Effects of Intrarenal Infusion of 17-Octadecynoic Acid on Renal Antihypertensive Mechanisms in Anesthetized Rabbits

Abstract

To characterize the role of cytochrome P450 metabolism of fatty acids in the renal response to increased renal perfusion pressure, we tested the effects of renal arterial infusion of 17-octadecynoic acid (17-ODYA, 450 nmol/min) on renal and systemic hemodynamic, and renal excretory responses to step-wise increases in renal perfusion pressure (RPP) in anesthetized rabbits, using an extracorporeal circuit for renal autoperfusion. Inhibition of cytochrome P450-dependent fatty acid metabolism was estimated by comparing the metabolism of arachidonic acid in microsomes prepared from the kidneys of control and 17-ODYA-treated animals. Step-wise increases in RPP decreased mean arterial pressure, which previous studies have indicated is attributable to the release of a depressor hormone from the renal medulla. Elevations in RPP also increased renal blood flow and glomerular filtration rate, and the absolute and fractional excretions of urine and sodium. Intrarenal infusion of 17-ODYA reduced the metabolism of arachidonic acid to 20-hydroxyeicosatetraenoic acid by 41%, but it did not significantly influence the responses to increased renal perfusion pressure. We conclude that either the responses elicited by increased renal perfusion pressure in anesthetized rabbits do not depend on cytochrome P450-dependent fatty acid metabolism, or that cytochrome P450 activity must be inhibited by more than was achieved in the present study (41%), before functional effects on the response to increased renal perfusion pressure are observed.

Arachidonic acid, cytochrome P450, medullipin, pressure natriuresis, renal perfusion pressure

When renal perfusion pressure (RPP) is increased, two major renal antihypertensive mechanisms are initiated: pressure natriuresis^1,2 and the release of putative depressor hormones from the renal medulla.^3,4 There is extensive evidence that these renal antihypertensive mechanisms act in concert with other cardiovascular homeostatic factors in the control of arterial pressure.^2,4–6

The hormonal antihypertensive response to increased RPP has been a matter of intense study for the past 45 years, yet the nature of this substance remains to be characterized biochemically.^4,6 Recently, evidence has accumulated for the involvement of cytochrome P450 enzyme systems in its expression. The rapid normalization of blood pressure after unclipping the renal artery of a 1-kidney, 1-clip hypertensive rat is potentiated by induction of cytochrome P450 systems by phenobarbital,⁷ and by the dual inhibitor of cyclooxygenase and lipoxygenase BW755C.⁸ The latter presumably potentiates the metabolism of arachidonic acid through the cytochrome P450 ω-hydroxylase and epoxygenase systems.^9,10 Furthermore, administration of inhibitors of cytochrome P450 ω-hydroxylase and epoxygenase systems (eg, ketoconazole and 17-octadecynoic acid), nonspecific inhibitors of arachidonic acid metabolism (eg, eicosatetraynoic acid), or removal of the liver prevent or delay the blood pressure-lowering effect of unclipping the renal artery of 1-kidney, 1-clip hypertensive rats.^8,11 Cytochrome P450 inhibitors also abolish the depressor response in rats to intravenous administration of renal venous effluent from high pressure-perfused rat isolated kidneys, either when the perfused kidney is treated with ketoconazole,¹² or when the assay rat is treated with SKF 525A.¹³ Taken together, these and other data^3,4,6 have been interpreted to indicate that a lipid pro-hormone (medullipin I) is synthesised within the kidney (presumably the medulla) and converted to the active depressor substance (medullipin II) within the liver. Both the synthesis of medullipin I in the kidney and its conversion to medullipin II in the liver appear to be dependent on the actions of cytochrome P450.⁶

When perfusion pressure of a blood-perfused rabbit (and also dog and rat) kidney is increased using an extracorporeal circuit, the release of a depressor substance can also be demonstrated.^4,14 However, as yet there is only circumstantial evidence that these phenomena are mediated by medullipin as characterized by the experiments of Muirhead et al.^3,4 Therefore, one of the aims of the present study was to determine whether, like medullipin, the depressor effect of increased RPP in the anesthetized rabbit is dependent on renal cytochrome P450 enzyme systems.

Renal cytochrome P450 systems also appear to influence intrarenal hemodynamics and tubular function. In vitro cytochrome P450 inhibitors dilate vessels of the rat renal microvasculature,¹⁵ inhibit vasoconstriction to a range of agents in the isolated perfused rat kidney,^16,17 and attenuate the myogenic responses of dog renal arcuate arteries and rat afferent arterioles to elevated perfusion pressure.^18,19 Furthermore in vitro, products of cytochrome P450-dependent arachidonic acid metabolism have been shown to constrict the renal vasculature (20-hydroxyeicosatetraenoic acid [20-HETE]²⁰; 12-HETE²¹), dilate the renal vasculature (unidentified products of arachidonic acid²²), and inhibit ion transport in the rabbit loop of Henle (unidentified products of arachidonic acid²³). In anesthetized rats, cytochrome P450 inhibition inhibits tubuloglomerular feedback,²⁴ selectively increases medullary blood flow and increases fluid and sodium excretion,¹⁰ augments hyperperfusion and hyperfiltration responses to uninephrectomy,²⁵ and increases loop of Henle chloride transport.²⁶ Products of cytochrome P450-dependent arachidonic acid metabolism have also been shown to alter renal hemodynamic and excretory function in vivo.^26,27

Taken together with observations of altered renal cytochrome P450-dependent metabolism of arachidonic acid in genetic models of hypertension,^{15,26,28–32} these observations suggest that cytochrome P450 enzymes within the kidney may play an important role in modulating the renal endocrine and exocrine responses to altered RPP. In the present study we have tested this hypothesis using an extracorporeal circuit in anesthetized rabbits, which we have used previously to examine both the hormonal^33–36 and excretory³⁷ response to elevating RPP. Cytochrome P450 fatty acid ω-hydroxylase was inhibited by intrarenal infusion of the suicide substrate inhibitor 17-octadecynoic acid (17-ODYA).^10,38

Methods

Animals

Twelve rabbits (1.8 to 3.1 kg [mean weight, 2.4]) of either sex (5 male, 7 female) and either of a multicolored English strain (n = 2) or of the New Zealand White strain (n = 10) were studied. Rabbits were randomly assigned to the two treatment groups (vehicle or 17-ODYA; Sigma Chemical Co., St. Louis, MO). The rabbits were allowed food and water ad libitum until the experimental procedures began. At the conclusion of the experiment they were killed with an intravenous overdose of pentobarbitone sodium (300 mg). The experiments were done in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes³⁹ and were approved in advance by the Monash University Standing Committee on Ethics in Animal Experimentation.

The experimental procedures involved 1) pentobarbitone anesthesia; 2) right nephrectomy; 3) establishing an extracorporeal circuit to allow left RPP to be altered without direct effects on systemic hemodynamics; 4) an equilibration period of 30 min, during which renal artery pressure was set to 65 mm Hg; 5) starting an intrarenal infusion of either 17-ODYA (450 nmol/min) or its vehicle, which continued for the duration of the experiment, 6) a further 60-min equilibration period; and 7) a series of four to five 20-min experimental periods during which RPP was set at progressively higher levels over the range from 65 to 160 mm Hg.

Extracorporeal Circuit

This circuit has been described in detail previously.^33,37 Briefly, blood was withdrawn from the distal aorta by means of a roller pump (Masterflex model 7521-45, Barnant Co., Barrington, IL) and returned to the animal both through the renal artery and the vena cava. A Starling resistor was incorporated into the venous limb so that graded reductions in the flow of blood through this limb (and therefore increases in pressure and flow in the renal limb) could be achieved. The circuit was primed with 10% wt/vol dextran 40 and 50 IU/mL heparin in 154 mmol/L NaCl solution (16 mL).

Surgery

Catheters were placed in the central ear arteries and marginal ear veins in each ear under local anesthesia (Xylocaine, 0.5% wt/vol lignocaine, Astra Pharmaceuticals, North Ryde, New South Wales, Australia). Induction of general anesthesia was by intravenous administration of pentobarbitone sodium (90 to 150 mg; Nembutal, Boehringer Ingelheim, Artarmon, New South Wales, Australia) and was immediately followed by endotracheal intubation and artificial respiration (Phipps & Bird small animal respirator, Richmond, VA). PaO₂ was maintained between 95 and 110 mm Hg, whereas PaCO₂ was maintained between 25 and 30 mm Hg. Anesthesia was maintained throughout the surgery and the experiment by pentobarbitone infusion (30 to 50 mg/h).

Surgery was performed on a heated table. During surgery, 154 mmol/L NaCl solution was infused intravenously at a rate of 0.18 mL/kg per min. First, the right kidney was removed through a right flank retroperitoneal incision, and frozen at −70°C for later analysis of microsomal arachidonic acid ω-hydroxylase activity. This wound was closed with sutures and the rabbit was placed in an upright crouching position for exposure of the left renal artery and ureter, and the distal aorta and vena cava, through a left retroperitoneal incision. A Silastic catheter (0.5 mm inner diameter [ID], 0.95 mm outer diameter [OD]) was inserted into the ureter for urine collection. At this point, the rabbit was heparinized intravenously (15,000 IU sodium heparin; Fisons Pharmaceuticals, Sydney, Australia) and cannulae were inserted into the aorta below the level of the inferior mesenteric artery (2.60 mm ID, 3.00 mm OD) and into the vena cava (1.58 mm ID, 2.16 mm OD). A catheter was then placed in the renal artery (0.80 mm ID, 1.60 mm OD), and perfusion of the kidney through the extracorporeal circuit was commenced. Renal ischemic time in the vehicle- and 17-ODYA-treated groups averaged 3 min 59 sec and 4 min 2 sec, respectively. The kidney was denervated by stripping the nerves from the renal artery.

Immediately after establishment of the extracorporeal circuit, RPP was set and maintained at 60 to 70 mm Hg. To minimize fluid loss the rabbit's wounds were covered with gauze soaked in 154 mmol/L NaCl solution, which was then covered with silicone gel (Wacker-Chemie, Munich, Germany; 10 parts RTV-2E604A, 1 part RTV-E604B, and 1 part KATALY.OL). Esophageal temperature was measured throughout and maintained at 37° to 38.1°C during the period of experimental observation by the combination of a heated table, a heat exchanger incorporated into the extracorporeal circuit just before the roller pump, and a thermostatically controlled infrared lamp (Digi-Sens 60648 Temperature Controller with H-03057-00 heating lamp; Cole Parmer, IL). Bolus doses of [³H]inulin (4 μCi) (NEN Research Products, Sydney, New South Wales, Australia), LiCl (25 mg) (Merck, Darmstadt, Germany), and paraminohippuric acid (PAH, 10 mg; Sigma) were then administered. The infusion of 154 mmol/L NaCl solution (0.18 mL/kg per min) was replaced with 10% vol/vol polygeline (Haemaccel, Hoechst, Melbourne, Victoria, Australia) containing 200 IU/mL sodium heparin, 0.25 mg/mL LiCl, 0.3 μCi/mL [³H]inulin, and 1 mg/mL PAH. [³H]inulin was purified before use, by dialysis in 1000 volumes of 154 mmol/L NaCl (Spectra/Por cellulose ester 500 molecular weight cutoff; Spectrum, Houston, TX).

Experimental Protocol

Thirty minutes after the extracorporeal circuit was established intrarenal infusion of either 17-ODYA (450 nmol/min) or its vehicle (0.1 mL/min, 10% wt/vol 2-hydroxypropyl-β-cyclodextrin [Research Biochemicals Incorporated, Natick, MA] plus 10% vol/vol ethanol in 250 mmol/L NaHCO₃) commenced, and continued for the remainder of the experiment. After a further 60 minutes, the experimental manipulations commenced. The RPP was set at 65, 85, 105, 130, and 160 mm Hg, respectively, at the start of each of five 20-min periods. Once set, RPP was not adjusted during the 20-min experimental period. After a 5-min equilibration period at the start of each 20-min period, urine produced by the left kidney was collected during the last 15 min. Arterial blood for clearance and plasma renin activity measurements was collected before the start of the first 15-min clearance period, and at the end of the second, fourth, and fifth clearance periods. Blood volume was replaced by an equivalent volume of 10% polygeline solution. At the completion of the final clearance period the left kidney was removed and frozen at −70°C for later analysis of cytochrome P450-dependent arachidonate metabolism activity.

Recording of Hemodynamic Variables

Mean arterial pressure (MAP) was measured by connecting the ear artery catheter to a pressure transducer (Cobe) placed at the level of the rabbit's heart. Heart rate (HR) was measured by a tachometer activated by the pressure pulse. Pressure in the renal limb of the circuit (RPP) was measured by connecting a sidearm catheter, 3 mm proximal to the tip of the cannula inserted into the renal artery, to a pressure transducer placed at the same height as that used to measure MAP. Previously we have found that pressure measured directly by renal artery puncture at the end of similar experiments provides values about 4 mm Hg lower than through the side arm catheter.³⁷ Levels of RPP were not corrected for this systematic error. Blood flow through the renal limb (renal blood flow; RBF) was measured with an in-line ultrasonic flow probe (type 4N, Transonic Systems Inc., Ithaca, NY) connected to a model T108 flowmeter. The signals were amplified and recorded on a Neotrace pen recorder (Neomedix Systems, Sydney, Australia) and relayed to an Olivetti M280 computer equipped with an A-D converter that provided 20-sec means of MAP (mm Hg), HR (beats/min), RPP (mm Hg), and RBF (mL/min).

Analysis of Urine, Blood, and Tissue Samples

For clearance measurements, 1-mL blood samples were withdrawn from an ear artery. Hematocrit was measured by the capillary tube method, and the remaining blood centrifuged at 4°C for 15 min at 3000 rpm. Blood samples (1 mL) were also collected for later measurement of plasma renin activity.⁴⁰ The plasma was aspirated and frozen at −20°C for later analysis. Urine was collected into preweighed containers and aliquots frozen for later analysis.

[³H]inulin clearance was used to estimate glomerular filtration rate (GFR) as previously described.³⁷ Sodium and potassium concentrations were measured by flame photometry (Instrumentation Laboratory 943, Milan, Italy).

The ability of 17-ODYA to inhibit cytochrome P450-dependent metabolism of arachidonate was estimated by determination of the rate of ω-hydroxylation of arachidonate in preparations of kidney microsomes. Previously described methods were used for preparation of kidney microsomes, and analysis of their ability to convert [¹⁴C]arachidonate into [¹⁴C]20-HETE.¹⁰

Analysis of Results

All statistical analyses were performed using the computer software SYSTAT.⁴¹ The data regarding the effects of 17-ODYA on the responses to increased RPP were analyzed by repeated measures analysis of variance. This allowed us to partition the effects of increasing RPP (P_RPP in Table 1), and the RPP-independent (P_treatment in Table 1) and RPP-dependent (P_{treatment*RPP} in Table 1) effects of 17-ODYA-treatment. To protect against the increased risk of comparison-wise type 1 error resulting from compound asymmetry, P values were conservatively adjusted using the Greenhouse-Geisser correction.⁴² The rate of 20-HETE formation by microsome preparations from the kidneys of the vehicle and 17-ODYA rabbits were compared by Student's t test. These data were further analyzed by analysis of covariance, between the rate of 20-HETE formation by the kidney microsome preparations and the rate at which arterial pressure decreased during perfusion of the kidney at increased pressure.

Table 1.

Outcome of Repeated Measures Analyses of Variance for the Data Depicted in Figures 23 to 4

Variable	PRPP	Ptreatment	Ptreatment*RPP
Renal perfusion pressure	<.001	.37	.94
Mean arterial pressure (MAP)	.01	.34	.20
Rate of change of MAP	.001	.12	.32
Heart rate	.09	.13	.46
Hematocrit	<.001	.78	.65
Renal blood flow	<.001	.48	.78
Glomerular filtration rate	<.001	.67	.30
Renal vascular resistance	.005	.35	.58
Filtration fraction	.002	.43	.31
Urine flow	<.001	.41	.47
Fractional urine excretion	<.001	.25	.53
Sodium excretion	<.001	.48	.54
Fractional sodium excretion	<.001	.61	.74

P values for the effect of increased renal perfusion pressure (P_RPP) and for the interaction between treatment (17-octadecynoic acid or its vehicle) and increased renal artery pressure (P_{treatment*RPP}) were derived from F_3,30 and corrected for compound asymmetry using the Greenhouse-Geisser correction (see Methods). P values for the effects of treatment independent of renal artery pressure (P_treatment) were derived from F_1,10.

Results

Responses to Increased RPP

When RPP was increased step-wise from 65 to 160 mm Hg, MAP was reduced in a stimulus-dependent manner (Figures 1 and 2, Table 1). The rate at which MAP decreased during the course of the experiment was also strongly dependent on the level of RPP (Figure 2, Table 1). HR did not change appreciably over the course of the experiment (Figure 2, Table 1). Hematocrit responded in a biphasic manner to increased RPP; being slightly reduced (by 0.7 ± 0.3%) when RPP was increased from 65 to 85 mm Hg, but returning to baseline levels at higher RPP (Figure 2, Table 1). This likely reflects the balance between the hemodiluting effects of the constant infusion of plasma expander (see Methods) and the hemoconcentrating effects of the diuresis and natriuresis accompanying increased RPP (see below).

Levels of renal artery pressure, renal blood flow, and mean arterial pressure across the course of each experiment. The lines show the levels of each variable for each individual animal. Note that in two of the rabbits treated with vehicle the full experimental protocol could not be completed, as mean arterial pressure decreased to levels incompatible with continuation of the experiment during the penultimate experimental period.

Figure 1.

Effects of renal arterial infusion of 17-octadecynoic acid on systemic hemodynamic variables during step-wise increases in renal perfusion pressure. Each point is the average coordinate of six rabbits, except for the levels at the greatest renal artery pressure (160 mm Hg) in vehicle-treated animals (shown by the dotted line), where n = 4 because the experimental protocol could not be completed in two of the rabbits (see Figure 1). (○) Vehicle treatment (0.1 mL/min; 10% wt/vol 2-hydroxypropyl-β-cyclodextrin, 10% vol/vol ethanol in 250 mmol/L NaHCO₃). (•) 17-octadecynoic acid treatment (450 nmol/min). The outcomes of repeated measures analyses of variance, testing for the effects of renal artery pressure, and for the renal artery pressure-dependent and -independent effects of 17-octadecynoic acid treatment are shown in Table 1.

Figure 2.

Renal hemodynamics were also altered by increased RPP (Figure 3, Table 1). The RBF and GFR were increased in a monophasic stimulus-dependent manner. Changes in renal vascular resistance and filtration fraction followed biphasic patterns. Renal vascular resistance increased when RPP was increased from 65 to 85 mm Hg, but at greater RPP it was reduced. Filtration fraction increased with increasing RPP up to a pressure of 110 mm Hg, but was reduced as RPP was further increased.

Effects of renal arterial infusion of 17-octadecynoic acid on renal hemodynamic variables during step-wise increases in renal perfusion pressure. Lines, symbols, and statistical analyses are as for Figure 2.

Figure 3.

The rates of renal excretion of urine and sodium, expressed either in absolute terms or as a proportion to their filtered loads, was increased by increased RPP in a stimulus-dependent manner (Figure 4, Table 1).

In vehicle-treated rabbits, plasma renin activity averaged 8.4 ± 2.7 ng angiotensin I/mL per h when RPP was set at 65 mm Hg. Plasma renin activity was reduced to 6.5 ± 1.6, 5.1 ± 1.5, and 5.8 ± 1.3 ng angiotensin I/mL per h when RPP was set at 85, 130, and 160 mm Hg respectively.

Effects of renal arterial infusion of 17-octadecynoic acid on renal excretory variables during step-wise increases in renal perfusion pressure. Lines, symbols, and statistical analyses are as for Figure 2.

Figure 4.

Effects of 17-ODYA on Cytochrome P450-Dependent Arachidonate Metabolism

There was a tendency for the rate of 20-HETE formation in microsomes from the left (high pressure perfused) kidney of vehicle-treated rabbits to be less than that in microsomes from their right (nonperfused) kidney (27 ± 7%; P = .06) (Table 2). Nevertheless, there were significant differences in the rate of 20-HETE formation between the left and right kidneys of 17-ODYA-treated rabbits (46 ± 7%; P = .007), and between the left kidney of vehicle- and 17-ODYA-treated rabbits (41% difference between the means; P = .02) (Table 2).

Table 2.

20-Hydroxyeicosatetraenoic Acid (20-HETE) Formation by Microsomes Prepared From Experimental Rabbit Kidneys

	20-HETE Formation (pmol/min/mg protein)
Treatment	Right Kidney	Left Kidney
Vehicle (n = 5)	190 ± 32	133 ± 20
17-Octadecynoic acid (n = 6)	147 ± 15	79 ± 7*†

*

P < .05 for difference between vehicle and 17-octadecynoic acid treatment.

†

P < .01 for difference between left and right kidney. Note that kidneys were not removed from one of the vehicle-treated rabbits, so n = 5 for this group.

Effects of 17-ODYA on Responses to Increased RPP

The responses of MAP, HR, hematocrit, RBF, GFR, renal vascular resistance, filtration fraction, urine flow, sodium excretion, fractional urine excretion, and fractional sodium excretion to increased RPP were indistinguishable in the vehicle- and 17-ODYA-treated groups of rabbits (Figures 1, 2,3,4, Table 1). Plasma renin activity was also similar in the two groups of rabbits, and in the 17-ODYA-treated group averaged 9.0 ± 2.8, 6.1 ± 1.6, 4.9 ± 0.9, and 7.3 ± 1.6 ng angiotensin I/mL per h, respectively when RPP was 65, 85, 130, and 160 mm Hg. There was, however, a slight tendency for the rate of change of MAP to be attenuated in the 17-ODYA-treated rabbits when RPP was set at 130 mm Hg (Figure 2). Despite the fact that the overall response of MAP to increased RPP was not different in the two groups of rabbits (P = .32), the response when RPP was set at 130 mm Hg was further investigated by covariant analysis of the rate of change of MAP with the levels of 20-HETE production by microsomes prepared from the right and left kidneys of the experimental rabbits. We found no relationship between the rate of formation of 20-HETE (by microsome preparations from either the right or left kidney) and the rate of change of arterial pressure when RPP was set at 130 mm Hg (P ≥ .15; Figure 5).

Scatterplot of the rate of change of mean arterial pressure at a renal artery pressure of 130 mm Hg for each rabbit versus the rate of 20-hydroxyeicosatetraenoic acid (20-HETE) formation in (A) right kidneys (removed from each rabbit before establishment of the extracorporeal circuit and treatment with vehicle (○) or 17-octadecynoic acid (•)) and (B) the left kidneys that were treated with 17-octadecynoic acid (■) or its vehicle (□) and subjected to increased renal perfusion pressure. Note that kidneys were not taken from one of the vehicle-treated rabbits, so n = 5 for this group.

Figure 5.

Discussion

The major finding of the present study was that an intrarenal infusion of the suicide substrate inhibitor of cytochrome P450-dependent arachidonate metabolism, 17-ODYA, that reduced the rate of formation of 20-HETE by about 40%, had no significant effect on the decrease in blood pressure and changes in renal function produced by increases in RPP in anesthetized rabbits.

As previously observed,^33–37,43 we found that increasing RPP using an extracorporeal circuit initiated two renal antihypertensive mechanisms: increased urine and sodium excretion (pressure diuresis/natriuresis) and a depressor response that appears to be dependent on a humoral mechanism mediated within the renal medulla.^33,35,37 Both of these phenomena were stimulated in a RPP-dependent manner; the greater the level of RPP the greater the level of urine and sodium excretion and the greater the rate at which MAP was reduced. The increased urine flow and sodium excretion in response to increased RPP appear to be attributable both to increased GFR, and reduced tubular reabsorption, which were also changed in a RPP-dependent manner. The depressor response appears to be independent of the increased urine and sodium excretion, as hemoconcentration, and by inference, reduction in plasma volume, was minimal. Indeed, the level of hematocrit at the completion of the experiment, when RPP was ∼130 to 160 mm Hg, was not different from that at the beginning of the experiment, when RPP was ∼65 mm Hg.

There is evidence that the depressor response to increased RPP is initiated by release of a hormonal factor from the renal medulla, as it is independent of the autonomic nervous system but prevented by chemical destruction of the renal medulla with 2-bromoethylamine.³³ As we⁴ and others¹⁴ have found previously, the depressor response to increased RPP was not associated with increased HR, providing at least circumstantial evidence that autonomic reflexes do not increase sympathetic outflow in response to the reduced MAP. Taken together with the observation of reduced sympathetic nerve activity after unclipping renal hypertensive rats,^4,14 these observations indicate that the putative renal medullary depressor hormone has a sympathoinhibitory action.

The chemical nature of this hormone remains unknown, but it is not a prostanoid, platelet activating factor, or nitric oxide,^34,36,37 nor is it attributable to reduced activity of the renin/angiotensin system.³⁴ However, a body of evidence from studies in rats suggests that a neutral lipid substance (or group of substances), dubbed medullipin I by Muirhead,³ is released by increased RPP, including that induced by removal of the constricting clip in renovascular hypertension.^4,6 It has been proposed that this substance is a cytochrome P450-dependent metabolite of cellular lipids.³ This hypothesis recently received further support with the observation that renal arterial infusion of 17-ODYA (33 nmol/min) prevents the rapid normalization of arterial pressure after unclipping 1-kidney, 1-clip hypertensive rats.¹¹ 17-ODYA is an irreversible inhibitor of cytochrome P450 isozymes involved in long-chain fatty acid metabolism.^38,44 The hemodynamic effects of a range of cytochrome P450-dependent metabolites of arachidonic acid were also tested in this previous study, but none of these were found to resemble medullipin I in their effects.¹¹ Thus, the rapid antihypertensive effect of unclipping renovascular hypertensive rats appears to be dependent on cytochrome P450 lipid metabolism, but the substrate for this metabolism is unlikely to be arachidonic acid.

At present there is no evidence to suggest that the factors responsible for rapid normalization of arterial pressure after unclipping renovascular hypertensive rats are the same as those responsible for the depressor response to increased RPP in rabbits. Therefore, we tested the effects of intrarenal infusion of 17-ODYA (450 nmol/min) on the latter to determine whether it might also involve a cytochrome P450-dependent lipid. In previous experiments in rats, infusion of 17-ODYA (made up in an albumin solution) into the renal cortical interstitium at a dose of 16.5 nmol/min for 30 min increased papillary but not cortical blood flow, increased urine flow and sodium excretion, and reduced the ω-hydroxylase activity of subsequently prepared renal microsomes by 61 ± 9%.¹⁰ In contrast, renal arterial infusion of a dose of 33 nmol/min also increased urine and sodium excretion, but failed to significantly inhibit ω-hydroxylase activity in the renal cortex. This is presumably because 17-ODYA binds strongly to plasma proteins including the albumin used in these experiments, and therefore, was not filtered. To overcome this problem in the present study, we dissolved 17-ODYA in a solution containing 10% wt/vol 2-hydroxypropyl-β-cyclodextrin.⁴⁵ This is a ringed polymer of D-(+)-glucopyranose that has an apolar cavity and a polar external surface. It forms inclusion complexes with lipophilic molecules (such as 17-ODYA) that dissociate with dilution, as occurs when these solutions are injected into the bloodstream. Because the molecular size of 2-hydroxypropyl-β-cyclodextrin (average molecular weight 1542) allows it to be filtered by the kidney, we reasoned that this vehicle should provide access for 17-ODYA to both the tubular and vascular compartments. We also infused a much higher dose (450 nmol/min) directly into the renal artery for 60 min before the start of experimental measurements, and continued the infusion for the duration of the experiment (a further 80 to 100 min). This treatment reduced the ω-hydroxylase activity of microsomes prepared from the experimental kidneys by about 40%.

Despite the fact that 17-ODYA treatment significantly inhibited the metabolism of arachidonic acid by P450, it had no effect on the depressor response to increased RPP. Even at a RPP of 130 mm Hg, where there was at least a tendency for the rate of change of MAP to be less in the 17-ODYA-treated rabbits than in the vehicle-treated rabbits, no relationship could be established between the residual levels of P450 ω-hydroxylase activity and the rate of change of arterial pressure. This observation seems at odds with previous studies demonstrating that renal arterial 17-ODYA prevents the rapid normalization of arterial pressure after unclipping in 1-kidney, 1-clip hypertensive rats. This discrepancy has a number of possible explanations, including that renal cytochrome P450 activity was not inhibited sufficiently to prevent the formation of the renal medullary depressor hormone. There is also evidence that P450 metabolites of fatty acids are rapidly reincorporated and stored in membrane phospholipids.⁴⁶ Thus, a prolonged inhibition of P450 metabolism may be required to deplete pools of the medullary lipid. This would be particularly likely if the enzymatic step in the synthesis of the renal medullary depressor hormone catalyzed by renal cytochrome P450s is not the rate-limiting step in the synthetic pathway. However, because we have no information regarding the nature of the renal medullary depressor hormone in these experiments, this supposition is purely speculative. On the other hand, it must be noted that the renal arterial dose of 17-ODYA that prevented the rapid normalization of arterial pressure after unclipping in rats¹¹ does not inhibit the formation of ω-hydroxylase metabolites by renal microsomes in vitro.¹⁰ Thus, in the present study biochemical indices of cytochrome P450 activity were reduced more than in previous experiments showing clear physiologic effects. These observations suggest that the factors responsible for the rapid normalization of arterial pressure after unclipping in rats may be different from those responsible for the depressor response to increased RPP in the rabbit extracorporeal circuit model.

As well as having a proposed endocrine function (see above), there is considerable evidence that products of renal cytochrome P450-dependent fatty acid (particularly arachidonic acid) metabolism act locally to influence myogenic responses in the renal microcirculation. However, in the present study 17-ODYA treatment had no effect on the relationships between RPP and RBF, GFR and filtration fraction. This is consistent with previous studies in rats in which neither cortical interstitial nor renal arterial 17-ODYA affected total RBF or GFR.¹⁰ This is not to say that cytochrome P450 fatty acid metabolism does not have an important impact on the renal circulation, as 17-ODYA treatment has been shown to selectively increase papillary blood flow.¹⁰

There is also evidence that cytochrome P450-dependent fatty acid metabolism influences fluid and sodium reabsorption, perhaps by effects on papillary blood flow.¹⁰ However, under the present experimental conditions 17-ODYA treatment had no effect on the pressure diuresis/natriuresis relationship.

In summary, intrarenal infusion of 17-ODYA (450 nmol/min for 140 to 160 min) had no effect on the relationships between RPP and systemic hemodynamic, renal hemodynamic and renal excretory function in anesthetized rabbits equipped with an extracorporeal circuit for renal autoperfusion. These observations differ from those of previous studies, which have provided support for the view that cytochrome P450-dependent products of long-chain fatty acid (particularly arachidonic acid) metabolism can influence the renal response to increased RPP, at least in the rat. We cannot exclude the possibility that the present observations are attributable to the need to inhibit cytochrome P450 fatty acid metabolism by more than the 41% achieved in the present experiments, before functional consequences are observed. This seems unlikely, however, as previous studies have observed functional effects of renal arterial 17-ODYA infusion in rats under conditions where biochemical evidence of cytochrome P450 inhibition was lacking.^10,11 It is also possible that the present observations reflect species differences, between rats and rabbits, in the roles of cytochrome P450-dependent fatty acid metabolites in the control of renal exocrine and endocrine function. In particular, it may be that the hormonal antihypertensive response to unclipping renovascular hypertensive rats is mechanistically different from that induced by a sudden increase in RPP in the extracorporeal circuit model in rabbits. These issues require clarification by further study.