Role of NO in endothelin-regulated drug transport in the
renal proximal tubule
Sylvia
Notenboom1,3,
David S.
Miller2,3,
Paul
Smits1,
Frans G. M.
Russel1, and
Rosalinde
Masereeuw1
1 Department of Pharmacology and Toxicology, University
Medical Center Nijmegen, 6500 HB Nijmegen, The Netherlands;
2 Laboratory of Pharmacology and Chemistry, National
Institute of Environmental Health Science, National Institutes of
Health, Research Triangle Park, North Carolina 27709; and
3 Mount Desert Island Biological Laboratory, Salisbury Cove,
Maine 04672
 |
ABSTRACT |
We previously
demonstrated in intact killifish renal proximal tubules that endothelin
(ET), acting through an ETB receptor and protein kinase C
(PKC), reduced transport mediated by multidrug resistance-associated protein 2 (Mrp2), i.e., luminal accumulation of
fluorescein methotrexate (FL-MTX) (Masereeuw R, Terlouw SA, Van Aubel
RAMH, Russel FGM, and Miller DS. Mol Pharmacol 57:
59-67, 2000). In the present study, we used confocal microscopy
and quantitative image analysis to measure Mrp2-mediated transport of
FL-MTX in killifish tubules as an indicator of the status of this
ET-fired, intracellular signaling pathway. Exposing tubules to sodium
nitroprusside (SNP), a nitric oxide (NO) donor, signaled a reduction in
luminal accumulation of FL-MTX, which suggested pathway activation.
NG-monomethyl-L-arginine
(L-NMMA), an NO synthase inhibitor, blocked the action of
ET-1 on transport. Because SNP effects on transport were blocked by
bisindoylmaleide, a PKC-selective inhibitor, but not by RES-701-1,
an ETB-receptor antagonist, generation of NO occurred after
ETB receptor signaling but before PKC activation. NO
generation was implicated in the actions of several nephrotoxicants, i.e., diatrizoate, gentamicin, amikacin, HgCl2, and
CdCl2, each of which decreased Mrp2-mediated transport by
activating ET signaling. For each nephrotoxicant, decreased FL-MTX
transport was prevented when tubules were exposed to
L-NMMA. ET-1 and each nephrotoxicant stimulated NO
production by the tubules, as determined by a fluorescence-based assay.
Together, the data show that NO generation follows ET binding to the
basolateral ETB receptor and that, in activating the
ET-signaling pathway, nephrotoxicants produce NO, a molecule that could
contribute to subsequent toxic effects.
multidrug resistance-associated protein 2; calcium; endothelin signaling; protein kinase C; xenobiotic transport; nitric
oxide
 |
INTRODUCTION |
METABOLISM AND EXCRETION
provide a first line of defense against the wide variety of
potentially toxic chemicals to which we are continually exposed. In the
kidney, the proximal tubule is responsible for the excretory transport
from blood to urine of xenobiotics, xenobiotic metabolites, and waste
products of metabolism. As a result of its rich transport function, the
proximal tubule is also an important target for toxic effects (2,
27).
To accomplish this excretory function, epithelial cells in the proximal
tubule possess multiple plasma membrane transporters that use ATP and
transmembrane ion gradients to drive active drug secretion into urine.
Among the proteins implicated in this process is a member of the
ATP-binding cassette superfamily of membrane transporters: multidrug
resistance protein 2 (Mrp2). This transport protein is present at high
levels in the luminal membrane of proximal tubule cells and handles a
wide range of chemicals, from large, lipophilic organic anions to
polypeptides (12, 22).
Using intact killifish (Fundulus heteroclitus) renal
proximal tubules, a fluorescein methotrexate (FL-MTX) derivative, and confocal microscopy, we recently demonstrated that Mrp2 was regulated by endothelin (ET), which acted through a basolateral ETB
receptor and protein kinase C (PKC) (13). By activating
this signaling pathway, ET rapidly decreased cell-to-lumen transport of
the fluorescent Mrp2 substrate FL-MTX. Interestingly, a similar
decrease in transport was seen when tubules were exposed to several
known nephrotoxic drugs (aminoglycoside antibiotics and radiocontrast
agents), and that decrease was found to be caused by activation of
ET-ETB-PKC signaling (13, 25). The
nephrotoxicants caused release of ET from the tubules, and the hormone
acted by an autocrine mechanism to signal a decrease in Mrp2-mediated
transport. ET release appeared to be Ca2+ dependent in that
elevated medium Ca2+ triggered the pathway, and the effects
of nephrotoxicants and elevated medium Ca2+ were abolished
by nifedipine, a Ca2+ channel blocker (25).
The precise sequence of events by which ET regulates Mrp2 transport is
unclear. However, there is reason to believe that nitric oxide (NO)
could be part of this ET-signaling cascade in the kidney (18). NO is produced by NO synthase (NOS). Both NOS and ET
were found to be localized to the same nephron segments, including the
proximal tubule (16, 18). In the vascular wall, it is well
documented that ET can trigger the endothelial release of NO by means
of stimulation of the ETB receptors (4, 5,
26). Recent data suggest comparable cross talk between ET and NO
in renal tubular cells (19, 20). In addition, NO has been
implicated in cyclosporin A- and FK506-induced nephrotoxicity in renal
proximal tubules (6).
In the present study, we used killifish renal proximal tubules to
demonstrate that release of NO is an intermediate step in ET signaling.
The data also show that ET-activated NO production is also an early
event in the action of nephrotoxicants, thus providing a possible
mechanistic link between signaling and toxicity.
 |
METHODS |
Chemicals.
The chemicals included FL-MTX, bisindolylmaleide (BIM),
NG-monomethyl-L-arginine
(L-NMMA),
4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM)
diacetate (Molecular Probes, Eugene, OR); RES-701-1, an
ETB-receptor antagonist (Peninsula Laboratories, Belmont,
CA); sodium nitroprusside (SNP; Calbiochem, San Diego, Ca); and
HgCl2, CdCl2, gentamicin, amikacin, and
diatrizoic acid (Sigma, St. Louis, MO). All other chemicals
were obtained at the highest purity available.
Animals and tissue preparation.
Killifish were collected by local fishermen in the vicinity of Mount
Desert Island, ME, and maintained at the Mount Desert Island Biological
Laboratory in tanks with natural flowing seawater. Renal tubular masses
were isolated in a marine teleost saline, based on that of Forster and
Taggart (3), containing (in mM) 140 NaCl, 2.5 KCl, 1.5 CaCl2, 1.0 MgCl2, and 20 Tris at pH 8.0. All
experiments were carried out at 18-20°C. Under a dissecting microscope, each mass was teased with fine forceps to remove adherent hematopoietic tissue. Individual killifish proximal tubules were dissected and transferred to a foil-covered Teflon chamber (Bionique) containing 1.5 ml of marine teleost saline with 1 µM FL-MTX and added
effectors. The chamber floor was a 4 × 4-cm glass coverslip to
which the tubules adhered lightly and through which the tissue could be
viewed by means of an inverted microscope. Tubules were incubated at
room temperature for 30 min until steady state was reached for FL-MTX.
Analysis of tubule extracts by high-performance liquid chromatography
showed no metabolic degradation of FL-MTX when killifish proximal
tubules were incubated for periods of at least 1 h (12,
23).
Confocal microscopy.
The chamber containing renal tubules was mounted on the stage of an
Olympus FluoView inverted confocal laser scanning microscope and viewed
through a ×40 water-immersion objective (numerical aperature
1.15). Excitation was provided by the 488-nm line of an argon ion
laser. A 510-nm dichroic filter and 515-nm long-pass emission filter
were used. Neutral density filters and low laser intensity were used to
avoid photobleaching. With the photomultiplier gain set to give an
average luminal fluorescence intensity of 1,500-3,000 (on a scale
of 0-4,096), tissue autofluorescence was undetectable. To obtain
an image, dye-loaded tubules in the chamber were viewed under reduced,
transmitted light illumination, and a single proximal tubule with
well-defined lumen and undamaged epithelium was selected. The plane of
focus was adjusted to cut through the center of the tubular lumen, and
an image was acquired by averaging four scans. The confocal image was
viewed on a high-resolution monitor and saved to an optical disk or Zip
disk (Iomega). In previous studies, it has been shown that
there is a linear relationship between fluorescence intensity and dye
concentration (15). However, because of the many
uncertainties in relating cellular fluorescence to actual compound
concentration in cells and tissues with complex geometry, data are
reported here as average measured pixel intensity rather than estimated
dye concentration. Fluorescence intensities were measured from stored
images using Scion image version 1.8 for Windows (12, 14).
Briefly, two or three adjacent cellular and luminal areas were selected
from each tubule, and the average pixel intensity for each area was
calculated. The values used for that tubule were the means of all
selected areas.
NO production by tubules was measured using an indicator, DAF-FM, that
increases fluorescence intensity on exposure to NO. For the
experiments, tubules were loaded for 1 h in medium containing 10 µM DAF-FM diacetate. This nonfluorescent derivative is membrane permeant and, on entering cells, is hydrolyzed to DAF-FM. After loading, tubules were transferred to confocal chambers containing medium without (control) and with effectors. After 5 min, confocal images were collected (as for FL-MTX) and saved to a Zip disk. Average tubule fluorescence was measured from the stored images as
described above.
Data analysis.
Values are given as means ± SE. Mean values were considered to be
significantly different when P < 0.05, by use of the
unpaired t-test, or by a one-way ANOVA followed by
Bonferroni's multiple comparison test. Software used for statistical
analysis was GraphPad Prism (version 3.00 for Windows; GraphPad
Software, San Diego, CA).
 |
RESULTS |
The present experiments were conducted using isolated renal
proximal tubules from a marine teleost fish, the killifish. This has
proven a powerful model for the study of secretory transport in an
intact proximal tubule (21). As in mammalian proximal tubules, killifish express high levels of Mrp2 in the luminal membrane
of renal proximal tubule cells. Moreover, intact killifish tubules
exhibit Mrp2-mediated transport of a number of fluorescent substrates,
e.g., FL-MTX, that can be visualized and measured using confocal
microscopy (12-14). Figure
1A shows a typical confocal image of a control killifish tubule after 30-min (steady-state) incubation in medium with 1 µM FL-MTX. The fluorescence distribution pattern is the same as that shown previously, i.e., fluorescence intensity in lumen > cells > medium (12, 13).
We have demonstrated that this pattern is indicative of a two-step
process involving uptake at the basolateral membrane mediated by an as
yet uncharacterized transporter for large organic anions and secretion
into the lumen mediated by a teleost form of Mrp2 (for data on
substrate and inhibitor specificities as well as immunostaining with
Mrp2 antibodies, see Refs. 13 and 25).

View larger version (57K):
[in this window]
[in a new window]
|
Fig. 1.
Representative confocal images of killifish proximal tubules after
incubation in marine teleost saline with 1 µM fluorescein
methotrexate (FL-MTX) for 30 min in the absence (A) and
presence (B) of the nitric oxide (NO) donor sodium
nitroprusside (SNP). Treatment with 100 µM SNP reduced luminal
fluorescence, indicating that FL-MTX secretion on multidrug resistance
protein 2 (Mrp2) was inhibited.
|
|
Figure 1 also shows confocal images of FL-MTX transport in tubules
exposed to the NO donor SNP. In previous experiments using ET-1, a
decrease in luminal accumulation of FL-MTX and no effect on cellular
accumulation have been taken to indicate decreased transport on Mrp2
(13). Similar to the effects of ET-1, 100 µM SNP
decreased luminal, but not cellular, accumulation of FL-MTX (Fig.
1B). Quantitation of images showed that 50-100 µM SNP
reduced steady-state luminal accumulation of FL-MTX by about 50% (Fig. 2A). The action of SNP was
rapid, causing a significant reduction of luminal fluorescence within
10 min of exposure and a sustained decrease over the 30-min experiment
(Fig. 2B). SNP did not affect cellular fluorescence.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 2.
The NO donor SNP reduces transport of FL-MTX on Mrp2.
A: effects of 10-100 µM SNP on steady-state (30-min)
FL-MTX accumulation. B: time course of FL-MTX accumulation
in control tubules and tubules exposed to 100 µM SNP from time zero
on. SNP significantly reduced luminal fluorescence intensity at all
times (P < 0.001). Values are means ± SE for
6-16 tubules from a single fish. **Significantly lower than the
control value, P < 0.001.
|
|
Figures 1 and 2 indicate that SNP and ET-1 have similar effects
on Mrp2-mediated transport of FL-MTX. To determine whether NO release
was a consequence of ET-1 exposure, tubules were exposed to 10 nM ET-1
in the absence or presence of L-NMMA, an NOS inhibitor. Figure 3 shows that L-NMMA by
itself had no effect on FL-MTX transport but attenuated the effect of
ET-1. Thus ET-1 activated NOS. In killifish tubules, ET-1 interacts
with a basolateral ETB receptor and reduces Mrp2-mediated
transport by acting through PKC (13). In this study the
effects provided by ET-1 were prevented using the ETB
receptor-antagonist RES-701-1 or PKC-selective inhibitors BIM,
calphostin C, or staurosporine. To determine where NO generation was
placed in the signaling pathway, we examined the abilities of
RES-701-1 and the PKC-selective inhibitor BIM to attenuate the
effects of SNP on FL-MTX transport. RES-701-1 did not alter the
effects of SNP on transport (Fig.
4), but BIM protected completely (Fig.
5), indicating that NO generation came
after ETB receptor binding but before PKC activation.

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 3.
Inhibition of NO synthase (NOS) blocks the action of
endothelin-1 (ET-1) on FL-MTX transport. Tubules were incubated for 30 min in medium containing 1 µM FL-MTX alone (control) or with 10 nM
ET-1, 50 µM
NG-monomethyl-L-arginine
(L-NMMA), or ET-1 plus L-NMMA. Confocal images
were collected and analyzed as described in METHODS. Values
are means ± SE for 11-20 tubules from a single fish.
**Significantly lower than the control value, P < 0.001.
|
|

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 4.
The ETB receptor antagonist
RES-701-1 does not protect against inhibition of Mrp2-mediated
FL-MTX transport by NO. Tubules were incubated for 30 min in medium
containing 1 µM FL-MTX alone (control) or with 100 nM SNP, or SNP
plus 100 nM RES-701-1. Confocal images were collected and analyzed
as described in METHODS. Values are means ± SE for
10-11 tubules from a single fish. **Significantly lower than the
control value, P < 0.001.
|
|

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 5.
A protein kinase C (PKC)-selective inhibitor
bisindolmaleide (BIM) protects against inhibition of Mrp2-mediated
FL-MTX transport by NO. Tubules were incubated for 30 min in medium
containing 1 µM FL-MTX alone (control) or with 100 µM SNP, 100 nM
BIM, or SNP plus BIM. Confocal images were collected and analyzed as
described in METHODS. Values are given as means ± SE
for 9-15 tubules from a single fish. **Significantly lower than
the control value, P < 0.01.
|
|
We recently found that ET release and subsequent signaling in
killifish tubules could be activated by two types of external stimuli:
elevated extracellular Ca2+ and several structurally
unrelated nephrotoxicants (25). The present experiments
indicate that each acts through NO. Figure 6 shows that L-NMMA prevented
the decrease in luminal FL-MTX accumulation caused by increasing the
medium Ca2+ concentration from 1.5 mM in controls to 3 mM
in treated tubules. Table 1 shows the
results of a series of experiments in which nephrotoxicants reduced
FL-MTX transport mediated by Mrp2. The data for the aminoglycoside
antibiotics gentamicin and amikacin and for the radiocontrast agent
diatrizoate confirm previous findings (25). For these
drugs, effects on Mrp2-mediated transport were shown to be caused by
stimulation of ET release by the tubules and subsequent ET action
through the ETB receptor and PKC (25). Additional experiments with killifish tubules have disclosed a similar
mechanism of action for low concentrations of heavy-metal salts
HgCl2 and CdCl2 (Terlouw SA and Miller DS,
unpublished observations). Table 1 shows that, for all these
nephrotoxicants, including HgCl2 and CdCl2,
inhibition of NOS by L-NMMA prevented the reduction in
Mrp2-mediated transport. Thus the effects of elevated medium Ca2+ and the nephrotoxicants appear to involve generation
of NO.

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 6.
L-NMMA protects against inhibition of
Mrp2-mediated FL-MTX transport by elevated medium Ca. Tubules were
incubated for 30 min in medium containing 1.5 mM Ca and 1 µM FL-MTX
(control); 5 mM Ca and 1 µM FL-MTX; and 5 mM Ca, 1 µM FL-MTX, and
50 µM L-NMMA. Confocal images were collected and analyzed
as described in METHODS. Values are given as means ± SE for 18 tubules from a single fish. **Significantly lower than the
control value, P < 0.001.
|
|
To test this supposition directly, we used a fluorescence-based
technique to measure NO generation in intact killifish tubules. The
technique relies on the formation of a highly fluorescent product when
DAF-FM reacts with NO. The indicator is introduced to the tubule as a
nonfluorescent ester, DAF-FM diacetate, which is membrane permeable and
hydrolyzed intracellularly to DAF-FM. Figure
7 shows that the average fluorescence
intensity was low in control tubules but increased by several times in
tubules exposed to SNP (100 µM). Roughly the same magnitude of
increase was also found for tubules exposed to diatrizoate,
CdCl2, amikacin, and gentamicin at concentrations that
reduce FL-MTX transport mediated by Mrp2. ET-1 also generated NO (Fig.
8A), and this production was
blocked by the NOS inhibitor L-NMMA. Finally, Fig.
8B shows that NO production induced by gentamicin was
blocked by the ETB receptor antagonist RES-701-1.
Together these data demonstrate that ET-1 and the nephrotoxicants
stimulated NO production in the tubules.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 7.
The NO-donor SNP (100 nM) and several nephrotoxicants (10 µM diatrizoate, 10 µM CdCl2, 10 µM amikacin, and 10 µM gentamicin) stimulate NO production. Tubules were preincubated for
1 h in medium containing 10 µM
4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM) diacetate.
After loading, tubules were transferred to confocal chambers containing
medium without (control) and with effectors. After 5 min, confocal
images were collected and analyzed as described in METHODS.
Values are given as means ± SE for 11-14 tubules from two
fish. **Significantly higher than the control value, P < 0.001.
|
|

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 8.
A: stimulation of NO production caused by
exposure to 10 nM ET-1 was blocked 50 µM L-NMMA.
B: stimulation of NO production caused by exposure to 10 µM gentamicin (Gent) could also be blocked by 100 nM RES-701-1
(B). Tubules were preincubated for 1 h in medium
containing 10 µM DAF-FM diacetate. After loading, tubules were
transferred to confocal chambers containing medium without (control)
and with effectors. After 5 min, confocal images were collected and
analyzed as described in METHODS. Values are given as
means ± SE for 11-14 tubules from two fish. *Significantly
higher than the control value, P < 0.01. **Significantly higher than the control value, P < 0.001.
|
|
 |
DISCUSSION |
Using killifish renal proximal tubules, we previously demonstrated
that ET, acting through an ETB receptor and PKC, reduced transport mediated by Mrp2, i.e., luminal accumulation of FL-MTX (13). In the present study, we used Mrp2-mediated
transport of FL-MTX to monitor activity of this ET-fired, intracellular signaling pathway; the purpose was to examine further the chain of
events that connect ETB receptor binding at the basolateral membrane to reduction of transport at the luminal membrane. We show
here that exposing tubules to SNP, an NO donor, also signaled a
reduction in transport. Importantly, exposure to L-NMMA, an NOS inhibitor, blocked the action of ET-1 on transport. Because the
effects of SNP on transport were blocked by BIM, a PKC selective inhibitor, but not by RES-701-1, an ETB receptor
antagonist, generation of NO occurred after receptor signaling but
before PKC activation. Several additional observations are consistent
with this proposed sequence of events. Experiments using a
fluorescence-based NO assay showed that ET-1 and the nephrotoxicants
stimulated NO production by intact tubules. For ET-1, NO production was
blocked by an inhibitor of NOS; for the nephrotoxicant, gentamicin, NO
production was blocked by an ETB receptor antagonist. A
scheme of the proposed signaling pathway is shown in Fig.
9.

View larger version (71K):
[in this window]
[in a new window]
|
Fig. 9.
Scheme illustrating the proposed sequence of events by which
nephrotoxicants reduce Mrp2-mediated transport in isolated renal
proximal tubules. Nephrotoxicants cause a transient opening of calcium
channels, which increases intracellular Ca concentration and stimulates
ET release. The hormone binds to a basolateral ETB
receptor, which activates NOS, increases NO production, and activates
PKC. PKC activation rapidly reduces transport on Mrp2.
|
|
The present data are the first to describe an ET-NO-PKC signaling axis
in renal proximal tubules. In agreement with our findings, activation
of NOS was found after ETB receptor stimulation in the
thick ascending limb, and this was associated with inhibited chloride
transport (19, 20). In OK cells, an opossum kidney proximal tubule cell line, Liang and Knox (9) described an NO-PKC-Na+,K+-ATPase signaling pathway. Such a
pathway was not present in LLC-PK1 cells, a proximal tubule
cell line from pig (9). Obviously, species differences or
differences in culture conditions could underlie the differences
in signaling found in the two cell lines.
Liang and Knox (9) have suggested that NOS might
activate PKC through guanylate cyclase. Results indicating cGMP
generation after NO synthesis confirm this suggestion (24,
30). Interestingly, it was recently shown that one member of the
MRP family, viz. MRP5, transports cGMP (7). It is possible
that Mrp2 transports the cyclic nucleotide as well, and cGMP might
compete with FL-MTX for efflux, resulting in a decreased FL-MTX
transport. In this regard, our preliminary experiments show that, in
killifish tubules, 8-bromoguanosine 3',5'-cyclic monophosphate
is a potent inhibitor of FL-MTX transport from cell to lumen, and at
least a portion of that inhibition is removed when tubules are
pretreated with an inhibitor of protein kinase G (Notenboom S and
Miller DS, unpublished observations). The latter suggests
activation of guanylate cylclase by PKC rather than the opposite,
because the effect of NO generation by the NO donor SNP was completely
reversed by inhibiting PKC. Whether guanylate cyclase is directly or
indirectly responsible for a diminished transport of organic anions by
Mrp2 will be investigated in further research.
Although it is not clear whether proximal tubules are capable of
generating NO under basal conditions, they certainly can produce large
amounts of the substance when stimulated (10). In this
regard, proximal tubules express several NOS isoforms, including
inducible and endothelial NOS, and NO production increases on exposure
to a variety of stimuli, including lipopolysaccharide, cytokines,
hypoxia, and several nephrotoxic chemicals (10). The
present results indicate that, when killifish proximal tubules were
exposed to radiocontrast agents, aminoglycoside antibiotics, or
heavy-metal salts, NO production increased. In addition, Mrp2-mediated transport decreased for each compound and these decreases were prevented when the tubules were also exposed to an NOS inhibitor, L-NMMA. We have recently found that each tested
nephrotoxicant reduced Mrp2-mediated transport in killifish tubules by
inducing ET release and activating ETB receptor-PKC
signaling (13, 25). Nephrotoxicant action appeared to be
Ca2+ dependent, because nifedipine, an L-type
Ca2+ channel blocker, protected its action and because
elevated medium Ca2+ also stimulated ET release and
signaling. The present results indicate that exposure of
tubules to nephrotoxicants or elevated medium Ca2+
also produces NO as an intermediate step in signaling (Fig. 9).
Production of NO as a consequence of nephrotoxicant-triggered signaling
provides a toxicological context in which to consider the present and
previous results. NO production has been implicated in renal diseases
and the action of several nephrotoxic compounds (8). For
example, hypoxic/ischemic injury is prevented by
NG-nitro-L-arginine methyl ester and
enhanced by SNP (29), and inducible NOS appears to be the
critical isoform involved (11, 17). In renal proximal
tubule cells, NO induces apoptosis and potentiates
immunosupressant-induced apoptosis (1, 6). ET signaling and NO production are implicated in HgCl2-induced
acute renal failure (28). In this regard, although
short-term exposure of killifish tubules to the relatively low
concentrations of nephrotoxic compounds used in the present study
produced no evidence of cellular toxicity [as measured by the ability
to actively transport fluorescein on the classical organic anion system
(Ref. 25 and Notenboom S. and Miller DS, unpublished
observations)], our initial experiments show that longer
exposures are clearly toxic and that L-NMMA can protect
(Notenboom S and Miller DS, unpublished observations). Experiments are under way to determine whether toxicity is a result of
signaling through the ET-ETB receptor-NOS-PKC pathway or
whether a separate sequence of events leads to NO production and
subsequent toxicity.
 |
ACKNOWLEDGEMENTS |
This study was supported by travel grants from the Dutch Kidney
Foundation and Dr. S. van Zwanenbergstichting.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: R. Masereeuw, Dept. of Pharmacology and Toxicology 233, Univ. Medical Center Nijmegen, PO Box 9101, 6500 HB Nijmegen, The Netherlands (E-mail: R.Masereeuw{at}ncmls.kun.nl).
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. Section 1734 solely to indicate this fact.
10.1152/ajprenal.00173.2001
Received 31 May 2001; accepted in final form 11 October 2001.
 |
REFERENCES |
1.
Amore, A,
Emancipator SN,
Cirina P,
Conti G,
Ricotti E,
Bagheri N,
and
Coppo R.
Nitric oxide mediates cyclosporine-induced apoptosis in cultured renal cells.
Kidney Int
57:
1549-1559,
2000[ISI][Medline].
2.
Burckhardt, G,
and
Wolff NA.
Structure of renal organic anion and cation transporters.
Am J Physiol Renal Physiol
278:
F853-F866,
2000[Abstract/Free Full Text].
3.
Forster, RP,
and
Taggart JV.
Use of isolated renal tubules in the estimation of metabolic processes associated with active cellular transport.
J Cell Comp Physiol
36:
251-270,
1950[ISI].
4.
Hirata, Y,
Hayakawa H,
Suzuki E,
Kimura K,
Kikuchi K,
Nagano T,
Hirobe M,
and
Omata M.
Direct measurements of endothelium-derived nitric oxide release by stimulation of endothelin receptors in rat kidney and its alteration in salt-induced hypertension.
Circulation
91:
1229-1235,
1995[Abstract/Free Full Text].
5.
Hocher, B,
Thone-Reineke C,
Bauer C,
Raschack M,
and
Neumayer HH.
The paracrine endothelin system: pathophysiology and implications in clinical medicine.
Eur J Clin Chem Clin Biochem
35:
175-189,
1997[ISI][Medline].
6.
Hortelano, S,
Castilla M,
Torres AM,
Tejedor A,
and
Bosca L.
Potentiation by nitric oxide of cyclosporin A and FK506-induced apoptosis in renal proximal tubule cells.
J Am Soc Nephrol
11:
2315-2323,
2000[Abstract/Free Full Text].
7.
Jedlitschky, G,
Burchell B,
and
Keppler D.
The multidrug resistance protein 5 functions as an ATP-dependent export pump for cyclic nucleotides.
J Biol Chem
275:
30069-30074,
2000[Abstract/Free Full Text].
8.
Kone, BC.
Nitric oxide in renal health and disease.
Am J Kidney Dis
30:
311-333,
1997[ISI][Medline].
9.
Liang, M,
and
Knox FG.
Nitric oxide activates PKC
and inhibits Na+-K+-ATPase in opossum kidney cells.
Am J Physiol Renal Physiol
277:
F859-F865,
1999[Abstract/Free Full Text].
10.
Liang, M,
and
Knox FG.
Production and functional roles of nitric oxide in the proximal tubule.
Am J Physiol Regulatory Integrative Comp Physiol
278:
R1117-R1124,
2000[Abstract/Free Full Text].
11.
Ling, H,
Gengaro PE,
Edelstein CL,
Martin PY,
Wangsiripaisan A,
Nemenoff R,
and
Schrier RW.
Effect of hypoxia on proximal tubules isolated from nitric oxide synthase knockout mice.
Kidney Int
53:
1642-1646,
1998[ISI][Medline].
12.
Masereeuw, R,
Russel FGM,
and
Miller DS.
Multiple pathways of organic anion secretion in renal proximal tubule revealed by confocal microscopy.
Am J Physiol Renal Fluid Electrolyte Physiol
271:
F1173-F1182,
1996[Abstract/Free Full Text].
13.
Masereeuw, R,
Terlouw SA,
Van Aubel RAMH,
Russel FGM,
and
Miller DS.
Endothelin B receptor-mediated regulation of ATP-driven drug secretion in renal proximal tubule.
Mol Pharmacol
57:
59-67,
2000[Abstract/Free Full Text].
14.
Miller, DS,
Letcher S,
and
Barnes DM.
Fluorescence imaging study of organic anion transport from renal proximal tubule cell to lumen.
Am J Physiol Renal Fluid Electrolyte Physiol
271:
F508-F520,
1996[Abstract/Free Full Text].
15.
Miller, DS,
and
Pritchard JB.
Indirect coupling of organic anion secretion to sodium in teleost (Paralichthys lethostigma) renal tubules.
Am J Physiol Regulatory Integrative Comp Physiol
261:
R1470-R1477,
1991[Abstract/Free Full Text].
16.
Mohaupt, MG,
Elzie JL,
Ahn KY,
Clapp WL,
Wilcox CS,
and
Kone BC.
Differential expression and induction of mRNAs encoding two inducible nitric oxide synthases in rat kidney.
Kidney Int
46:
653-665,
1994[ISI][Medline].
17.
Noiri, E,
Peresleni T,
Miller F,
and
Goligorsky MS.
In vivo targeting of inducible NO synthase with oligodeoxynucleotides protects rat kidney against ischemia.
J Clin Invest
97:
2377-2383,
1996[Abstract/Free Full Text].
18.
Plato, CF,
and
Garvin JL.
Nitric oxide, endothelin and nephron transport: potential interactions.
Clin Exp Pharmacol Physiol
26:
262-268,
1999[ISI][Medline].
19.
Plato, CF,
Pollock DM,
and
Garvin JL.
Endothelin inhibits thick ascending limb chloride flux via ETB receptor-mediated NO release.
Am J Physiol Renal Physiol
279:
F326-F333,
2000[Abstract/Free Full Text].
20.
Plato, CF,
Stoos BA,
Wang D,
and
Garvin JL.
Endogenous nitric oxide inhibits chloride transport in the thick ascending limb.
Am J Physiol Renal Physiol
276:
F159-F163,
1999[Abstract/Free Full Text].
21.
Pritchard, JB,
and
Miller DS.
Comparative insights into the mechanisms of renal organic anion and cation secretion.
Am J Physiol Regulatory Integrative Comp Physiol
261:
R1329-R1340,
1991[Abstract/Free Full Text].
22.
Schaub, TP,
Kartenbeck J,
Konig J,
Vogel O,
Witzgall R,
Kriz W,
and
Keppler D.
Expression of the conjugate export pump encoded by the Mrp2 gene in the apical membrane of kidney proximal tubules.
J Am Soc Nephrol
8:
1213-1221,
1997[Abstract].
23.
Schramm, U,
Fricker G,
Wenger R,
and
Miller DS.
P-glycoprotein-mediated secretion of a fluorescent cyclosporin analog by teleost renal proximal tubules.
Am J Physiol Renal Fluid Electrolyte Physiol
268:
F46-F52,
1995[Abstract/Free Full Text].
24.
Tack, I,
Marin-Castano E,
Bascands JL,
Pecher C,
Ader JL,
and
Girolami JP.
Cyclosporine A-induced increase in glomerular cyclic GMP in rats and the involvement of the endothelinB receptor.
Br J Pharmacol
121:
433-440,
1997[Abstract].
25.
Terlouw, SA,
Masereeuw R,
Russel FGM,
and
Miller DS.
Nephrotoxicants induce endothelin release and signaling in renal proximal tubules: effect on drug efflux.
Mol Pharmacol
59:
1433-1440,
2001[Abstract/Free Full Text].
26.
Tsukahara, H,
Ende H,
Magazine HI,
Bahou WF,
and
Goligorsky MS.
Molecular and functional characterization of the non-isopeptide-selective ETB receptor in endothelial cells. Receptor coupling to nitric oxide synthase.
J Biol Chem
269:
21778-21785,
1994[Abstract/Free Full Text].
27.
Van Aubel, RAMH,
Masereeuw R,
and
Russel FGM
Molecular pharmacology of renal organic anion transporters.
Am J Physiol Renal Physiol
279:
F216-F232,
2000[Abstract/Free Full Text].
28.
Yanagisawa, H,
Nodera M,
Umemori Y,
Shimoguchi Y,
and
Wada O.
Role of angiotensin II, endothelin-1, and nitric oxide in HgCl2-induced acute renal failure.
Toxicol Appl Pharmacol
152:
315-326,
1998[ISI][Medline].
29.
Yu, L,
Gengaro PE,
Niederberger M,
Burke TJ,
and
Schrier RW.
Nitric oxide: a mediator in rat tubular hypoxia/reoxygenation injury.
Proc Natl Acad Sci USA
91:
1691-1695,
1994[Abstract].
30.
Zhang, C,
and
Mayeux PR.
NO/cGMP signaling modulates regulation of Na+-K+-ATPase activity by angiotensin II in rat proximal tubules.
Am J Physiol Renal Physiol
280:
F474-F479,
2001[Abstract/Free Full Text].
Am J Physiol Renal Fluid Electrolyte Physiol 282(3):F458-F464