist, 2,5-dime- deprived of food, but received tap water ad libitum. thoxy-4-iodoamphetamine hydrochloride (DOI; RBI, Natick, MA), Experiments were performed at day 11. Previous experiments in were performed in control rats (n ϭ 5) as well as in cyclosporin our laboratory showed that effective renal plasma flow (RPF) as well A-treated rats (n ϭ 5) by means of intrarenal bolus injections (10, 30, as GFR of cyclosporin A-treated rats (n ϭ 12) were significantly 100, and 300 ng, doses without systemic effects), while RBF was lowered to approximately 70% of the values obtained in control rats continuously measured. Preliminary experiments showed that dose– (n ϭ 6), i.e., from 6.30 Ϯ 0.37 ml/min to 4.44 Ϯ 0.28 ml/min for RPF response curves to intrarenal DOI obtained in control or cyclosporin and from 2.07 Ϯ 0.11 ml/min to 1.41 Ϯ 0.07 ml/min for GFR (10). A-treated rats were completely blocked by ritanserin. ␣ The rats were anesthetized with an intraperitoneal injection of RBF measurements during bolus intrarenal injections of the 1- sodium pentobarbital (60 mg/kg; Nembutal, Ceva, Brussels, Belgium) receptor agonist phenylephrine (Sigma; 0.1, 0.3, 0.5, and 0.7 ␮g) were and placed on a heating table maintaining body temperature at 37°C. performed in cyclosporin A-treated rats (n ϭ 6) as well as in control The femoral artery and vein were cannulated with polyethylene PE50 rats (n ϭ 5) before and after ritanserin. tubings. Subsequently, isotonic saline was infused continuously into the femoral vein at a rate of 0.0425 ml/min. The femoral arterial Statistical Analyses pressure was monitored with a pressure transducer (Harvard Appara- The results are represented as the means Ϯ SEM. Wilcoxon tests tus, Millis, MA) attached to a recorder (Linearcorder F WR 3701, for paired or unpaired observations were used for point to point Graphtec Co., Yokohama, Japan). comparisons. A P value Ͻ 0.05 was considered significant. Linear regression analysis of the RPP to RBF (␦ %) data were performed. In Study of the Autoregulation of RBF cases of autoregulation of RBF, the set point was determined by Autoregulation of RBF was analyzed as described before (22,24). constructing two best fitting regression lines, one of them representing Briefly, the aorta and right renal artery were exposed by means of an the plateau phase. In the absence of autoregulation, one single regres- abdominal incision. A string was placed around the aorta above both sion line was obtained. renal arteries. By gradual compression of the aorta, the RPP was lowered by steps of 5 mmHg. A blood flow sensor with an inner Results diameter of 0.6 to 0.8 mm was placed on the right renal artery, Influence of Cyclosporin-Treatment on Autoregulation allowing RBF monitoring by an electromagnetic square wave flow- of RBF meter (Skalar Medical, Delft, The Netherlands) and continuous re- Figure 1 illustrates the lowering of RBF (expressed in per- cording (Linearcorder F WR 3701, Graphtec Co.). RPP, considered to centage of RBF at 110 mmHg) in response to a progressive be equal to the femoral artery BP, was gradually reduced to 70 mmHg decrease of RPP, in control and cyclosporin A-treated rats. by steps of 5 mmHg, and both RBF and RPP were continuously Autoregulation of RBF is obvious in the control group: a recorded. At every level of RPP, RBF was allowed to equilibrate for plateau phase is obtained between RPP values of 110 and 95 2 to 3 min. For each level of RPP, RBF was calculated as a percentage of the initial (at 110 mmHg) RBF. Cyclosporin-treated animals with mmHg, followed by a second declining curve between 95 and a basal mean arterial BP (MAP) below 110 mmHg were excluded. 70 mmHg. In the cyclosporin A-treated group, a single regres-

Experimental Protocols Intravenous administration of drugs was performed by means of the femoral vein. Ritanserin (ICN, Costa Mesa, CA) was administered intravenously as a 0.6 mg/kg bolus, followed by an infusion of 1.2 mg/kg per h in isotonic saline, infused at a rate of 0.0425 ml/min. In preliminary experiments, it was found that this dose of ritanserin completely blocks vasoconstriction in dose–response to intrarenal 5-HT in control as well as in cyclosporin A-treated rats. Ritanserin did not alter systemic BP in controls (116.3 Ϯ 2.9 mmHg before versus 115.0 Ϯ 3.1 mmHg after ritanserin, n ϭ 10) and cyclosporin A-treated rats (109.7 Ϯ 2.7 mmHg before versus 109.2 Ϯ 1.9 mmHg after ritanserin, n ϭ 12). Autoregulation of RBF was analyzed in controls (n ϭ 5) and in cyclosporin A-treated rats (n ϭ 7), during saline infusion and 60 min after the start of ritanserin administration. For intrarenal administration of drugs, a catheter was inserted into the right suprarenal artery, as described previously (22,24). Dose– response curves to serotonin (5-hydroxytryptamine; Sigma Chemical Co., St. Louis, MO) were performed in control rats (n ϭ 6) as well as Figure 1. F, autoregulation of renal blood flow (RBF) in control rats, in cyclosporin A-treated rats (n ϭ 7) by means of intrarenal bolus after 10 d of olive oil administration (n ϭ 6; RBF of 5.02 Ϯ 0.42 injections (0.1, 0.3, 0.5, and 0.7 ␮g, doses without systemic effects), ml/min at 110 mmHg). f, RBF versus renal perfusion pressure (RPP) while RBF was continuously measured. In addition, dose–response in cyclosporin A-treated rats, after 10 d of cyclosporin A administra- curves to intrarenal 5-HT were constructed before and 10 min after tion (n ϭ 8; RBF of 3.96 Ϯ 0.22 ml/min at 110 mmHg). *P Ͻ 0.05, G intrarenal administration of the NO-synthase inhibitor N -nitro-L- point to point comparison. 30 Journal of the American Society of Nephrology J Am Soc Nephrol 10: 28–34, 1999 sion line between RBF and RPP was drawn from 110 to 70 mmHg, indicating loss of autoregulation of RBF.

Dose–Responses to Intrarenal Phenylephrine ␣ Intrarenal bolus injections of the 1-agonist phenylephrine resulted in transient dose-dependent decreases of RBF. Figure 2A shows that the response of RBF (⌬%) to increasing doses of intrarenal phenylephrine is not different in cyclosporin A- treated rats compared with control-rats. As shown in Figure 2,

B and C, the 5-HT2 antagonist ritanserin has no influence on the dose–response curve obtained with phenylephrine in control and in cyclosporin A-treated rats, respectively. In the cyclosporin A-treated animals, the RBF response to 0.7 ␮g phenylephrine was omitted because this dose induced systemic hypertension.

Influence of Ritanserin on Autoregulation of RBF The influence of ritanserin on the autoregulatory capacity in kidneys from control and cyclosporin A-treated rats was studied. In control rats, ritanserin shifted the set point of autoregulation from a RPP of 95 mmHg (Figure 1) to 80 mmHg (Figure 3A). In cyclosporin A-treated rats, a plateau phase in the RBF versus RPP curve is found after acute administration of ritanserin, indicating restoration of autoregulation. At almost all points of RPP, the percentage RBF is significantly increased after administering ritanserin (Figure 3B).

Dose–Responses to Intrarenal Serotonin: Effect of NO-Synthase Inhibition The effect of increasing intrarenal doses of 5-HT on RBF were compared in control and cyclosporin A-treated rats. As shown in Figure 4, the responses were not different in both experimental groups (Figure 4). Furthermore, the possibility was explored that during 5-HT injection an enhanced activation of 5-HT1 receptors leading to an enhanced NO-mediated vasorelaxation might counteract and, thus, mask an enhanced 5-HT2-mediated vasoconstriction in the kidneys from cyclosporin A-treated rats. Therefore, the influence of the NO-synthase inhibitor L-NNA was studied on the renal vascular response of 5-HT in control as well as in cyclosporin A-treated rats. After L-NNA, 5-HT elicits a significantly stronger decrease of RBF in the cyclosporin A-treated rats (Figure 5B), whereas there was no additional decrease in control rats (Figure 5A). In the cyclosporin A-treated animals, the RBF response to 0.7 ␮g of serotonin was omitted because this dose induced systemic hypertension.

Dose–Responses to Intrarenal DOI In these experiments, the responses to increasing doses of Figure 2. Dose–response relationship of RBF (as a percentage of the DOI, a highly selective and potent 5-HT2 agonist (27), on RBF was studied. Figure 6 shows that DOI induces a significantly initial RBF) to intrarenal injections of increasing doses of phenylephrine (only doses without systemic effects) in control rats (n ϭ 5; f, more pronounced percentage fall in RBF in cyclosporin A- before ritanserin; Ⅺ, after ritanserin) or in cyclosporin A-treated rats treated rats, compared with control rats. (n ϭ 6; F, before ritanserin; E, after ritanserin). (A) RBF responses to phenylephrine in control (f) and cyclosporin A-treated (F) rats. Discussion (B) RBF responses to phenylephrine in control rats before (f) and RBF autoregulation maintains RBF during changes of RPP after (Ⅺ) ritanserin. (C) RBF responses to phenylephrine in cyclo- due to extrarenal causes (28). Autoregulation occurs indepen- sporin A-treated rats before (F) and after (E) ritanserin. J Am Soc Nephrol 10: 28–34, 1999 Cyclosporin, Serotonin, and Autoregulation 31

Figure 4. Dose–response relationship for RBF (as a percentage of the initial RBF) to injections of increasing serotonin doses into kidneys from control rats (f, n ϭ 6) or cyclosporin A-treated rats (Ⅺ, n ϭ 7).

vasoconstriction. This is similar to what was demonstrated previously in postischemic kidneys (24). Earlier experiments from our laboratory on normal rat kidneys showed that the limitation of the effective autoregulatory range to a set point of 95 mmHg was due to 5-HT. Indeed, after

5-HT2 antagonism by means of ketanserin, autoregulation of RBF was prolonged down to 75 mmHg (22). In contrast to

ketanserin, ritanserin is a more selective 5-HT2 blocker, having ␣ ␣ no 1-adrenolytic activity (31). The lack of 1-adrenolytic activity is confirmed in the present study by showing that ritanserin does not influence phenylephrine-induced vasoconstriction, neither in control nor in cyclosporin A-treated rats. The experiments with ritanserin confirm that in normal rat Figure 3. (A) RBF (as a percentage of the initial RBF) versus RPP in kidneys a 5-HT -receptor-mediated effect impairs autoregula- ϭ Ϯ 2 control rats after ritanserin infusion (n 5; RPP of 4.95 0.29 tion in the RPP range of 80 to 95 mmHg (compare Figure 1 and ml/min at 110 mmHg). (B) RBF (as a percentage of the initial RBF) Figure 3A). A similar effect has been observed with ritanserin versus RPP in cyclosporin A-treated rats before (, RPP of 3.43 Ϯ 0.35 ml/min at 110 mmHg) and after (f, RPP of 3.75 Ϯ 0.29 ml/min in the split hydronephrotic rat kidney (23). at 110 mmHg) ritanserin infusion (n ϭ 7). *P Ͻ 0.05, point to point In all cyclosporin A-treated animals, RBF autoregulation comparison. was lost. Autoregulation could be reestablished acutely after 1 h of ritanserin administration. The loss of autoregulation from 110 mmHg onward in cyclosporin A-treated rats, and its dently of renal innervation and depends to a large extent on the reappearance during ritanserin, cannot be attributed to a BP- intrinsic myogenic response to changes in transmural pressure induced shift of the autoregulatory range along the RPP axis, gradients in the preglomerular arteries. Tubuloglomerular feed- because in our experimental model of cyclosporin A nephro- back is responsible for further adjustments of vascular tone toxicity, MAP was not significantly altered, and because MAP (28). In the normal rat kidney, the autoregulatory vasodilation was not influenced by the administration of ritanserin. Al- mechanisms keep RBF constant until a threshold RPP level of though hypertension has been reported, it is known that hyper- 95 mmHg (22,24,29). Kaskel et al. (30) attributed the loss of tension is not a general finding in rat models of cyclosporin autoregulation in cyclosporin A-treated rats to a direct effect on A-induced nephrotoxicity (32–35). the preglomerular myogenic mechanism, rather than to an Reappearance of autoregulation by ritanserin administration impairment of tubuloglomerular feedback. indicates that an enhanced 5-HT2-mediated vasoconstriction is The most important finding of the present study is that involved in the loss of autoregulation after cyclosporin A. This administration of the selective 5-HT2-receptor antagonist ritan- is analogous to previous observations in a model of postisch- serin leads to a reappearance of RBF autoregulation in cyclo- emic loss of autoregulation (24). sporin A-treated rats. This finding indicates that the vasodila- In contrast to postischemic kidneys (24), however, intrarenal tory autoregulation capacity is not lost by cyclosporin A administration of 5-HT did not elicit a stronger vasoconstric- treatment, but only suppressed by a 5-HT2-receptor-mediated tion after cyclosporin A treatment. The lack of an increased 32 Journal of the American Society of Nephrology J Am Soc Nephrol 10: 28–34, 1999

sporin A-treated rats were compared. DOI is a known selective

5-HT2 agonist (27,40,41), and in our own preliminary experiments ritanserin completely blocked the DOI-induced renovas- cular constriction. During DOI injections, a more pronounced decrease in RBF was found after cyclosporin A treatment,

indicating an enhanced 5-HT2-receptor-mediated effect on renal vascular smooth muscle cells. That the RBF response to ␣ intrarenal phenylephrine, a pure 1-agonist, is unaltered by the cyclosporin A treatment further supports the suggestion of a

specific 5-HT2-receptor-mediated phenomenon. However, if the loss of RBF autoregulation in cyclosporin

A-treated rats could entirely be explained by enhanced 5-HT2- receptor activation, then we would also expect autoregulation to persist to 80 to 85 mmHg in these animals during ritanserin. Other unknown factors might be responsible for suppression of autoregulation in the RPP range 100 to 80 mmHg. A similar

discrepancy of the autoregulation set point during 5-HT2 antagonism was found between control and postischemic kidneys (24). Taken together, the present data indicate that in this model

of cyclosporin A nephrotoxicity, enhanced 5-HT2-mediated vasoconstriction is important for suppression of RBF autoreg-

ulation. Administration of ritanserin, a pure 5-HT2 antagonist, makes autoregulation reappear in these animals. This abrupt reappearance of autoregulation might be explained by the

blockade of an enhanced 5-HT2-receptor-mediated vasoconstriction, overwhelming the intrinsic vasodilatory autoregulation mechanism(s) during lowering of the RPP. In that respect, the present observation is analogous as described previously in Figure 5. Dose–response relationship for RBF (as a percent of the postischemic kidneys (24). This restoration of RBF autoregu- initial RBF) to injections of increasing serotonin doses (without lation by 5-HT antagonism may be of clinical relevance in ϭ 2 systemic effects) into kidneys from control rats (A; n 4) or cyclo- both postischemic and cyclosporin A-induced renal damage as ϭ f Ⅺ G sporin A-treated rats (B; n 8) before ( ) and after ( ) N -nitro- it is accepted that the loss of autoregulation of RBF is one of L-arginine administration. *P Ͻ 0.05, point to point comparison. the factors determining the duration of renal functional recov- ery in clinical acute renal failure. vasoconstriction to 5-HT was in apparent conflict with the beneficial effect of 5-HT2 antagonism on autoregulation. The possibility that the 5-HT2-mediated vasoconstrictor response, elicited during the 5-HT injections, might have been counteracted by a simultaneous enhanced vasodilatory effect was analyzed. Former studies performed in our laboratory on the same rat model (36,37) and experiments from others (38,39) suggested an enhanced vascular NO production after cyclosporin A treatment. Because serotonin is also a well known stimulator of NO production through 5-HT1-receptor activation, it was investigated whether enhancement of the

5-HT2 response could be revealed by NO blockade. After NO-synthase inhibition, the renal vasoconstrictory response to 5-HT was indeed reinforced in cyclosporin A-treated rats but not in control rats. These data indicate that while injecting

5-HT intrarenally, endogenous NO release mediated by 5-HT1- receptor activation attenuates the renal vasoconstrictory re- Figure 6. Dose–response relationship for RBF (as a percentage of the sponse elicited by 5-HT2-receptor activation in cyclosporin initial RBF) to injections of increasing doses of 2,5-dimethoxy-4- A-treated rats. iodoamphetamine hydrochloride (DOI) (without systemic effects) into To confirm this hypothesis, the RBF responses to increasing kidneys from control rats (f, n ϭ 5) or cyclosporin A-treated rats (Ⅺ, doses of intrarenally administered DOI to control or cyclo- n ϭ 8). *P Ͻ 0.05, point to point comparison. J Am Soc Nephrol 10: 28–34, 1999 Cyclosporin, Serotonin, and Autoregulation 33

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