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1 Department of Pharmacology, New York Medical College, Valhalla, New York 10595; and 2 Departamento Farmacologia y Toxicologia, Centro de Investigacion y de Estudios Avanzados del Instituto Politécnico Nacional, Mexico 07300
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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The effects of angiotensin II (ANG II) on tumor necrosis
factor-
(TNF) production were determined in freshly isolated tubules from the medullary thick ascending limb (MTAL). ANG II
(10
9 M) increased the
accumulation of TNF mRNA associated with enhanced production of TNF by
approximately five- to sixfold. ANG II also increased prostaglandin
E2
(PGE2) production by the MTAL in
a dose-dependent manner and exerted biphasic differential effects on
86Rb uptake, depending on the
exposure time of the tubules to the peptide and the doses used.
Low-dose ANG II (1011 M)
increased 86Rb uptake by MTAL
tubules after a "short-term" (15 min) challenge, whereas uptake
was inhibited after a "long-term" (3 h) incubation period.
High-dose ANG II (106 M)
inhibited MTAL 86Rb uptake,
irrespective of incubation time. Uptake of
86Rb was inhibited by ~60% in
MTAL tubules that were challenged for 3 h with ANG II. The inhibitory
action of ANG II was prevented by eliminating the participation of
either TNF with antisera to the cytokine or
PGE2 by inhibition of
cyclooxygenase with indomethacin. We conclude that ANG II regulates TNF
production in the MTAL, an interaction that affects
86Rb uptake via an
eicosanoid-dependent mechanism in this nephron segment.
prostaglandins; tumor necrosis factor-; kidney; cytokines
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INTRODUCTION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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THE MEDULLARY THICK ascending limb (MTAL) is pivotal in
the regulation of extracellular fluid volume and is responsible for establishing the osmotic gradient in the medulla, the critical event
for concentrating urine (15). Tumor necrosis factor- (TNF) produced
by the MTAL in response to lipopolysaccharide (LPS) stimulation, as
well as exogenous TNF, inhibited
86Rb uptake by this nephron
segment via a prostanoid-dependent mechanism, an effect consistent with
the reported natriuretic action of TNF (10, 32). Sublethal doses of TNF
administered to dogs caused a marked polyuria and natriuresis that was
prevented by inhibition of cyclooxygenase, indicating the essential
role of prostaglandins to the tubular action of TNF (32).
Angiotensin II (ANG II) is active at multiple sites in the kidney and contributes to the regulation of glomerular hemodynamics and electrolyte balance. Levels of this peptide in the proximal tubule are in the nanomolar range, compared with picomolar concentrations in the circulation, suggesting that ANG II may be produced by the proximal tubule and perhaps by other nephron segments and released into the tubular lumen (2). Receptors for ANG II are most prevalent in the proximal tubules (6); however, ANG II receptors also have been associated with type 1 interstitial cells, vasa recta bundles, and MTAL epithelial cells in the inner stripe of the outer medulla (22, 24, 30, 36). Although the proximal tubule is a major site of action for ANG II, several studies have indicated effects of this peptide on MTAL function. Bicarbonate transport has been reported to be increased in the MTAL after ANG II infusion, an effect that was abolished by coadministration of DuP-753, a selective AT1 receptor antagonist (4). ANG II also inhibited activity of the apical 70-pS K+ channel in the MTAL (20).
Because ANG II has been shown to affect ion transport in the MTAL and because TNF is produced by the MTAL, we addressed possible effects of ANG II on MTAL transport via a mechanism that involves TNF-dependent stimulation of prostaglandin E2 (PGE2) synthesis. Induction of TNF synthesis by the MTAL in response to LPS took upward of 4 h. We therefore determined not only concentration-dependent effects of ANG II but also short- (15 min) and long-term (3 h) effects of exposure of the MTAL to the peptide in terms of changes in 86Rb uptake. We were able to distinguish indomethacin-sensitive and -insensitive effects of ANG II on 86Rb uptake by the MTAL that were dependent on time of exposure to ANG II, as well as the ability of the peptide to promote synthesis of TNF. We postulate a novel mechanism operating within the MTAL that can be initiated by ANG II stimulation of TNF synthesis, resulting in expression of prostaglandin-mediated effects on 86Rb uptake.
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METHODS |
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Abstract
Introduction
Methods
Results
Discussion
References
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Animals
Male Sprague-Dawley rats (Charles River, Wilmington, MA) weighing 250-300 g were maintained on standard rat chow (Ralston Purina, Chicago, IL) and given tap water ad libitum.Reagents
Tissue culture media and collagenase (type 1A) were obtained from Life Technologies (Grand Island, NY). Reagent-grade chemicals, indomethacin, 17-octadecynoic acid (17-ODYA), and losartan were from Sigma (St. Louis, MO); guanidine isothiocyanate was purchased from Fluka (Ronkonkama, NY); and GeneScreen was from NEN-DuPont (Boston, MA). PD-123319 was purchased from Research Biochemicals International (Natick, MA).Isolation of MTAL Tubules
MTAL tubules were isolated as previously described (10). Briefly, male Sprague-Dawley rats were anesthetized with an intraperitoneal injection of pentobarbital sodium (0.65 mg/100 g body wt). The kidneys were perfused with sterile 0.9% saline, via retrograde perfusion of the aorta, and cut along the corticopapillary axis. The inner stripe of the outer medulla was excised, minced with a sterile blade, and incubated for 10 min at 37°C in a 0.75% collagenase solution gassed with 95% oxygen. The suspension was sedimented on ice, and the supernatant containing the crude suspension of tubules was collected. The collagenase digestion was repeated with the remaining undigested tissue. The combined supernatants were spun, resuspended in Hanks' buffer, and filtered through a 52-µm nylon mesh (Fisher Scientific, Springfield, NJ). The filtrate was discarded, and the tubules retained on the mesh were resuspended in Dulbecco's modified Eagle's medium (DMEM, Life Technologies). Combination of the perfusion and size-exclusion steps was done to eliminate blood elements (19, 31).Enzyme-Linked Immunosorbent Assay for TNF
The measurement of TNF by enzyme-linked immunosorbent assay (ELISA) was performed using a commercially available rat TNF ELISA kit purchased from Biosource (Camarillo, CA). The assay was specific for TNF and had been used extensively for the detection of TNF in tissue culture supernatants, plasma, serum, and urine. Briefly, microtiter 96-well plates coated with hamster anti-TNF monoclonal antibody were used to capture TNF present in the samples (diluted 1:2). The plates were washed to remove unbound material, and a horseradish peroxidase-conjugated goat polyclonal anti-TNF that binds the captured TNF was added. The plate was washed again, and substrate was added to initiate a peroxidase-catalyzed color change that was subsequently stopped by acidification. Absorbance was measured at 450 nm and was proportional to the concentration of TNF in the sample. Standard curves were obtained, and TNF concentrations in experimental samples were determined using the standard curve.Enzyme-Linked Immunoassay for PGE2
Prostaglandin concentration in the supernatant was measured in unextracted samples using solid-phase, enzyme-linked immunoassay (EIA). Microtiter plates were coated with 200 µl/well goat anti-rabbit immunoglobulin G at a concentration of 10 µg/ml. After an overnight incubation at room temperature, plates were washed three times with wash buffer (0.05% Tween 20, in phosphate-buffered saline pH 7.4), covered with microtest plastic film (Fisher), and stored at
4°C for a minimum of 1 h prior to their use. After removal of EIA buffer, 50-µl aliquots of enzymatic tracer, antibody, and either prostaglandin standard (4-2,000 pg/ml) or samples, diluted with EIA buffer, were placed in duplicate in the coated wells. Nonspecific binding was determined by replacing antibody with buffer,
and maximum binding was determined by equilibration in the absence of
prostanoids. After overnight equilibration at 4°C, plates were
washed three times with EIA buffer. Wells were filled with 200 µl
Ellman's reagent, which was stored as a concentrated stock solution of
69 mM acetylcholine iodide and 54 mM
5,5'-dithiobis(2-nitrobenzoic acid) in phosphate buffer at
20°C and diluted 1:100 as required. After shaking for
1-2 h, absorbance was read at 405 nm with automated subtraction of
readings at 630 nm to correct for nonspecific absorbance. Microtitration and washing steps were carried out with an automatic dispenser (Pro/Oette; Perkin-Elmer, Wilton, CT).
cDNA Probes
A 1.4-kb murine TNF cDNA probe (a gift from Dr. Bruce Beutler, Univ. of Texas Southwestern Medical School, Dallas, TX) was excised from the BamH I and Pst I sites of the vector pGEM4.Isolation of Total RNA/Northern Blot Analysis
Total RNA was isolated by lysing the tubules in guanidine isothiocyanate/sodium citrate buffer followed by phenol:chloroform extraction and ethanol precipitation, as previously described (11). Total RNA (10 µg) was electrophoresed in a 1% agarose/formaldehyde gel in 1× 3-(N-morpholino)propanesulfonic acid as the running buffer. RNA was then transferred to a nylon membrane (GeneScreen) and hybridized to random-primed 32P-labeled probes in a buffer containing 50% formamide, 10% dextran sulfate, 0.2% polyvinylpyrrolidone, 0.2% Ficoll, 0.2% bovine serum albumin, 1.0 M NaCl, 1.0% sodium dodecyl sulfate (SDS), 0.05 M tris(hydroxymethyl)aminomethane, pH 7.5, and 0.1% sodium pyrophosphate at 42°C for 18 h. Posthybridization washes of the membrane were carried out in 2× standard sodium citrate (SSC), 1.0% SDS at 65°C for 1 h and in 0.1× SSC at 25°C for 1 h. Then the probed blots were exposed at70°C to Kodak X-OMAT film.
86Rb Uptake
Freshly isolated MTAL tubules were incubated on ice for 20 min in 1 ml K+-free Hanks' balanced salt solution containing (composition in mM): 136.8 NaCl, 1.26 CaCl2, 0.49 MgCl2, 0.45 MgSO4, 0.77 NaH2PO4, 4.16 NaHCO3, and 5.5 glucose. After incubation, uptake was initiated by adding 86Rb (1 µCi; Amersham, sp act 500 mg/mCi) and 5 mM K+ to suspensions of intact tubules (100 µl) in a shaking water bath at 37°C. Isotope uptake was terminated after 5 min by pipetting 100-µl aliquots of the suspension into a stop solution (100 ml 75 mM KCl Hanks', 100 ml silicone, and 100 ml dioctylphthalamate) and centrifuged immediately through oil in a microcentrifuge at 13,000 g for 30 s. The supernatants were discarded, and the bottom of the tube containing the pellet was cut off and placed in a test tube. The radioactivity of the pellet was determined in a gamma counter. The ouabain-sensitive component of total 86Rb uptake was calculated by subtracting 86Rb uptake in the presence of ouabain (1 mM) from uptake in the absence of ouabain (10).Oxygen Consumption Measurements
The oxygen consumption of freshly isolated MTAL tubules resuspended in DMEM was measured with a Clark-type electrode inserted into a 0.3-ml temperature-controlled chamber. The amount of tubules added was titrated so that all the oxygen in the chamber was consumed in 30 min. The change in oxygen concentration in the chamber was monitored on a Soltec recorder. A constant slope was established during a control period, and additions to the chamber were made in volumes that did not exceed 1% of the volume of the measuring chamber. The rate of oxygen consumption was determined from the slope of the recorded curve by measuring its angle against the 100% oxygen baseline and calculating its tangent (5). Results are expressed as nanomoles of oxygen utilized per minute per milligram of protein.Statistical Analysis
The responses of control and treated MTAL tubules were compared by either unpaired Student's t-test or by one-way analysis of variance.| |
RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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ANG II Increases TNF Production by MTAL Tubules
Production of TNF by the MTAL was assessed using freshly isolated tubules placed into wells of a 24-well tissue culture plate. The tubules were incubated at 37°C in a tissue culture incubator in the absence or presence of ANG II (1011-107
M) for 3 h to allow significant amounts of TNF to accumulate; TNF
levels were determined by ELISA. As shown in Fig.
1, marginal elevations in TNF levels were
observed after challenging MTAL tubules with ANG II concentrations of
1011 and
109 M. TNF production by
tubules increased by approximately sixfold after challenge with
107 M ANG II (Fig. 1).
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ANG II-Mediated mRNA Accumulation in MTAL Tubules
Northern blot analysis was performed to assess TNF mRNA accumulation and to confirm that ANG II induced TNF production. MTAL tubules were incubated for 3 h in the absence or presence of ANG II (109 M). At the end of the
incubation period, total RNA was isolated from the tubules, and a
32P-labeled TNF cDNA probe was
used to determine the accumulation of TNF mRNA. TNF mRNA accumulation
in MTAL tubules increased approximately threefold after challenge with
109 M ANG II (Fig.
2).
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ANG II Stimulates MTAL PGE2 Production
ANG II has been shown to increase PGE2 production under a variety of experimental conditions (26). We have reported that TNF increased PGE2 synthesis by the MTAL (10). Synthesis of PGE2 by MTAL tubules was determined by incubating tubules in the absence or presence of ANG II (1011-107
M) for 3 h at 37°C. At the end of the incubation period, the levels
of PGE2 were determined by EIA.
ANG II, at each of the concentrations tested, increased
PGE2 formation by MTAL tubules (Fig. 3). Thus ANG II stimulates
PGE2 synthesis at concentrations that have been reported in tubular fluid (2).
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Na+-K+-ATPase
and
Na+-K+-2Cl
Cotransporter Activity in MTAL Tubules
cotransporter account for a large component of oxygen consumption by
the MTAL and are subject to modulation by arachidonic acid (AA)
metabolites (8, 9). The activity of the
Na+-K+-ATPase
and
Na+-K+-2Cl
cotransporter was assessed to demonstrate the functional integrity of
isolated MTAL tubules and to establish conditions to investigate interactions of TNF and AA metabolites with these transport mechanisms. Ion transport mechanisms in isolated MTAL tubules were examined by
measuring 86Rb uptake as an index
of K+ transport (8). The ability
of furosemide and ouabain to inhibit Na+-K+-2Cl
and
Na+-K+-ATPase
activity, respectively, was determined. Furosemide (0.1 mM) and ouabain
(1 mM) inhibited 86Rb uptake by
~58 and 66%, respectively (Fig. 4),
indicating that the functions of two important transport mechanisms are
retained in freshly isolated MTAL tubules.
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The functional integrity of isolated MTAL tubules also was assessed by
measuring oxygen consumption, which, to a large extent (~50%), is
dependent on the activity of
Na+-K+-ATPase
driven by Na+ entry through the
Na+-K+-2Cl
cotransporter. Addition of ouabain (1 mM) and determination of the
slope of oxygen consumption revealed a decrease in oxygen consumption,
compared with untreated tubules, of ~40-45% (control, 105 ± 15, vs. ouabain, 60 ± 5 nmol
O2 · min1 · mg
protein1;
P < 0.01, n = 4). Addition of furosemide (0.1 mM), an inhibitor of the cotransporter, decreased oxygen consumption by
~30-40% (control, 140 ± 25, vs. furosemide, 98 ± 15 nmol
O2 · min1 · mg
protein1;
P < 0.01, n = 4). As ouabain and furosemide
inhibited 86Rb uptake and oxygen
consumption,
Na+-K+-ATPase
and the
Na+-K+-2Cl
cotransporter, respectively, were functional in isolated rat MTAL
tubules. Moreover, these data suggest that tubular lumina were in
direct contact with inhibitors contained in the extracellular buffer,
since furosemide inhibited the
Na+-K+-2Cl
cotransporter, which is located on apical membranes in the MTAL.
Time- and Dose-Dependent Effects of ANG II on MTAL Ouabain-Sensitive 86Rb Uptake
The effects of ANG II on ion transport in the MTAL were determined by incubating isolated tubules in the absence or presence of ANG II and measuring ouabain-sensitive 86Rb uptake. A low concentration of ANG II (1011 M) added to MTAL
tubules for 15 min produced a statistically significant increase in
86Rb uptake (Fig.
5). Intermediate concentrations of ANG II
(1010-108
M) were without effect, whereas the higher concentrations
(107
and106 M) decreased uptake
(Fig. 5). A temporal effect also was evident when MTAL tubules were
exposed to ANG II. As shown in Fig. 6, incubation of MTAL tubules with ANG II
(1011 M) for 15 min
increased ouabain-sensitive 86Rb
uptake, whereas, after a 3-h exposure, this concentration of the
peptide did not increase uptake (Fig. 6). On the other hand, a higher
concentration of ANG II
(106 M) inhibited
86Rb uptake by MTAL tubules
irrespective of the time of exposure (15 min vs. 3 h) (Fig. 6). These
data are consistent with the biphasic response to ANG II observed in
the proximal tubule after short exposure times to ANG II (14, 27) and
suggest that the short- (15 min) and long-term (3 h) effects of ANG II
may operate through different mechanisms in the MTAL. As both
cyclooxygenase (COX)- and cytochrome
P-450 (CYP450)-derived metabolites of
arachidonic acid have been shown to affect
86Rb uptake in the MTAL
(8-10), their possible contribution to the effects of ANG II
on86Rb uptake was addressed.
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Analysis of the Contribution of COX- and CYP450-AA Products to ANG II-Mediated Ouabain-Sensitive 86Rb Uptake
Short-term (15 min) regulation. To determine whether the short-term (15 min) effects of ANG II were dependent on COX metabolites, 86Rb uptake was determined in MTAL tubules that were preincubated with the COX inhibitor, indomethacin, for 15 min and then challenged with low and high concentrations of the peptide for 15 min. Indomethacin (1 µM) did not affect stimulation of 86Rb uptake induced by the low concentration of ANG II (1011 M) (Fig.
7), nor did it affect the inhibitory action
of the high concentration of ANG II
(106 M) on
86Rb uptake (Fig. 7). These data,
taken together, suggest that prostaglandins did not participate in
short-term (15 min) effects of ANG II on 86Rb uptake. However,
preincubation of MTAL tubules with 17-ODYA (1 µM) did prevent
inhibition of 86Rb uptake in
response to challenge for 15 min with the high concentration of ANG II
(106 M) but did not prevent
the stimulatory action of the low concentration of ANG II
(1011 M) on
86Rb uptake (Fig. 7). These
findings are consistent with the reported ability of high
concentrations of ANG II to increase
20-hydroxy-5,8,11,14-eicosatetranoic acid (20-HETE) production in the
short term (20).
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Long-term (3 h) regulation. A putative eicosanoid-dependent mechanism involving TNF and PGE2 that determines the long-term (3 h) effects of ANG II was addressed, since we have shown that changes in 86Rb uptake mediated by TNF and PGE2 in response to LPS occurred after a period of several hours and were COX dependent but CYP450 independent (10). As ANG II increased TNF and PGE2 production by the MTAL, we evaluated the cytokine and prostaglandin contribution to the effects of ANG II on 86Rb uptake. The contribution of prostanoids and TNF to the effects of ANG II on 86Rb uptake was determined by preincubating MTAL tubules with either indomethacin or a polyclonal monospecific anti-TNF antisera, respectively, for 20 min before addition of ANG II for 3 h. After a 3-h incubation period, both high and low concentrations of ANG II inhibited ouabain-sensitive 86Rb uptake by ~50% (Fig. 8). Addition of either indomethacin or its vehicle, 0.01% ethanol, caused a 10-15% decrease in 86Rb uptake compared with control tubules incubated in the absence of ANG II. Indomethacin (1 µM) prevented the decrease in 86Rb uptake produced by either high or low concentrations of ANG II (Fig. 8). Moreover, indomethacin reset the basal uptake to a level that is higher than in the absence of indomethacin, which may reflect attenuation of an inhibitory effect of PGE2 on ouabain-sensitive 86Rb uptake in tubules that were challenged with ANG II (Fig. 8). Addition of anti-TNF also attenuated the inhibitory effects of both high and low concentrations of ANG II on 86Rb uptake, suggesting that TNF contributed to the ANG II-induced decrease in 86Rb uptake by an autocrine mechanism (Fig. 8). We have shown that anti-TNF alone did not affect basal ouabain-sensitive 86Rb uptake and that the dilution of anti-TNF chosen (1:50) specifically and completely inhibited TNF bioactivity released from MTAL tubules (21). These data suggest that TNF and PGE2 may mediate the effects of ANG II on 86Rb uptake when tubules are exposed to ANG II for 3 h.
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DISCUSSION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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We have demonstrated in freshly isolated MTAL tubules that the effects of ANG II on 86Rb uptake, an index of K+ transport, were critically related to the concentration of ANG II and to the duration of exposure of the peptide to the tubules. ANG II, when incubated with MTAL tubules for 3 h, increased PGE2 formation and promoted accumulation of TNF mRNA, the latter associated with enhanced production of TNF. Importantly, production of TNF and PGE2 in response to ANG II was linked to a functional correlate, that is, ANG II-induced inhibition of 86Rb uptake, as pretreatment with either indomethacin or a neutralizing concentration of anti-TNF antisera, prevented the inhibitory effects on 86Rb uptake produced by a 3-h exposure to either high or low concentrations of ANG II. We had obtained direct evidence to support a prostanoid effect on 86Rb uptake by the MTAL as addition of PGE2 to primary cultured MTAL cells reduced 86Rb uptake (10).
The presence of both a COX and CYP450 pathway of AA metabolism has been
described in the MTAL (8, 10). Inhibition of COX by indomethacin did
not modify the short-term (15 min) effects of ANG II, given in either
high or low concentrations, on
86Rb uptake by MTAL tubules.
However, indomethacin inhibited the effects of both concentrations of
ANG II after a 3-h exposure to the tubules, indicating that expression
of a prostanoid-dependent mechanism occurred only after prolonged
exposure to the peptide. We have reported that both LPS and TNF
inhibited ouabain-sensitive 86Rb
uptake in the MTAL through a prostaglandin-dependent mechanism that was
expressed after several hours (10).
PGE2 was first shown by Stokes and
Kokko (29) to inhibit transport by the MTAL segment, possibly by
inhibiting
Na+-K+-ATPase
activity, which has been described in several nephron segments,
including the MTAL (16, 29, 34). A recent definitive study has
localized the primary effect of
PGE2 to the
Na+-K+-2Cl
cotransporter in cultured murine MTAL cells, an event that led to
subsequent inhibition of the Na+
pump (18). Moreover, both TNF and interleukin-1 (IL-1) produce natriuresis through stimulation of
PGE2 synthesis, an effect that is
consistent with the observations in the present study.
A CYP450-mediated action of the high concentration of ANG II is evident
in the short term because it was prevented by a mechanism-based inhibitor of CYP450, 17-ODYA, but not by indomethacin. The likely CYP450 product mediating this effect of ANG II is 20-HETE, a potent inhibitor of the
Na+-K+-2Cl
cotransporter in the MTAL (8), which has been shown to be a major
product of AA metabolism in the MTAL and is released from MTAL tubules
challenged with ANG II (20). The stimulatory action produced in the
short term by a low concentration
(1011 M) of ANG II was not
affected by 17-ODYA nor indomethacin. Whether a lipid mediator
contributes to the stimulatory action of ANG II on
86Rb uptake by the MTAL will
require biochemical studies that profile AA metabolites formed by the
MTAL under these experimental conditions. Thus a
prostaglandin-independent effect of ANG II on
86Rb uptake by the MTAL can be
differentiated from a prostaglandin-dependent action determined by the
duration of exposure to ANG II.
As the long-term (3 h) inhibitory effect of ANG II on 86Rb uptake was prevented by indomethacin, TNF-mediated inhibition of 86Rb uptake may be linked to an effect on metabolism of AA, possibly via the cytokine-inducible isoform of cyclooxygenase, COX-2. Furthermore, increased production of PGE2 by MTAL tubules in response to ANG II may reflect a TNF effect as the cytokine has been reported to increase phospholipase A2 mass and activity and promote expression of COX-2 (25, 35). Indeed, several effects of ANG II are mediated via interactions with eicosanoids and/or cytokines. ANG II activates phospholipase A2 in mesangial and proximal tubular cells, and TNF potentiates ANG II-induced PGE2 production by these cells (7, 26). Our finding that ANG II increases TNF production in the MTAL suggests that the cytokine is integral to the activity of ANG II on transport in this nephron segment. The ability of anti-TNF antibodies to prevent inhibition of ouabain-sensitive 86Rb uptake induced by ANG II is in keeping with the interpretation that increased synthesis of TNF is required to express the prostaglandin component in a mechanism that affects MTAL transport.
The capacity to produce TNF was first described for macrophages and T
cells (33). Production of TNF also has been demonstrated in proximal
tubules (17), mesangial cells (1), and other nonhematopoietic cells. We
have reported that the MTAL synthesized and released biologically
active TNF in response to stimulation with LPS (21). Moreover,
induction of TNF in this nephron segment is apparently not limited to
pathological conditions, such as endotoxemia and elevated levels of
LPS, because TNF production by the MTAL is increased by concentrations
of ANG II that have been reported in the tubular fluid (2). Human
peripheral blood monocytes also have been shown to produce TNF when
challenged with ANG II (13), and interactions of ANG II and cytokines
within the kidney are not limited to TNF, as ANG II stimulated IL-6
production by mesangial cells (23). Thus some effects of ANG II may be mediated, either directly or indirectly, by cytokines produced in
response to the peptide. ANG II in low concentrations promotes and in
high concentrations inhibits transport function, in keeping with the
physiological role of ANG II in the conservation of extracellular fluid
volume and the diuretic-natriuretic action of supraphysiological concentrations of ANG II, respectively. The greater part of the concentration range of ANG II used in the present study corresponded to
those concentrations found in tubular fluids, which are two to three
orders of magnitude greater than in plasma under normal conditions,
i.e., 1010 to
108 M vs.
1012 to
1011 M, respectively (2).
The high concentrations in tubular fluid occur despite avid metabolism
of ANG II by peptidases on the luminal surface of the proximal tubule
(28). Concentrations of ANG II as high as
107 M have been reported in
deep veins draining the renal interstitium. Moreover, the values cited
for ANG II concentrations in the tubular fluid are in accord with the
binding constants of ANG II receptors in the nephron (3). The renal
renin-angiotensin system (RAS), as represented in the nephron and
tubular fluid, may be regulated independently of the systemic
(circulating) RAS, a proposal supported by the response to volume
expansion, namely, plasma levels of ANG II were suppressed without
affecting ANG II levels in tubular fluid (2).
TNF-ANG II interactions are not limited to the kidney. We have shown that anti-TNF antisera increases mean arterial pressure in rats made hypertensive by chronic ANG II infusions, suggesting that TNF participates in a counter-regulatory mechanism that opposes the pressor effects of ANG II (12). These findings suggest that circulatory function may be affected by TNF formation, induced by ANG II at extrarenal sites, such as vascular smooth muscle and/or circulating cells. A recent report supports this view, that is, aortas from rats in which hemorrhagic shock was induced exhibited reduced contractility to phenylephrine, an effect reversed by adminstration of anti-TNF antisera (37). In contrast to the systemic effects of TNF evoked by pathological conditions, our findings support the concept that peptide-cytokine-eicosanoid interactions in the microenvironment of the MTAL, i.e., in a contained environment, participate in homeostatic mechanisms modulating the tubular actions of ANG II.
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ACKNOWLEDGEMENTS |
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We thank Melody Steinberg for editorial assistance.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Program Project Grants HL-34300 and HL-56423, and by a grant-in-aid from the American Heart Association (96015620).
Address for reprint requests: N. R. Ferreri, Dept. of Pharmacology, New York Medical College, Valhalla, NY 10595.
Received 24 January 1997; accepted in final form 24 September 1997.
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REFERENCES |
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Abstract
Introduction
Methods
Results
Discussion
References
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