Department of Cell and Molecular Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7545
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ABSTRACT |
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We performed micropuncture studies to determine the role of
thromboxane A2 in the exaggerated
tubuloglomerular feedback (TGF) activity in young spontaneously
hypertensive rats (SHR). Glomerular function was assessed by changes in
proximal tubular stop-flow pressure (SFP) produced by different rates
of orthograde perfusion through Henle's loop. Seven-week-old SHR
exhibited an exaggerated TGF activity compared with Wistar-Kyoto rats
(WKY) during euvolemia, confirming earlier studies. During control
periods, the feedback-induced maximal SFP response (SFP) was greater
in SHR (18-19 vs. 12-13 mmHg in WKY), whereas basal SFP and
proximal tubular free-flow pressure were similar in both
strains. In one series, the thromboxane A2 agonist U-46619 was added to
the tubular perfusate for a final concentration of
10
6 M. In WKY,
SFP was
increased by 100% to 26 mmHg. In contrast,
SFP in young SHR was
unaffected by the thromboxane A2
agonist. In other animals, the thromboxane synthase inhibitor pirmagrel (50 mg/kg) was injected intravenously to inhibit thromboxane
production. In SHR, pirmagrel decreased
SFP by 8.5 mmHg
and reduced reactivity. Less attenuation was observed in WKY;
SFP
was reduced by 3 mmHg, whereas reactivity was unchanged. In other
studies, tubular perfusion with the thromboxane receptor inhibitor
SQ-29548 (10
6 M) reduced
SFP more in SHR (7 vs. 3 mmHg in WKY) and also decreased reactivity
more in SHR (2.3 vs. 0.5 mmHg · nl
1 · min
1).
Coperfusion of SQ-29548 and U-46619 resulted in an 85% block of the
effect of U-46619 on
SFP. Tubular perfusion with the agonist U-46619
during thromboxane synthase inhibition markedly enhanced
SFP in both
strains, with a greater effect in WKY. These results suggest that
elevated levels of thromboxane A2
in young SHR contribute to the exaggerated TGF control of glomerular
function in SHR during the developmental phase of hypertension.
genetic hypertension; juxtaglomerular apparatus; glomerular capillary pressure; macula densa; thromboxane inhibition
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INTRODUCTION |
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THE DEVELOPMENT of hypertension in the spontaneously hypertensive rat (SHR) is associated with several intrarenal abnormalities. Renal vascular resistance is elevated, whereas glomerular filtration rate (GFR) and renal blood flow are reduced in 7-wk-old SHR compared with normotensive Wistar-Kyoto rats (WKY) (14). Furthermore, tubuloglomerular feedback (TGF) control of glomerular capillary pressure is enhanced during the developmental phase of hypertension in SHR (7, 8, 12, 13). This exaggerated TGF activity is accompanied by decreased single-nephron GFR and increased preglomerular resistance (14). The reduced GFR and renal vasoconstriction may contribute to the salt and water retention observed in young SHR (3). In adult SHR with established hypertension, renal blood flow, GFR, and TGF activity are normalized (1, 13). An exaggerated TGF activity and salt and water retention may cause a shift in the pressure-natriuresis relationship and contribute to the progression of increased arterial pressure in these animals (3).
TGF activity is also altered in other models of genetic hypertension. The Milan young prehypertensive rats exhibited enhanced TGF responses compared with their normotensive controls (4). The mechanism(s) underlying the exaggerated TGF activity in these models of essential hypertension is not known. We have previously shown that blockade of ANG II, a well-known modulator of TGF activity, tends to normalize TGF responses in 7-wk-old SHR (7, 8). Administration of an AT1-receptor antagonist (losartan or candesartan) markedly reduces TGF activity in hypertensive-prone animals. During euvolemic conditions, AT1-receptor blockade normalizes the TGF response in young SHR to WKY values, whereas it has a minimal effect in normotensive rats. Attenuation of TGF control of glomerular function is also observed in SHR during acute volume expansion, although TGF activity remains enhanced compared with that of volume-expanded WKY (6). Furthermore, inhibition of ANG II action, via AT1 receptors, during volume expansion does not completely revert TGF responses in SHR to WKY values, indicating the involvement of additional modulating factors in the enhanced TGF activity in SHR during the development of hypertension.
In addition to ANG II, several other hormones and paracrine factors have been shown to modulate TGF activity. One factor of special interest with regard to SHR is thromboxane A2, a vasoconstrictor arachidonic acid product derived via the cyclooxygenase pathway. Previous studies on normotensive Sprague-Dawley rats showed that a stable analog of thromboxane A2 enhances maximal TGF responses (32). In this study, maximal TGF responses were reduced during thromboxane synthesis inhibition or thromboxane receptor antagonism, suggesting that endogenous thromboxane A2 plays a role in modulating TGF. However, other investigators have used thromboxane synthesis inhibitors or receptor antagonists and did not observe any changes in TGF activity in normal hydropenic rats (15, 23).
At the whole kidney level, thromboxane intra-arterial infusion decreases renal blood flow and GFR in normotensive animals at doses that do not substantially affect mean arterial blood pressure (2, 35). In 6-wk-old SHR, bolus injections of thromboxane A2 mimetic introduced into the renal artery cause greater reductions in renal blood flow than in WKY during cyclooxygenase inhibition (11). The greater vascular reactivity to thromboxane in young SHR during cyclooxygenase blockade may be due to 1) a more effective buffering action of vasodilatory metabolites such as prostaglandin I2 or prostaglandin E2, 2) elevated basal levels of thromboxane A2 in young SHR, or 3) a combination of the two. In fact, increased production of thromboxane in SHR has been found in isolated glomeruli and kidneys, as well as in renal excretion in whole animals (19, 24, 27). Both young and adult SHR display enhanced thromboxane formation compared with WKY; however, this strain difference in thromboxane generation becomes less pronounced in older animals (19).
Studies investigating the possible role of thromboxane in the development of hypertension in SHR have yielded conflicting results. Chronic inhibition of thromboxane synthesis and thromboxane receptor blockade, alone or in combination, have been shown to improve kidney function and decrease mean arterial pressure in the long term (24) or transiently for 1 wk (26) or have had no effect (16).
The purpose of the present study was to determine the role of thromboxane A2 in the exaggerated TGF activity in young SHR. TGF responses were assessed by stop-flow pressure (SFP) measurements during changes in late proximal tubular perfusion rate. We evaluated the effect of exogenous thromboxane A2 by adding the stable analog U-46619 to the tubular perfusate. We assessed the involvement of endogenous thromboxane A2 using the thromboxane synthesis inhibitor pirmagrel and the thromboxane receptor antagonist SQ-29548. We also tested the effect of the thromboxane A2 mimetic on TGF activity during reduced levels of endogenous thromboxane A2.
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METHODS |
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We performed experiments on 7-wk-old SHR and age-matched WKY obtained from our Chapel Hill breeding colonies using standard methodology for our laboratory. The rats were allowed free access to food and water until the day before the experiment, when food, but not water, was removed. The rats were anesthetized by an intraperitoneal injection of pentobarbitol sodium (65 mg/kg body wt) and body temperature was kept at 37.5°C by placement of the animals on a servo-regulated heating table. We performed a tracheotomy to facilitate spontaneous breathing. The left jugular vein was cannulated for supplementary doses of anesthetic and for administration of isoncotic BSA (4.7 g/dl). The initial rate of albumin administration was 50 µl/min, to replace losses during surgery (1.25 ml/100 g body wt), and the experimental rate was 5 µl/min for the duration of an experiment. Previous studies in our laboratory have demonstrated that this protocol maintains hematocrit and plasma protein concentration at presurgical levels during control conditions of euvolemia (7, 13). We cannulated the right femoral artery to obtain blood samples and to record mean arterial pressure (Statham P23Db transducer). The right femoral vein was cannulated for saline infusion at a rate of 10 µl/min. We cannulated the bladder to allow free urine flow. The left kidney was exposed through an abdominal midline incision and a lateral subcostal incision, dissected free from adhering tissue, and placed in a Lucite cup. The kidney was loosely surrounded with saline-soaked cotton, and the cup was filled with a 3% agar solution. We covered the kidney surface with saline to prevent drying.
After surgery, the animals were allowed to stabilize for 1 h before
observations were started. We determined TGF characteristics in
superficial nephrons by proximal tubular SFP determinations, an index
of glomerular capillary pressure, using standard micropuncture methods
combined with perfusion of Henle's loop, as described previously (4,
10). Random proximal convolutions were punctured with a sharpened glass
pipette, with an outer diameter (OD) of 3-4 µm, filled with a
2-M NaCl solution stained with FD&C green no. 3 dye. We
connected the pipette to a servo-null device (Department of Physiology,
University of North Carolina, Chapel Hill, North Carolina) to measure
proximal tubular pressure during free-flow conditions. A second pipette
(OD 7-9 µm), connected to a microperfusion pump (Klaus
Effenberger, Pfaffing/Attel, Germany), was positioned in the last
accessible coil of the proximal convolution and used to perfuse
Henle's loop in the orthograde direction with artificial tubular
fluid. The composition of the artificial tubular fluid solution was (in
mM) 140 NaCl, 4 NaHCO3, 5 KCl, 2 CaCl2, 1 MgCl2, 7 urea, and 2 g/l FD&C
green no. 3 dye. We used a third pipette (OD 7-8 µm) to inject
an immobile wax block in a segment of the midproximal tubule to obtain
SFP and to isolate the pressure and perfusion pipettes. The SFP was
measured in an early proximal convolution while the loop of Henle was
perfused at different rates between 0 and 40 nl/min, starting either at
40 or 0 nl/min. The perfusion rate was changed in steps of 2.5-5
nl/min and kept at each rate for 1-5 min, as required to observe a
stable response. The maximal decrease in SFP (SFP) was recorded at
high rates of perfusion (30-40 nl/min). We calculated the
perfusion rate that elicited a half-maximal decrease in SFP, designated
as turning point (TP), and slope or reactivity (R) by best-fit
regression analysis of data for each nephron using SigmaPlot software
(SPSS Scientific, Chicago, IL) and the logistic equation
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Series A: Thromboxane
A2 agonist.
We performed measurements of SFP during a control period using
artificial tubular fluid (ATF) for orthograde tubular perfusion and
during an experimental period when the stable thromboxane A2 receptor agonist U-46619
(Cayman Chemicals, Ann Arbor, MI) was added to ATF for a final
concentration of 106 M.
Series B: Thromboxane synthase inhibition. TGF determinations were made during a control period after intravenous injection of vehicle and during thromboxane synthesis inhibition produced by intravenous administration of the thromboxane synthase inhibitor pirmagrel (CGS-13,080, 50 mg/kg in 200 µl saline; Novartis, Summit, NJ). This dose has been reported to decrease urinary thromboxane B2 excretion by 51% (31). In the experimental period, which lasted 60 min, TGF determinations were begun 15 min after pirmagrel administration.
Series C: Thromboxane receptor
antagonist.
We added the thromboxane receptor antagonist SQ-29548 (Cayman
Chemicals) to the perfusate in the experimental period to yield a final
concentration of 106 M. A
stock solution of SQ-29548 was prepared in ethanol (10 mg/ml), and
dilutions were made in saline on the day of an experiment. The efficacy
of receptor blockade was tested by coperfusion of SQ-29548 and U-46619
in 11 nephrons in 5 WKY rats.
Series D: Thromboxane synthase inhibition and thromboxane agonist. To evaluate the influence of basal levels of thromboxane, SFP measurements were made when thromboxane synthesis was inhibited throughout the experiment. After a control period, U-46619 was added to the perfusate. Pirmagrel was administered, as described above, 15 min before the start of the control period. To obtain a sustained reduction in endogenous levels of thromboxane, we gave a second dose 15 min before initiating experimental observations.
We tested all measurements by Student's t-test for strain difference and intervention effect using SigmaStat software (SPSS Scientific, Chicago, IL). Values are means ± SE. P < 0.05 was considered statistically significant. ![]() |
RESULTS |
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Age, hematocrit, left kidney weight, and body mass of WKY and SHR
within each protocol were similar (Table
1). Mean arterial pressure was higher in
7-wk-old SHR compared with WKY. Hematocrit remained stable throughout
the experiments. Mean arterial blood pressure was stable throughout the
assessment of TGF activity and was statistically unchanged over the
entire duration of the experiments. Systemic administration of the
thromboxane synthase inhibitor pirmagrel did not affect mean arterial
pressure in either strain.
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Control proximal tubular SFP and free-flow pressure were
similar in the two strains during all experimental series (Table 2). Proximal free-flow pressure averaged
11-13 mmHg. SFP varied between 39 and 43 mmHg. Sigmoidal-shaped
SFP response curves during euvolemia and tubular perfusion with U-46619
are shown in Fig. 1. SFP measurements
during the control period revealed exaggerated TGF activity in young
euvolemic SHR, confirming previous results from this and other
laboratories (7, 12, 13). The exaggerated TGF activity was evidenced by
a greater maximal SFP response in young SHR (18 vs. 13 mmHg in WKY) and
a lower turning point (13 vs. 16 nl/min in WKY). Young
euvolemic SHR also displayed greater reactivity, as evidenced by a
steeper slope at the turning point (5.4 vs.
2.5
mmHg · nl
1 · min).
In the experimental period, the SFP response during high perfusion
rates with the thromboxane A2
analog was substantially increased in WKY (26 vs. 13 mmHg during
control conditions). However, neither turning point (17 vs. 16 nl/min)
nor reactivity (
4.1 vs.
2.6
mmHg · nl
1 · min;
P = 0.2) were affected statistically
by U-46619 perfusion. The thromboxane mimetic decreased reactivity
(
3.0 vs.
5.4
mmHg · nl
1 · min)
in SHR but had no discernable effect on the other TGF parameters.
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Figure 2 shows the effect of systemic
inhibition of thromboxane synthase by pirmagrel on SFP responses. SFP
responses during control conditions are in close agreement with those
obtained during the first series (Table 2). SHR exhibited greater
SFP (19 vs. 12 mmHg) and reactivity (
5.7 vs.
2.0
mmHg · nl
1 · min),
and lower turning point (13 vs. 16 nl/min). Pirmagrel administration
markedly attenuated TGF responses in SHR. A smaller reduction of the
maximal SFP response was observed in WKY (3.1 vs. 8.5 mmHg or
25% vs. 47% of the control response). As can be seen in Fig.
2, the thromboxane A2 synthase
inhibitor had a larger effect on TGF in the physiological range of
tubular flow rate (~15 nl/min). In SHR, reactivity
decreased from
5.7 to
1.8 mmHg · nl
1 · min,
whereas the turning point was unchanged. In WKY, the turning point and
reactivity remained at 16 nl/min and
2.0
mmHg · nl
1 · min,
respectively. Thus decreased thromboxane production shifted the TGF
response curves upward in both strains, but more prominently so in SHR.
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Figure 3 shows the TGF responses during a
control period of no drug and during an experimental period with the
thromboxane receptor antagonist SQ-29548. During control conditions,
TGF responses are enhanced in SHR compared with WKY, and TGF parameters
agree well with those obtained in the first and second experimental series of the present study (Table 2). Luminal application of SQ-29548
resulted in attenuated maximal SFP responses. The maximal SFP response
was reduced more by the receptor antagonist in SHR (from 19 to 12 mmHg)
than in WKY (from 13 to 9 mmHg). The turning point remained unchanged
in both groups of rats. Thromboxane receptor blockade reduced TGF
reactivity in SHR (5.4 to
3.1
mmHg · nl
1 · min)
with no effect in WKY (
2.2 vs.
1.7
mmHg · nl
1 · min).
The effectiveness of thromboxane receptor blockade was documented in
WKY (Fig. 4). The mimetic U-46619 increased
the maximal SFP response from 13 to 26 mmHg. This effect was reversed by the receptor antagonist. Coperfusion of U-46619 and SQ-29548 reduced
the stimulatory effect of U-46619 on maximal SFP responses by 10 mmHg,
representing 85% blockade.
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In the next series, we administered pirmagrel to inhibit thromboxane
synthesis throughout an experiment, including control and experimental
periods. In the control period, TGF response curves were similar in SHR
and WKY (Table 2 and Fig. 5). No
significant differences were noted in maximal SFP responses (9 vs. 12 mmHg), turning point (15 nl/min), or reactivity (2.0
mmHg · nl
1 · min).
These feedback-mediated responses agree closely with those obtained
under identical conditions in series B
(Table 2). Loop of Henle perfusion with U-46619 during thromboxane
synthase inhibition caused substantial enhancement of maximal SFP
responses in both strains. In SHR, maximal SFP responses increased (22 vs. 12 mmHg), although no significant changes were observed in
reactivity or turning point. In WKY, loop perfusion with U-46619
markedly increased
SFP (27 vs. 9 mmHg), caused a small decrease in
turning point (13 vs. 15 nl/min), and had no significant effect on
reactivity (3.8 vs.
2.0
mmHg · nl
1 · min).
Thus the thromboxane mimetic had a greater effect on maximal TGF
responses and on reactivity in WKY.
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DISCUSSION |
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The present study provides new information about the role of
thromboxane A2 in the exaggerated
TGF activity found in young SHR. To facilitate comparisons among the
four different series of experiments, the major effects of stimulation
and inhibition of this system on SFP responses at 15 and 40 nl/min
perfusion rates and on feedback reactivity are summarized in Fig.
6. Systemic administration of the
thromboxane synthase inhibitor pirmagrel reduced feedback reactivity at
intermediate loop of Henle flow rates and blunted the maximal SFP
response elicited by supranormal flow rates. Attenuation of these
measures of TGF activity was more pronounced in SHR. Tubular perfusion
with the thromboxane A2 receptor
antagonist SQ-29548 yielded similar results. Accordingly, significant
reductions in feedback reactivity and SFP responses at mid- and high
perfusion rates were noted in SHR, more so than in WKY. On the other
hand, tubular perfusion with the stable thromboxane A2 analog U-46619 caused a
substantial enhancement of the maximal SFP response in young WKY but
not in SHR. During thromboxane synthesis inhibition with pirmagrel,
perfusion of the receptor agonist U-46619 caused a greater SFP response
at 15 and 40 nl/min perfusion rate in both strains, with a more
pronounced effect in WKY. Collectively, these results show that the
thromboxane mimetic had a smaller stimulatory effect on TGF in SHR and
that endogenous thromboxane A2
enhanced TGF control of preglomerular resistance, assessed as SFP
responses and reactivity in young SHR during basal conditions.
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Our results verify enhancement of maximal TGF responses by tubular perfusion with U-46619 in WKY, consistent with a previous report on normotensive Sprague-Dawley rats based on measurements of SFP and single-nephron GFR during basal conditions (32). This effect appears to be independent of the route of administration in as much as intravenous infusion, peritubular capillary infusion, or tubular perfusion of the agonist caused similar changes in TGF responses. The influence of endogenous thromboxane A2 on TGF activity in normotensive animals during normal conditions may be negligible, however. Earlier studies have demonstrated that thromboxane receptor antagonism or synthase inhibition has no effect on SFP responses in normotensive animals (15, 23). More recently, mice with a targeted deletion of the only thromboxane receptor known to date were found to have normal TGF responses (25). Basal thromboxane A2 production is known to be increased in pathological states such as chronic hydronephrosis, a case in which thromboxane synthase inhibition improves renal blood flow and excretory function of hydronephrotic kidneys (18, 22). Thromboxane synthesis inhibition has been found to attenuate the enhanced TGF activity in volume-expanded hydronephrotic kidneys (23). These findings are consistent with our present data, indicating a greater effect of thromboxane synthase inhibition and receptor blockade on TGF activity during the pathogenic phase of hypertension in young SHR.
The mechanism(s) by which thromboxane A2 receptor stimulation enhances TGF is not known. One possibility is that thromboxane A2 affects TGF at the sensing step by altering Na-K-2Cl cotransport by macula densa cells. Evidence from micropuncture measurements indicates that thromboxane A2 stimulates chloride reabsorption in the loop of Henle, defined in micropuncture studies as the nephron segment between late proximal tubule and earliest accessible distal tubule on the kidney surface (32). If the action were limited to the ascending limb of Henle's loop, a decreased NaCl delivery to macula densa cells would be predicted to reduce TGF control of vascular resistance and increase glomerular capillary pressure. However, stimulated NaCl reabsorption by macula densa cells may offset the expected decrease in NaCl delivery. Coperfusion of U-46619 with the inhibitor of the Na-K-2Cl cotransporter furosemide blunts, but does not completely block, the stimulatory effect of thromboxane receptor stimulation (32). In most cases, TGF responses are completely abolished by furosemide at the concentration used in that study. This observation raises the possibility that U-46619 may enhance TGF responses by a direct vasoconstrictor action on afferent arterioles. To this end, the thromboxane A2 mimetic is known to reduce glomerular plasma flow rate in normal rats in vivo and to preferentially constrict the afferent arteriole in the hydronephrotic kidney in vitro (2, 17). Immunolocalization of thromboxane receptors indicates that both mechanisms are conceivable. Immunoreactive staining has been found on the luminal border of the thick ascending limb of Henle's loop, as well as in renal arteries and arterioles (29). However, there was no mention of immunoreactive staining of the macula densa region located at the end of the thick ascending limb of Henle's loop. It is conceivable that our results regarding a stronger effect of U-46619 perfusion on TGF in WKY than in SHR is due to differences in thromboxane receptor density or signal transduction. One receptor ligand binding study has shown a greater density of thromboxane receptors in isolated glomeruli from young SHR compared with WKY (10). At this point, there is little information known about the effects of intrarenal thromboxane A2 concentration on vascular and tubular receptor transduction and regulation. However, platelet and aortic membrane thromboxane A2 receptor density increased in pigs after a 6-wk treatment with a thromboxane receptor antagonist or synthesis inhibition by aspirin (9).
There is a high probability that the observed effects of U-46619 are due to its binding to and stimulation of a specific thromboxane receptor because the mimetic has been shown to have excellent specificity with essentially no cross-reactivity with other receptors. In this regard, a recent study shows that the systemic pressor effect of U-46619 is absent in homologous mice lacking the thromboxane receptor because of gene targeting (25). To test the efficacy of receptor inhibition in our animals, we coperfused nephrons with the mimetic and the receptor antagonist SQ-29548. We found that receptor inhibition blocked 85% of the TGF enhancing effect of the thromboxane A2 mimetic. A similar degree of blockade was found by other investigators using the same protocol and pharmacological agents in a study in which single-nephron GFR was measured in normotensive rats (32). It is noteworthy that this receptor antagonist completely blocks the U-46619-induced intracellular calcium increase in vascular smooth muscle cells at an equimolar dose (20).
The present data demonstrate a greater attenuation of TGF in SHR during blockade of thromboxane synthase or receptor inhibition, suggesting that elevated levels of endogenous thromboxane A2 exert a stimulatory influence on TGF-mediated control of glomerular function during basal euvolemic conditions. This view is supported by our finding that the weak U-46619 effect in SHR can be reversed by thromboxane synthase inhibition. Consistent with this notion, a number of studies have shown enhanced thromboxane generation in SHR (16, 19, 24, 27). Animal studies reveal that urinary excretion rate and serum levels of the stable metabolite thromboxane B2 are increased in SHR (16, 24). An enhanced thromboxane production has also been shown in vitro in isolated perfused kidneys and in isolated glomeruli from 6- to 8-wk-old SHR (19, 27).
Despite the large body of evidence for enhanced thromboxane A2 production in SHR, studies investigating beneficial effects on blood pressure or kidney function of thromboxane synthase or receptor inhibition have reported variable results. Grone et al. (16) have shown that chronic or acute treatment with thromboxane synthase or receptor inhibitor, alone or in combination, has no effect on blood pressure, renal hemodynamics, or GFR in 6- to 8-wk-old SHR under euvolemic conditions. The present data show predominant changes due to thromboxane A2 blockade in maximal TGF responses, a measure of TGF activity reflecting a decrease in TGF control of glomerular function when tubular flow rates are supernormal. However, thromboxane A2 appears to have a weaker influence on turning point and reactivity assessed at intermediate tubular flow rates, in agreement with the lack of change in GFR when ambient tubular flow is in the normal range. In conscious SHR, administration of a combined thromboxane synthase and receptor inhibitor, via osmotic minipumps, is reported to moderately reduce systolic arterial pressure between 6 and 11 wk of age (5). The hypotensive effect was most likely due to blockade of thromboxane receptors because thromboxane production, as measured by urinary excretion of thromboxane B2, was unaffected by treatment. Other studies have indicated a temporary decrease in blood pressure during thromboxane synthase inhibition, without preventing the final development of hypertension at 8 wk of age (26, 28). Reasons for the discrepancies among the studies are not clear. But it has been suggested that the reduced blood pressure reported in some studies using thromboxane synthase inhibitors may be due to nonspecific actions of the drug or to increased production of vasodilator prostaglandins (16, 26, 30). These explanations do not hold for the study reporting hypotensive effects by a highly selective thromboxane receptor inhibitor, however (5).
Another possibility is an interaction between the effect of thromboxane and ANG II. ANG II is known to enhance production of prostaglandins and thromboxane (21, 34). Conversely, thromboxane A2 has been reported to reduce plasma renin activity (33). Evidence from some laboratories suggests that acute and chronic ANG II-induced increases in mean arterial pressure and renal vascular resistance and reductions in GFR are blunted by treatment with thromboxane synthase or receptor inhibitors (21, 34). However, no interaction between ANG II and thromboxane A2 on TGF activity was found in normotensive animals when combinations of inhibitors were given systemically (31). We have previously shown that the exaggerated TGF activity in young SHR can be normalized by selective AT1-receptor antagonists (7, 8). At face value, these results were interpreted to mean that ANG II had a direct action on the juxtaglomerular apparatus to enhance TGF. The fact that inhibition of the thromboxane system had similar effects introduces the notion of an interaction between ANG II and thromboxane. One may speculate that the attenuation of TGF activity observed during AT1-receptor blockade may be due to the effect of reducing ANG II stimulation of thromboxane A2 production. There are, however, some subtle differences between the effects of thromboxane A2 and ANG II on TGF. Thromboxane A2 synthase or receptor inhibition mainly affects feedback reactivity and maximal TGF responses, whereas AT1-receptor blockade increases the turning point, in addition to reducing reactivity and maximal TGF responses (7, 8). These differences may be explained by the various mechanisms responsible for ANG II- and thromboxane A2-induced modulation of TGF activity. One possible scenario is that thromboxane A2 has a dual effect, primarily enhancing TGF responses at the macula densa sensing site, but also increasing the TGF-induced vasoconstriction in cooperation with ANG II. This hypothesis can explain why an interaction is not readily discernable through the use of TGF determinations and why ANG II-induced changes in vascular tonus are attenuated by thromboxane inhibition. Nevertheless, several other alternatives are plausible; for example, the modulating effect of thromboxane A2 on TGF activity may be mediated by ANG II.
In summary, our results show a marked enhancing effect on maximal TGF responses of the stable thromboxane A2 analog U-46619 in 7-wk-old WKY, but not in age-matched SHR. During control conditions, TGF activity is exaggerated in 7-wk-old SHR. This relationship was reversed by tubular perfusion of the thromboxane A2 mimetic as the increase in TGF activity in WKY exceeded that in SHR. Conversely, thromboxane synthesis inhibition and receptor blockade attenuated TGF activity to a greater extent in SHR. Inhibition of thromboxane A2 production and its action in SHR reduce feedback control of glomerular function close to that in WKY. Thus the differences in the effect of or response to the thromboxane A2 mimetic appear to be due to, at least in part, an elevated thromboxane production in young SHR. Increased endogenous levels of thromboxane A2 contribute to the enhanced TGF activity in young SHR, which may lead to a rightward shift in the pressure-natriuresis curve, promoting the development of hypertension.
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ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Research Grant HL-02334. Pirmagrel was kindly provided by Novartis Pharmaceuticals (Summit, NJ).
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: W. J. Arendshorst, Dept. of Cell and Molecular Physiology, CB# 7545, Rm. 152 Medical Sciences Research Bldg., Univ. of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7545 (E-mail: arends{at}med.unc.edu).
Received 4 December 1998; accepted in final form 12 February 1999.
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