Departments of 1 Pediatrics, 2 Internal Medicine, and 3 Biochemistry, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235-9063
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ABSTRACT |
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The kidney has the highest abundance of
cytochrome P-450 of all extrahepatic organs. Within the kidney,
the highest concentration of cytochrome P-450 is found in the
proximal tubule. Whether 20- or
19(S)-hydroxyeicosatetraenoic acid (HETE), the major
P-450 metabolites of arachidonic acid in the proximal tubule,
affect transport in this segment has not been previously investigated. We examined the direct effects of 20- and 19(S)-HETE on volume absorption (Jv) in the rabbit proximal straight
tubule (PST). Production of 20-HETE by rabbit PST was demonstrated by
incubating microdissected tubules with
[3H]arachidonic acid and separating the lipid
extract by HPLC. There was significant conversion of
[3H]arachidonic acid to 20-HETE in control
tubules that was inhibited by 105 M
N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS). Addition of exogenous 20-HETE had no effect on PST volume transport. However, inhibition of endogenous production of 20-HETE using DDMS stimulated transport. In the presence of DDMS, 20-HETE inhibited PST
Jv. 19(S)-HETE in the bathing solution
stimulated PST Jv alone and in the presence of
DDMS. Thus
- and
-1-hydroxylase products of arachidonic acid have
direct effects on PST transport. Endogenous production of 20-HETE may
play a role in tonic suppression of transport and may therefore be an
endogenous regulator of transport in the proximal tubule.
in vitro microperfusion; cytochrome P-450; -hydroxylase; hypertension; sodium-potassium-adenosinetriphosphatase; hydroxyeicosatetraenoic acids
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INTRODUCTION |
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THE MAMMALIAN RENAL CORTEX is an abundant source of
cytochrome P-450 isozymes (21, 32). Within the kidney, the
highest concentration of cytochrome P-450 is found in the
proximal tubule (12). The isozymes present in the rat proximal tubule
have recently been shown to be in the CYP4A class and metabolize
arachidonic acid predominately by the -hydroxylase pathway (17).
Microsomes from rabbit renal cortex have also been shown to metabolize
arachidonic acid by the
-hydroxylase pathway to 20- and
19(S)-hydroxyeicosatetraenoic (HETE) acids (23).
Metabolites of the renal cytochrome P-450 -hydroxylase
system have multiple effects on renal function. Infusion of 20-HETE into rat renal arteries resulted in natriuresis without affecting glomerular filtration rate or systemic blood pressure (34). Inhibition
of endogenous
-hydroxylase activity by infusion of 17-octadecynoic
acid into rat renal arteries also caused an increase in urine flow rate
and sodium excretion (38). These results are difficult to interpret,
because these compounds are known to alter renal hemodynamics and
affect transport in multiple nephron segments (1, 3, 13, 14, 16, 22,
27, 31). 20-HETE constricts canine renal arcuate arteries (22) and rat
renal arterioles (16) and increases renal vascular resistance (1). The
vascular effects of 20-HETE also play a role in autoregulation and
tubuloglomerular feedback (36, 37). 20-HETE has been shown to modulate
thick ascending limb sodium chloride transport (3, 13, 14, 31). Cells
from the thick ascending limb produce 20-HETE in culture, and
production is stimulated by arginine vasopressin and calcitonin (13,
31). 20-HETE inhibits transport in the thick ascending limb by blocking
the Na-K-2Cl cotransporter (3, 14). This inhibitory effect of 20-HETE
on transport may be part of the signal transduction mechanism for the
inhibition of transport by pharmacological doses of angiotensin II in
this segment (4).
The proximal tubule is a major site of renal cytochrome P-450 (12, 17, 22). It has been estimated that the majority of 20-HETE produced by the kidney comes from the proximal tubule (20). The effects of 20-HETE on proximal tubule transport remain unknown. The purpose of the present study was to directly examine the effect of 20- and 19(S)-HETE on volume transport in the in vitro microperfused rabbit proximal straight tubule (PST).
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METHODS |
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Production of 20-HETE.
The production of 20-HETE from rabbit PST was measured using a
modification of the method developed by Ito and Roman (18). Briefly,
PST from rabbit kidneys were dissected as described below and
transferred to a test tube in Hanks' solution. The tubules were
permeabilized with three freeze-thaw cycles using liquid nitrogen to
snap freeze the tissue, followed by thawing in warm water. The tubules
were then centrifuged at 4°C at 2,000 rpm for 5 min. The
supernatant was aspirated, and the cells were resuspended in 1 ml of a
buffer containing (in mM) 100 potassium phosphate (pH 7.4), 10 MgCl2, and 1 EDTA. [3H]arachidonic
acid (4 µCi/ml; New England Nuclear, Boston, MA), 1 mM NADPH, 10 mM
isocitrate, and 0.4 U/ml isocitrate dehydrogenase (as an NADPH
generating system) were then added to each tube. The tubes were then
incubated at 37°C for 60 min with 100% O2 blown over
the tops. Three tubes were control with vehicle added, and three tubes
were treated with 105 M
N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS). One tube
contained the solutions without tubules to assess the rate of
nonenzymatic conversion to 20-HETE. The reaction was terminated by
adding 250 µl of 1 M formic acid. The lipids were then extracted by
adding 2 ml of chloroform, vortexing, centrifuging at 2,000 rpm for 5 min, and then drying under nitrogen. The samples were then resuspended
in 20 µl of ethanol and separated by HPLC using a C18
column (150 × 2.1 mm, 3 µm, ODS,
Hypersil; ThermoQuest, San Jose, CA). The mobile phase was
an acetonitrile:water:acetic acid (62.5:37.5:0.5) gradient to 100%
acetonitrile in 20 min. Retention times for 20-HETE and arachidonic
acid were determined by running standards under the same conditions.
The fraction that came off at 7 min corresponded to 20-HETE, and the
fraction at 19 min corresponded to arachidonic acid. Using this system,
we found it was not possible to separate 19- from 20-HETE. The aqueous phase was saved to determine the protein concentration by bicinchoninic acid assay (BCA; Pierce Chemical, Rockford, IL).
In vitro microperfusion. Superficial PST (S2 segment) from New Zealand White rabbits were perfused in vitro as previously described (11, 26). Briefly, PST were dissected in cooled (4°C) modified Hanks' solution containing (in mM) 137 NaCl, 5 KCl, 0.8 MgSO4, 0.33 Na2HPO4, 0.44 KH2PO4, 1 MgCl2, 10 Tris · HCl, 0.25 CaCl2, 2 glutamine, and 2 L-lactate. This solution was bubbled with 100% O2 and had a pH of 7.4. Tubules were then transferred to a 1.2-ml thermostatically controlled (37-38°C) bathing chamber and perfused with concentric glass pipettes. The perfusion solution simulated an ultrafiltrate of plasma and contained (in mM) 115 NaCl, 25 NaHCO3, 2.3 Na2HPO4, 10 sodium acetate, 1.8 CaCl2, 1 MgSO4, 5 KCl, 8.3 glucose, and 5 alanine. The bathing solution was similar, but contained 6 g/dl of albumin. All solutions were bubbled with 95% O2 and 5% CO2 at 37°C and had a pH of 7.4. The osmolalities of the perfusion and bathing solutions were adjusted to 295 mosmol/kgH2O by the addition of water or NaCl. The bathing solution was exchanged at a rate of 0.5 ml/min to keep the osmolality and pH constant.
The control period began after a 60-min incubation period. Volume absorption (Jv; in nl · min ![]() |
RESULTS |
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20-HETE production.
Rabbit proximal tubules were shown to metabolize arachidonic acid to
20-HETE. The control rate of conversion was 47.7 ± 3.4 (0.18 ± 0.02% · µg
protein1 · 60 min
1). DDMS significantly reduced this
conversion rate to 17.7 ± 1.8% · tube
1 · 60 min
1 (0.05 ± 0.01% · µg
protein
1 · 60 min
1; P < 0.01, n = 3). The nonenzymatic conversion of arachidonic acid to 20-HETE was
minimal
(9.5% · tube
1 · 60 min
1) and was approximately the same as
the DDMS-treated tubules. Thus rabbit PST are capable of producing
20-HETE and this is inhibited by DDMS.
In vitro microperfusion.
The first series of experiments was designed to determine whether
20-HETE had a direct effect on PST Jv. During the
experimental period in these series, 20-HETE
(106 M or
10
5 M) was added to the bathing
solution. 20-HETE had no direct effect on Jv
[control, 0.50 ± 0.10 vs. 10
6 M
20-HETE, 0.50 ± 0.12 nl · min
1 · mm
1;
n = 4, P = not significant (NS); and control, 0.45 ± 0.12 vs. 10
5 M 20-HETE, 0.46 ± 0.14 nl · min
1 · mm
1;
n = 6, P = NS]. There was also no change in the
PD (control,
1.2 ± 0.3 vs. 10
6
M 20-HETE,
1.2 ± 0.2 mV; and control,
2.2 ± 0.9 vs.
10
5 M 20-HETE,
2.3 ± 0.8 mV;
P = NS).
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DISCUSSION |
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The present study examined the direct effects of -hydroxylase
products of arachidonic acid on proximal tubule volume transport. We
demonstrated that rabbit PST are capable of converting arachidonic acid
to 20-HETE. This conversion is inhibited by DDMS. Addition of exogenous
20-HETE, the major product of
-hydroxylase, had no direct effect on
PST volume transport. Inhibition of 20-HETE production with the
-hydroxylase inhibitor, DDMS, stimulated volume transport in the
PST. This implies that the high endogenous production of 20-HETE may
play a role in suppressing transport rates. In the presence of DDMS,
exogenous 20-HETE had a significant effect to inhibit transport. The
-1 product of arachidonic acid, 19(S)-HETE, stimulated
transport regardless of whether DDMS was present. This indicates that
19(S)-HETE directly stimulates transport without having to
affect 20-HETE metabolism. Thus the
-hydroxylase products of
arachidonic acid play a role in the control of volume transport in the PST.
Although the effects of these compounds on proximal tubule transport
have not been previously examined, their effects on Na-K-ATPase activity have been studied (15, 25, 27). 20-HETE inhibits Na-K-ATPase
in this segment and is thought to play a role in the effect of
parathyroid hormone and dopamine to inhibit transport in this segment
(25, 27). 19(S)-HETE, the major -1 product of
-hydroxylase, has been shown to stimulate rat renal Na-K-ATPase activity (15). Although Na-K-ATPase activity in the proximal tubule is
a determinant of solute and volume transport, effects on the
Na-K-ATPase do not always correlate with effects on
Jv. Dopamine, for example, has been shown to
inhibit proximal convoluted tubule Na-K-ATPase but has no direct effect
on transport in this segment (5, 9, 10). Thus the relationship between
regulating Na-K-ATPase activity and Jv rates is
complex and indicates the importance of directly examining proximal
tubule transport.
Changes in Jv rates in the present study were not associated with changes in PD. This is in contrast to previous studies in which increases in Jv rate correlated with increases in the PD (6) and decreases in transport correlated with decreases in the PD (8, 29). This suggests that the mechanism by which the Jv rates were increased may be due to changes in electroneutral transport and not due to direct changes in the Na-K-ATPase (7).
The proximal tubule reabsorbs between 60 and 70% of the glomerular
ultrafiltrate (30). Thus small changes in the volume absorption rate in
this nephron segment lead to large changes in overall fluid balance of
the organism. Inhibiting -hydroxylase activity in this nephron
segment led to an increase in transport by 16-28%, which could
then cause volume overload and hypertension. This indicates that renal
P-450
-hydroxylase may be involved in extracellular fluid
volume and blood pressure regulation.
The role of the renal P-450 system in the development of hypertension has been complex. The Dahl salt-sensitive rat (SS) is a model of hypertension that has been extensively studied, and renal cytochrome P-450 abnormalities may be involved in the development of hypertension in this model (28). In vivo perfusion studies indicate that the SS rats reabsorb more sodium in the loop of Henle than the salt-resistant (SR) or Lewis rats, indicating that tubular transport is higher in these animals (33, 35). Addition of 20-HETE to the perfusate in these studies reduced the chloride transport rates (35). More recently, thick ascending limbs from SS animals were shown to have higher transport rates that were reduced with 20-HETE than SR animals (19). Thus 20-HETE is a key factor in regulating sodium and chloride transport and volume regulation.
The above in vivo and in vitro studies examined the role of 20-HETE on thick ascending limb of Henle transport (19, 33, 35). Since the highest concentration of renal P-450 is in the proximal tubule, the effect of 20-HETE production on transport in this segment was important to examine (12). The present study directly demonstrates that endogenous production of 20-HETE exerts an effect to inhibit transport in the PST. When the endogenous production was blocked, exogenous administration of 20-HETE inhibited transport. These findings are consistent with the above studies linking a low production rate of 20-HETE with the development of hypertension.
The present study demonstrated direct effects of 20- and
19(S)-HETE on proximal tubule transport. The addition of
20-HETE inhibited proximal tubule transport only when endogenous
production was inhibited with DDMS. 19(S)-HETE was capable of
stimulating volume transport in the absence and presence of DDMS. Thus
the -hydroxylase products of arachidonic acid play a role in
regulating proximal tubule transport.
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ACKNOWLEDGEMENTS |
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We are grateful for the technical assistance provided by Amber Lisec and the secretarial assistance of Janell McQuinn.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-02232 (R. Quigley), DK-41612 (M. Baum), and DK-38226 (R. Quigley and J. R. Falck), and by the Robert A. Welch Foundation (J. R. Falck).
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 and other correspondence: R. Quigley, Dept. of Pediatrics, UT Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9063 (E-mail: RQUIGL{at}medmet.swmed.edu).
Received 28 April 1999; accepted in final form 19 January 2000.
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