Role of prostaglandin cyclooxygenase and

Acta Physiol Scand 2003, 177, 93–99

Role of prostaglandin cyclooxygenase and cytochrome P450 pathways in the mechanism of natriuresis which follows hypertonic saline infusion in the rat E. Kompanowska-Jezierska, A. Walkowska and J. Sadowski Laboratory of Renal and Body Fluid Physiology, M. Mossakowski Medical Research Centre of the Polish Academy of Sciences, Warsaw, Poland

Received 22 April 2002, accepted 18 September 2002 Correspondence: Dr E. Kompanowska-Jezierska, Laboratory of Renal Physiology, Medical Research Centre, Polish Academy of Sciences, Pawin˜skiego 5, 02-106 Warsaw, Poland.

Abstract Aim: The prostaglandin cyclooxygenase (COX) and P450 cytochrome (CYP450) pathways of arachidonic acid metabolism are functionally interrelated and both engaged in control of sodium excretion; the study focused on their contribution to the natriuresis which follows hypertonic saline infusion in the rat. Methods: In anaesthetized rats, clearance studies were conducted, supplemented with laser-Doppler measurements of the cortical and medullary blood flow (CBF, MBF), and measurement of medullary tissue admittance (Y), an index of interstitial ion concentration. Results: Indomethacin (Indo), 5 mg kg)1 i.v. paradoxically enhanced the natriuresis secondary to intra-aortic suprarenal 5% saline load, further increasing sodium excretion by 385 73% (P < 0.01). After acute clotrimazole, 10 mg kg)1 i.v. an inhibitor of CYP450 epoxygenase, the increase in natriuresis was smaller and did not differ from that observed after the drug’s ethanol solvent. In rats pre-treated with clotrimazole for 3 days, hypertonic saline loading increased sodium excretion (UNaV) to 0.94 0.22 lmol min)1, compared with a significantly greater (P < 0.05) increase to 2.76 0.48 lmol min)1 measured in untreated controls. Indo increased UNaV twofold, similarly in the clotrimazole and in the control group; in the absence or presence of clotrimazole treatment, COX blockade significantly decreased MBF and increased Y. Conclusion: The data indicate that blockade of the CYP450 epoxygenase significantly impairs excretion of sodium in rats acutely loaded with hypertonic NaCl solution. The paradoxical post-Indo natriuresis is preserved in clotrimazole treated rats, which speaks against the role of CYP450 pathway in the response. Keywords clotrimazole, hypertonic saline, indomethacin, intrarenal circulation, sodium excretion.

In a recent study, we showed that in rats acutely loaded with salt, inhibition of prostaglandin (PG) cyclooxygenase (COX) resulted in paradoxical natriuresis, in contrast to antidiuresis and antinatriuresis usually observed at standard conditions (Kompanowska-Jezierska et al. 1999). It should be considered that the ultimate effect of Ó 2003 Scandinavian Physiological Society

COX blockade may not simply depend on elimination of direct vascular and/or tubular effects of PGs. It is known that the three main pathways of arachidonic acid metabolism: those of COX, cytochrome P450 (CYP450) epoxygenase and hydroxylase, and lipoxygenase, show interaction. CYP450 metabolites may be further 93

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transformed by COX, a prostanoid may inhibit the activity of CYP450 enzymes, and/or an inhibition of one metabolic route may divert the substrate to others and thereby enhance their activity (McGiff 1991, Makita et al. 1996). As high lipoxygenase activity in the kidney appears to be detected mostly during inflammatory processes, interaction of the two other pathways and functional consequences thereof may be considered more important in the normal organ. This work attempts to provide an indirect evidence that an infusion of hypertonic saline may result in a rapid enhancement of CYP450 epoxygenase activity, similar to that reported in studies with animals on highsalt diet (Oyekan et al. 1999). If so, we wanted to find out if the paradoxical natriuresis which followed subsequent Indo administration was possibly due to further activation of CYP450 epoxygenase secondary to COX blockade. Epoxygenase products (EETs) were reported to limit NaCl reabsorption in the proximal and distal tubule by inhibiting the Na+, K+-ATPase and Na+, K+, Cl– cotransporter (Makita et al. 1996, Maier & Roman 2001). This could be important as the natriuretic CYP450 pathway metabolites could help eliminate excess salt under positive NaCl balance and would thereby have the potential to prevent the development of hypertension. Indeed, a failure to induce epoxygenase in response to NaCl load, as observed in Dahl salt sensitive rats, was associated with the development of hypertension; Sprague–Dawley and Dahl salt resistant rats were able to induce epoxygenase and remained normotensive (Makita et al. 1994). In this work the role of acute hypertonic saline loading as a potential factor stimulating CYP450 epoxygenase and a possible interaction of COX and CYP450 pathways under such conditions was studied in clearance experiments with anaesthetized rats. Simultaneous measurement of the electrical admittance (Y) of the medullary tissue, an index of NaCl concentration in (and delivery to) the interstitium (Sadowski & Portalska 1983), provided indirect information on the expected changes of NaCl transport in the medullary segment of the thick ascending limb of Henle’s loop (MALH). The studies were supplemented by separate measurements of the cortical and medullary blood flow (CBF, MBF), a valuable addition as both PGs and CYP450 products have well documented vascular effects (Imig 2000, Campbell & Harder 1999).

Vertebrate Animals. The animals were anaesthetized with intraperitoneal thiobutobarbital (Inactin, Byk Gulden, Konstanz, Germany), 100 mg kg)1 body weight. Throughout the surgical preparation and experimental procedures body temperature was maintained at about 37 °C by means of a heated pad. In order to compensate for fluid losses, during surgical procedures 3% bovine albumin in Ringer solution was infused at 2 mL h)1. A cannula was placed in the trachea to ensure free airways; the femoral vein was cannulated for infusion of fluids. One of the femoral artery catheters was pushed into the aorta to position the tip just above left renal artery, for suprarenal aortic infusion of hypertonic saline (see below). The other catheter was used for measurement of renal perfusion pressure (RPP) and blood sampling. The left kidney was exposed from a subcostal flank incision and placed in a plastic holder similar as that used for micropuncture. A screw-controlled snare was placed on the aorta above the left renal artery for control of RPP, and a polyethylene catheter was placed in the ureter for urine collection. Subsequently, a set combining a needle laserDoppler probe for MBF measurement and an admittance cell for measurement of tissue electrical admittance (both measured in the inner medulla) was inserted into the kidney along the cortico-papillary axis. The characteristics of the set and the exact placement procedure were described before (Sadowski et al. 1997). Another laser-Doppler probe, Perimed PF 407, was placed on the kidney surface for measurement of cortical (superficial) blood flow. The probes were suspended on flexible cables fixed to a frame above the kidney; there was no need for rigid fixation.

Methods

Admittance measurement

Experiments were performed with male Wistar rats weighing 280–320 g, maintained on dry pellet diet and given free access to water. The study was approved by the Regional Ethical Committee No. 1, Warsaw, in compliance with the European Convention for the Protection of

The principle and the main features of the technique were described elsewhere (Sadowski & Portalska 1983, Sadowski et al. 1997). It was shown that electrical admittance (Y, reciprocal impedance) values recorded from the medulla of an in situ kidney are linearly related

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Blood flow measurement The medullary and surface flow probes were connected to a laser-Doppler perfusion monitor, PeriFlux 4001 (Perimed AB, Jarfalla, Sweden). The system measures tissue perfusion with blood (Doppler flux) defined as the product of the number of blood cells moving and their mean velocity, within the area less than 1 cubic mm beneath the tip of the probe. The results are expressed in arbitrary perfusion units (PU) or in volts of the analogue output (1000 PU ¼ 10 V). Thus, only relative changes are measured but owing to a calibration procedure the results can be compared between animals.

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to all ions and to Na+ concentration in the extracellular compartment.

Experimental procedures Directly after insertion of the Y/MBF measuring set, the infusion of albumin in Ringer solution was replaced by an infusion which delivered 6 lCi of [methoxy-3H] inulin in 1.2 mL of isotonic saline solution per hour, preceded by a priming dose of 6 lCi. Simultaneously, a suprarenal aortic infusion of isotonic saline at 2 mL h)1 was started. After two 30-min control periods this infusion was replaced by an infusion of 5% saline. Protocol I. After an 1.5-h of suprarenal aortic infusion of 5% saline at 2 mL h)1 (three urine collection periods), indomethacin (Sigma) or clotrimazole (1-[ochlorotrityl]-imidazole, Sigma) or their respective solvents were infused i.v. during 5 min and one or two post-infusion clearance periods were made. Eight rats were used in each group. Indomethacin, 5 mg kg)1 body weight, was dissolved in 1.5 mL alkalinized isotonic saline. Clotrimazole, an inhibitor of cytochrome P450 epoxygenase, was given in the dose of 10 mg kg)1 body weight, dissolved in 1.5 mL of 25% ethanol. In this group, after post-clotrimazole urine collection indomethacin was given in the usual way, and one or two additional clearance periods were made. In two additional groups of eight rats each, effects of 1.5 mL of alkalinized saline (the vehicle for indomethacin) or of 25% ethanol (the vehicle for clotrimazole) were examined. In all groups 5% saline infusion was continued during the injections and in post-injection periods. Protocol II. Three groups, eight rats in each, were studied. In two groups rats were pre-treated either with clotrimazole, 80 mg kg)1 body weight per day injected i.p. as a suspension in linseed oil, on 3 days preceding the proper experiment, or pre-treated with the oil solvent alone. The dosage was earlier found to suppress P450 epoxygenase activity in kidney microsomes (Makita et al. 1994). The third group served as an additional untreated control. In each group rats received indomethacin after 1.5 h of intra-aortic 5% saline infusion. After experiments the rats were killed with an overdose of the anaesthetic. The position of the Y electrode tip (deep in the inner medulla) and of the tip of the laser-Doppler probe (close to the border with the outer medulla) was verified at the kidney’s cross-section.

Analytical procedures Urine volumes were determined gravimetrically. Plasma and urine tritiated inulin activities were measured in an LKB 1211 liquid scintillation counter (Sweden) and the Ó 2003 Scandinavian Physiological Society

Æ Role of renal eicosanoids in salt loading natriuresis

glomerular filtration rate (GFR) was determined as the clearance of 3H inulin (Cin). Osmolalities were measured using a vapour pressure osmometer (Wescor 5500, Logan, Utah, USA) and sodium concentration by an ion selective electrode analyser (Analyser Industries, Dreischor, The Netherlands).

Statistics The significance of changes within one group over time was first evaluated by repeat measurement analysis of variance (anova), followed by Student’s test for dependent variables. Differences in mean values between groups were first analysed by one-way anova followed by a modified Student’s t-test for independent variables, using Bonferroni’s correction for multiple comparisons. The standard error of mean (SEM) was used as the measure of data dispersion.

Results The RPP was maintained constant at 110–120 mmHg in all experimental groups. After 1 h of isotonic saline infusion at 2 mL h)1 the infusate was replaced by 5% saline given at the same rate. Such infusion increased plasma Na+ concentration 10–13 mm. Mean UNaV increased in individual groups from the isotonic saline baseline of 0.35–0.61 lmol min)1 (range of means) to 1.97–2.12 lmol after 1.5 h of hypertonic saline. The respective changes in V were from 3.6–6.0 to 12.0– 14.7 lL min)1. All these increments were significant at the 0.05 level or less. CBF, MBF and GFR did not change. The medullary interstial ion concentration (mean tissue Y) increased from 851–1010 to 969– 1050 lS (significant at P < 0.05). Per cent changes in renal function in response to indomethacin and clotrimazole in rats pre-loaded with 5% saline are shown in Figure 1. After Indo, UNaV increased almost fivefold, compared with a twofold change seen after the vehicle. Clotrimazole induced a threefold increase in UNaV, compared with more than a twofold increase after ethanol vehicle. Remarkably, the difference in the response between the drug and the vehicle was significant for Indo but not for clotrimazole. Between-group comparison showed that the increase in UNaV after Indo was significantly greater than that after clotrimazole. The pattern of changes in V was similar as in UNaV, except that the increases after the drugs were smaller and the between-group difference between the responses to Indo and to clotrimazole was not significant. Clotrimazole did not substantially affect medullary tissue admittance, in contrast to a major increase after Indo. This change was significantly greater than that after Indo vehicle, clotrimazole or clotrimazole vehicle. There was a major decrease in MBF after Indo, 95



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Figure 1 Responses of rats acutely loaded with hypertonic saline to the blockade of prostaglandin synthesis with indomethacin (Indo, 5 mg kg)1, i.v.) or to the blockade of cytochrome P450 epoxygenase with clotrimazole (Clotr) (Protocol I). Per cent changes (mean SEM) are shown for sodium and water excretion (UNaV, V), glomerular filtration rate (GFR), medullary blood flow (MBF), and medullary tissue admittance (Y). Veh – saline vehicle of Indo; 25% ethanol was the vehicle for Clotr. *Significantly different from pretreatment control at P < 0.05; different from response to vehicle at P < 0.05; different from the response to Clotr at P < 0.05.

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and an unexpected modest decrease after ethanol vehicle for clotrimazole. In order to find out if the major natriuretic response to Indo would be preserved after previous administra96


tion of clotrimazole, in rats of the clotrimazole group experiments were continued and Indo was additionally administered in the usual dose (not shown in Fig. 1). Indo tended to decrease UNaV ()23 15%, not significant) and decreased V by 38 11% (P < 0.05). In rats pre-treated with clotrimazole Indo induced a 30 3% increase in tissue Y and 27 7% decrease in MBF. In the same study no changes in CBF were seen whereas post-clotrimazole Indo administration decreased GFR 21 6% (P < 0.05). Data obtained within the protocol II (acute experiment performed in rats pre-treated with clotrimazole or oil vehicle for 3 days) are shown in Figure 2. At the start of experiments plasma Na+ concentration in clotrimazole treated rats was 129.7 1.3 mm, which was different (P < 0.005) from 124.7 0.9 seen in untreated controls. In the clotrimazole group, during 5% saline infusion an increase in UNaV was significantly smaller then in the oil treated group (P < 0.05). In each group Indo administration visibly increased UNaV above the baselines which were already ascending during 5% saline infusion; there was no significant difference in this response among the three groups. For urine flow the pattern of changes (not shown in the figure) was similar as for UNaV. In neither group did MBF change after hypertonic saline loading; it invariably decreased after Indo but the decrease was the smallest in the clotrimazole group. The CBF increased progressively in clotrimazole treated rats, from control of 501 30–540 29 PU after 1.5 h of 5% saline infusion (P < 0.05). In all three groups Y increased progressively, from 933–1023 lS (range of means) during isotonic saline infusion to 1076–1161 lS after 1.5 h of hypertonic saline (all three increases significant). Tissue Y appeared to increase more steeply after Indo than before, especially in the clotrimazole group.

Discussion We have shown recently that pre-loading of anaesthetized rats with hypertonic saline significantly modified Ó 2003 Scandinavian Physiological Society




Figure 2 Sodium excretion (UNaV) (a), glomerular filtration rate (GFR) (b), medullary blood flow (MBF, perfusion units) (c) and medullary tissue admittance (Y, lSiemens) (d) as affected by 5% saline infusion and subsequent administration of indomethacin (Indo, 5 mg kg)1, i.v). Three groups were studied: untreated, clotrimazole treated, and oil (clotrimazole vehicle) treated rats (Protocol II). 5% saline was infused at 2 mL h)1. The data are mean SEM. *Significantly different from isotonic saline control at P < 0.05; different from the pre-Indo value at P < 0.05; different from the mean increase from control in oil-treated rats.

well known responses to two experimental interventions. First, in so pre-loaded rats a very small volume expansion triggered a disproportionate natriuresis and diuresis. Second, the natriuresis paradoxically increased Ó 2003 Scandinavian Physiological Society

further after blockade of COX with indomethacin or meclophenamate (Kompanowska-Jezierska et al. 1999). The present study was aimed to examine the role of another route of arachidonic acid metabolism, the CYP450 pathway, in control of natriuresis secondary to hypertonic saline infusion. We also attempted to find out if the natriuretic effect of COX blockade performed on the background of hypertonic saline infusion could be because of interaction of COX and CYP450 pathways. What were the consequences of hypertonic saline infusion for intrarenal metabolism of arachidonic acid which could form a basis for the unusual renal response to PG blockade? This metabolism is known to be enhanced by high salt intake, however, it appears that the activation does not involve PG COX and the renal medullary activity of natriuretic PGE2 may actually decrease when salt balance is positive (Rathaus et al. 1987). On the other hand, the metabolism along the P450 pathway is greatly enhanced (Capdevila et al. 1992), of particular importance is stimulation of P450 epoxygenase whose activity increased to 400% of control. Recently, chronic saline loading was shown to greatly increase renal cortical and medullary epoxygenase activity whereas the activity of P450 x-hydroxylase was reduced, at least in the cortex (Oyekan et al. 1999). Thus, a major activation of the former enzyme can also be expected in the rats acutely loaded with hypertonic saline. The epoxygenase products, EETs, are known as inhibitors of proximal and distal tubular Na+ transport (Makita et al. 1996) and it is not unlikely that the natriuresis associated with hypertonic saline loading is mediated in part by these metabolites. We wanted to find out if high EETs synthesis resulting probably from NaCl loading would be further enhanced by subsequent inhibition of COX, leading to the post-Indo natriuresis observed in this study (Fig. 1). COX blockade may increase the synthesis of the metabolites of the CYP450 pathway by eliminating its PG modulatory inhibitor or, alternatively, because of a diversion of some of the COX substrate to the CYP450 pathway (McGiff 1991). Indeed, inhibition of COX was reported to induce bacterial P450 cytochrome (English et al. 1996). 97



In order to examine the possible role of activation of CYP450 epoxygenase in post-indomethacin natriuresis of hypertonic saline loaded rats, we inhibited this pathway with clotrimazole. Unexpectedly, acute intravenous administration of the drug actually tended to increase sodium and water excretion, however, effects were not significantly different from those of ethanol solvent (Fig. 1). Most probably, post-clotrimazole increase in excretion reflected an exaggerated response to a small volume expansion recently described by us in rats loaded with hypertonic saline (KompanowskaJezierska et al. 1999); the effect could have prevailed over a putative antinatriuresis secondary to inhibition of CYP 450 epoxygenase. On the other hand, when Indo was administered about 30 min after clotrimazole, no increase in sodium and water excretion was seen; in fact, UNaV tended to decrease and V decreased almost 40%. These data suggested that enhanced generation of EETs could, indeed, be responsible for post-indomethacin natriuresis and when this process was blocked, the effect was abolished. Alternatively, the abolishment could have been because of a simultaneous decrease in GFR. The observation that Indo injection preceded by clotrimazole administration (but not in absence of such pre-treatment) decreased GFR may suggest that P-450 epoxygenase products help maintain glomerular haemodynamics after elimination of vasodilator prostaglandins. However, as EETs were not measured, we could not be sure that acute hypertonic saline infusion did, indeed, stimulate their production, as was documented with chronic salt loading (Makita et al. 1996, Oyekan et al. 1999). Furthermore, the effectiveness of acute administration of clotrimazole could be questioned and the data suggested that a part of the drug’s effect was due to the ethanol solvent. Therefore, in another series of experiments, instead of acute administration of clotrimazole and indomethacin in sequence, we prepared rats by chronic clotrimazole treatment. Remarkably, these rats showed an elevated plasma Na+ concentration at the start of acute experiments, which indicates that they had retained Na+ during the 3 day clotrimazole treatment. During 5% saline infusion, these rats excreted less sodium and water compared with untreated controls (Fig. 2), which suggested that elimination of natriuretic EETs (inhibition of epoxygenase) was responsible for relative sodium retention. As in the data obtained within Protocol I, Indo enhanced the natriuresis secondary to hypertonic saline infusion; however, there was no indication that this response was modified by clotrimazole pre-treatment. Thus, the data did not substantiate the abolishment of post-Indo natriuresis by the blockade of CYP 450 epoxygenase, which was suggested by experiments in which clotrimazole and Indo were administered acutely in sequence (Protocol I). 98


The present study confirms our earlier observations on the distinct effect of COX blockade increasing Y and decreasing MBF (Kompanowska-Jezierska et al. 1999, Sadowski et al. 1997). The former effect is explained by elimination of the PGs antagonism of the vasopressin dependent NaCl transport in the MALH. Abolishment of this PG action must have resulted in increased delivery of NaCl to medullary interstitium measured as an increase in tissue Y. The post-Indo decrease in MBF adds to the evidence that medullary circulation is under tonic vasodilator control of prostaglandins, most probably those generated by COX-2 (Imig 2000, Harris & Breyer 2001). Neither the studies with the chronic blockade of epoxygenase with clotrimazole nor the above observations of the effects of PG blockade on medullary ionic hypertonicity and circulation explain the phenomenon of post-Indo natriuresis. Another controller (inhibitor) of tubular Na+ transport, 20-HETE, a product of CYP450 x-hydroxylase, could be involved (Maier & Roman 2001). It is possible that under chronic inhibition of CYP450 epoxygenase and reduced generation of EETs, the role of the hydroxylase pathway increases and a new balance of metabolic routes is established. Further studies, preferably involving measurement of active arachidonic acid metabolites in urine and renal tissue microdialysates, are required to characterize their role in control of renal haemodynamics and excretion. In summary, the studies indicate that acute loading of anaesthetized rats with hypertonic saline increases sodium excretion by a mechanism that is at least in part mediated by metabolites of P450 EETs; the generation of these metabolites is probably stimulated in response to salt loading. The data do not support the hypothesis that in rats infused with hypertonic saline the paradoxical natriuresis observed after COX blockade depends on enhanced generation and tubular action of EETs. Thus, the mechanism of the natriuresis requires further investigations.

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