© 1995 American Heart Association, Inc.
Articles |
Effect of Graded Hypoxia on the Induction and Function of Inducible Nitric Oxide Synthase in Rat Mesangial Cells
Presented in part as an abstract at the Annual Meeting of the American Society of Nephrology, Boston, Mass, November 1993.
From the Veterans Affairs Medical Center and University of Minnesota, Minneapolis.
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Abstract |
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Abstract Inducible nitric oxide synthase (iNOS) catalyzes the formation of nitric oxide (NO) from L-arginine and O2. Although some O2 is required for this reaction, it is uncertain whether biologically relevant levels of hypoxia alter this pathway. We examined the effects of graded hypoxia on several steps in the iNOS pathway in lipopolysaccharide (LPS)-stimulated rat glomerular mesangial cells: induction of iNOS mRNA, NO synthesis, NO oxidation to nitrite (NO2-) and nitrate (NO3-), and accumulation of cGMP. Cultured cells were incubated for 24 hours in airtight flasks containing O2 (21%, 10%, 2.5%, and 0%), CO2 (5%), and N2 (balance), resulting in media PO2 levels of 140±3, 85±1, 46±3 (moderate hypoxia), and 32±5 (severe hypoxia) mm Hg, respectively. During normoxia (PO2, 85 to 140 mm Hg) LPS increased iNOS mRNA with associated increases in NO synthesis, NO2- and NO3- accumulation, and intracellular cGMP levels. In the absence of LPS, there was minimal NO synthesis and no detectable iNOS mRNA. Even during severe hypoxia, LPS elevated NO2- and NO3- relative to levels in unstimulated cells (P<.05), although to a lesser extent than during normoxia (P<.05). The induction of iNOS mRNA by LPS was preserved in hypoxia, and intracellular cGMP levels were similar at all levels of oxygen tension, indicating that iNOS induction and function were not altered by moderate or severe hypoxia. However, moderate hypoxia did alter the partitioning and oxidation of NO, favoring the appearance of NO in the "headspace" (defined as the gas overlying the cells) and NO3- in the media. We conclude that although hypoxia can alter the partitioning and decomposition of NO, the induction of iNOS mRNA and the activity of its enzyme product are resistant to hypoxia (PO2, >32 mm Hg).
Key Words: inducible nitric oxide synthase • hypoxia • mesangial cells • cGMP • nitrite/nitrate
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Introduction |
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Nitric oxide (NO) is an endogenous mediator that functions as a vasodilator, inflammatory mediator, and neurotransmitter. NO is synthesized by the inducible isoform of nitric oxide synthase (iNOS) from the substrate L-arginine and molecular oxygen in macrophages,1 2 vascular smooth muscle cells,3 hepatocytes,4 5 and glomerular mesangial cells.6 The effects of hypoxia on NO synthesis are controversial, with some investigators finding impairment in hypoxia7 8 and others demonstrating enhancement or preservation of NO synthesis in spite of hypoxia.9 10 These inconsistencies relate to two factors. First, most studies fail to fully characterize the many steps in the L-arginine–NO pathway, from the transcription of the NO synthase (NOS) gene, activity of the NOS enzyme, postsynthetic oxidation and partitioning of NO, and finally the interaction of NO with guanylate cyclase (Fig 1
).
Second, imprecise definitions of hypoxia that confuse anoxia
with levels of hypoxia, which may be considered physiologically
relevant because they represent the extremes at which
humans can survive (>40 mm Hg),11 are often used.
Furthermore, although the effects of hypoxia have been studied
on constitutive NOS (cNOS) in arterial rings and isolated
lungs7 8 10 12 13 14 15 and on isolated cerebellar
cNOS,16 we are aware of no studies examining the effects
of hypoxia on the iNOS isoform. Since iNOS and cNOS differ in
their cofactor requirements and in the fact that induction of mRNA is a
critical regulation point for iNOS, it is important to determine the
effects of hypoxia on this isoform as well.
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We have previously shown that cultured rat mesangial cells possess iNOS, which can be induced by lipopolysaccharide (LPS) and cytokines, resulting in the time-dependent accumulation of NO in the "headspace" (defined as the gas overlying the cells), nitrite and nitrate (NO2-+NO3-) in the media, and cGMP within these cells.6 The present study evaluates the effects of graded hypoxia on iNOS mRNA induction, NO synthesis, postsynthetic NO oxidation and partitioning, and cGMP accumulation, independent of changes in blood flow and shear stress.
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Materials and Methods |
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Mesangial Cell Culture and Incubation
Rat glomerular mesangial cells were grown by using techniques that yield macrophage-free homogenous cultures, as previously characterized.17 18 Cells were grown from collagenase-treated glomerular explants, passed by trypsinization when confluent, and used after the second passage. For these experiments, actively growing near-confluent cells in 75-cm2 flasks were washed twice and incubated with Eagle's MEM without phenol red, which had been supplemented with 10% fetal calf serum (Hyclone) and contained 1 mmol/L L-arginine (Sigma Chemical Co).1 18 The media included Escherichia coli LPS (serotype 0127:B8, Difco Laboratories) at 10 µg/mL or equal volumes of saline (controls). All media and solutions were prepared in endotoxin-free, deionized, distilled water, which was periodically checked for endotoxin contamination by Limulus assay (Associates of Cape Cod) and discarded if levels exceeded 0.01 pg/mL. The flasks were filled with one of the gas mixtures described below and sealed with gastight lids, which were fitted with a rubber septum for aspiration of aqueous- and gas-phase samples. Incubations were carried out for 24 hours; after this time, the entire headspace volume was removed for measurement of NO. At the same time, aliquots of media were taken for measurement of PO2, pH (Corning Blood Gas Analyzer, Corning Scientific), and NO2-+NO3-, and the cells in each flask were extracted for measurement of intracellular cGMP. The four gas mixtures used in these studies contained O2 (21%, 10%, 2.5%, or <1%), CO2 (5%), and N2 (balance) (Table
).
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Measurement of NO,
NO2-+NO3-, and
cGMP in Mesangial Cell Cultures
NO in the headspace was measured by chemiluminescence. This
assay, based on the observation that ozone interacts with NO to
generate light,19 was performed with a chemiluminescence
analyzer (Sievers 270), as previously described.20
Picomolar amounts of NO can be detected, and the assay is quite
specific for NO.19 20 21 22 23 The entire headspace volume (
250
mL) was suctioned into the chemiluminescence analyzer by using
N2-flushed tubing. The resulting electrical signal,
measured in millivolts with an integration time of 0.06 second, was
converted to picomoles of NO by comparing the signal of the sample with
a calibration curve derived from gas NO standards.22
To measure total NO2-+NO3-, an aliquot of medium was centrifuged to remove cell fragments and was reduced by incubation with E coli nitrate reductase (thus converting NO3- to NO2-).24 NO2- was quantified by reaction with the Greiss reagent (1% sulfanilamide in acetic acid and 0.1% naphthylethylenediamine).18 23 24 In some experiments, samples were divided, and the amount of NO2- was measured both before and after the nitrate reduction step. NO3- was calculated as the difference between the total NO2-+NO3- (after reduction) and NO2- (measured before reduction). This assay can detect NO2- in a range of 5 to 500 µmol/L.
cGMP was extracted from the mesangial cells at the end of the
incubation period by removing all media, washing once, and then
exposing the cells to ice-cold 0.1N HCl for 1 hour. The extraction step
was carried out in the same gas mixture in which the flask had been
incubated. The HCl sample was removed and frozen at -70°C until
assaying for cGMP by radioimmunoassay as described
previously.18 The cell protein remaining in the flask
after HCl extraction was solubilized overnight in 1% SDS and measured
by the method of Lowry et al.25 The amount of
intracellular cGMP from each flask was factored for the amount of
mesangial cell protein in the same flask.18 In some
experiments in which cGMP was measured, the NO synthesis inhibitor
N
-nitro-L-arginine methyl ester
(L-NAME, at 10-3 mol/L) was included in the 24-hour
incubation with LPS to identify the NO-dependent portion of the
intracellular cGMP.
RNA Isolation and Detection of iNOS mRNA
After the incubation of mesangial cells as described above, RNA
was isolated by using the phenol chloroform method.6 26
Total RNA (10 µg per lane) was separated by electrophoresis in a 1%
agarose gel containing (mmol/L) MOPS 20, EDTA 1, and sodium acetate 5
(pH 7.0), along with 2.2 mol/L formaldehyde. After electrophoresis, the
RNA was transferred to nylon membranes (Duralon UV, Stratagene) and
fixed by UV cross-linking (Stratalinker Stratagene). Membranes were
prehybridized for 15 minutes at 65°C with Rapid-Hyb buffer (Amersham)
and hybridized at 65°C for 90 minutes with 32P-labeled
iNOS cDNA probe in the same buffer. Two 15-minute washes in 2x
standard saline citrate (SSC)/0.1% SDS were performed at room
temperature, and a third wash was performed at 60°C for 40 minutes.
These were followed by two washes in 0.1x SSC/0.1% SDS for 40 minutes
at 60°C. Hybridized membranes were exposed to x-ray film, without
enhancing screen, for 3 days. Autoradiographs and ethidium
bromide–stained membranes were photographed under UV light, as
previously described.6 27 The 28S and 18S ribosomal RNA
was quantified from the negative by using computer-assisted
videodensitometry to verify the absence of degradation and the presence
of equivalent loading and transfer of the RNA to the membrane. In
addition, the densitometry signal for iNOS mRNA on the autoradiograph
was corrected for that of the 28S RNA on the membrane for each lane, so
that changes in iNOS mRNA signal were corrected for any variability in
loading or transfer of the RNA.27
The cDNA probe for iNOS used in these studies was cloned from mouse macrophages and kindly provided by Drs Carl Nathan and Qiao-wen Xie (Cornell University Medical College).2 The probe was labeled with [32P]dCTP (6000 Ci/mmol, NEN Dupont) by using the random oligonucleotide-primer method (Promega). Only probe with a specific activity of >1x109 cpm/mg DNA was used in these studies.
In Vitro Cell-Free Studies of NO Partitioning and Oxidation
A series of experiments was performed to establish the effect of
O2 tension on the oxidation and partitioning of authentic
NO, independent of the mesangial cell. The purpose of these studies was
to determine the effects of hypoxia alone, independent of
possible effects of hypoxia on NO synthesis that could occur
because of hypoxic alteration of NOS activity or hypoxic inhibition of
production of radicals and peroxides by the mesangial cell. A bolus of
authentic NO (0.5 mL of 0.2 mmol/L NO) was added to a glass test tube.
The 10-mL tube, sealed with a rubber stopper, contained 5 mL of
nitrite-poor water (Omnisolve, Curtin Matheson Scientific). The
PO2 of the liquid was controlled by 10
minutes of vigorous bubbling of the water in the vented tubes with
gases containing O2 (95%, 10%, or 2.5%), CO2
(5%), and N2 (balance), resulting in
PO2 values of 500, 80, and 20 mm Hg,
respectively (n=5 each). The headspace in the tube contained 5 mL of
the same gas that had been used to bubble the water. Measurements of NO
were performed on 0.1 mL of headspace and water that were removed in a
gastight syringe 5 minutes after the addition of NO. NO in the media
was added to the glass reflux chamber of the Sievers NO analyzer and
stripped into the gas phase by bubbling the sample with helium for 1
minute. The headspace was then aspirated into the analyzer, and the NO
signal was measured, as previously described.22
Drugs
All drugs and supplies were obtained from Sigma and were of
reagent grade unless otherwise designated in the text.
Statistics
Values are expressed as mean±SEM. Intergroup differences were
assessed by a simple ANOVA with post hoc testing using a Fisher's
protected least significant difference test. A value of
P<.05 was considered statistically significant. Statistics
were performed using STATVIEW 4.01 (Abacus
Concepts) on a Macintosh computer.
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Graded Hypoxia
The media PO2 and pH values resulting from 24 hours of incubation in each of the four gas mixtures are shown in the Table
. Media pH was similarly acidic for all gas mixtures.
No morphological changes in the cells or increases in lactate
dehydrogenase release were noted in the moderately or severely hypoxic
cells compared with the normoxic cells (data not shown).
NO,
NO2-+NO3-, and
cGMP
NO was not detectable in the headspace of control cells incubated
for 24 hours with media only in a normoxic atmosphere (media
PO2, 145 mm Hg) but was stimulated
>500-fold after 24 hours of incubation with LPS (Fig 2A), similar to a previous report.6 The
increase in headspace NO occurred to a similar degree in normoxia and
severe hypoxia (Fig 2A). As expected, total
NO2-+NO3- levels were
also significantly stimulated after 24 hours of incubation with LPS.
However, unlike headspace NO, total
NO2-+NO3- levels were
significantly less in LPS-treated cells in severe hypoxia
compared with the normoxic cells (Fig 2B). It is important to
note that the combination of NO2- and
NO3- in the media is the predominant pool of
NO 24 hours after iNOS induction in mesangial cells (nanomolar
quantities), whereas the NO gas is present in only picomolar
amounts. These initial studies have suggested that hypoxia
alters the L-arginine–NO pathway but did not report
whether this was due to decreased iNOS enzyme activity, impaired
induction of iNOS, or other factors that could alter media
NO2-+NO3-.
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P<.01 vs LPS at
PO2 of 140 mm Hg.
To differentiate among these possibilities and to define the threshold
PO2 at which hypoxic changes in NO may occur,
the effects of intermediate O2 tensions on NO and total
NO2-+NO3- were
measured. As shown in Fig 3A, NO accumulation in the
headspace is lowest in severe hypoxia
(PO2, 32 mm Hg). The greatest amount
of headspace NO was found in cells with moderate hypoxia
(PO2, 46 mm Hg) and then decreased
again at the two highest oxygen tensions we tested. Total
NO2-+NO3- in the
media was lowest in moderate and severe hypoxia and increased
proportionately at higher PO2 levels (Fig 3B). cGMP levels (shown in Fig 3C) were similar in cells incubated with
LPS at all four levels of oxygen tension. Since cGMP is a second
messenger in mesangial cells for substances other than NO, we also
measured cGMP in cells during normoxia and severe hypoxia after
incubation with LPS and the NO synthesis inhibitor L-NAME
(10-3 mol/L). The mean percent inhibition by L-NAME, ie,
the NO-dependent portion of cGMP, was similar in normoxia and severe
hypoxia (36.3±25% and 33.6±6%, respectively; n=3
experiments). Thus, we conclude that neither the total intracellular
cGMP after LPS nor the NO-dependent portion of the cGMP is altered by
hypoxia.
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Cell-Free In Vitro Experiments
To better understand the effects of hypoxia on the
oxidation and partitioning of NO, we assessed these parameters in a
cell-free in vitro model. When a fixed amount of authentic NO was
introduced into deoxygenated water, the greatest recovery of NO in the
headspace and media occurred under hypoxic conditions (Fig 4). This confirms that hypoxia, independent of
effects on iNOS activity, facilitates the partitioning of NO to the
headspace. This direct effect of hypoxia on NO partitioning may
explain why headspace NO is increased in renal mesangial cell cultures
during moderate hypoxia, whereas
NO2-+NO3-
accumulation is diminished (as shown in Fig 3).
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Alterations in the Oxidation Products of NO
In some experiments, we also measured media
NO2- and NO3-
separately during graded hypoxia. We found that not only did
hypoxia reduce the total
NO2-+NO3- but it also
altered the relative amounts of NO2- and
NO3- found in the media (Fig 5). In normoxia, (PO2,
140 mm Hg), NO3- made up roughly 60% of the
total
NO2-+NO3-,
whereas NO2- accounted for 40%. However, as
media PO2 decreased from 140 to 32 mm Hg,
the relative contribution of NO2- to total
NO2-+NO3- diminished
to <10% of total
NO2-+NO3- (Fig 5).
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iNOS mRNA
We have previously shown that mRNA for iNOS is not detectable by
Northern analysis of total RNA from unstimulated rat mesangial
cells but is markedly induced by 24 hours of incubation with LPS (10
µg/mL).6 In the present study, we examined the
effect of hypoxia on induction of iNOS mRNA by LPS. Fig 6 shows the iNOS signal in mesangial cell RNA from two
separate mesangial cell experiments, each with identical
conditions. Cells were incubated with LPS (10 µg/mL) for 24 hours in
the same four levels of oxygen as for the experiments described above.
Fig 6A is the autoradiograph after hybridization with cDNA probe
against iNOS; Fig 6B shows the membrane stained with ethidium bromide.
The same total amount of RNA (15 µg) was loaded in each lane.
Densitometry was performed on the autoradiograph and the stained
membrane from each of these experiments, and the iNOS signal was
corrected for the 28S RNA signal. The results, shown in Fig 6C,
demonstrate that the induction of iNOS mRNA by LPS was similar at all
four oxygen tensions tested in both experiments. Furthermore, we have
confirmed these findings in two additional experiments (data not
shown).
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Discussion |
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We have examined the effects of reduced oxygen availability on the induction of iNOS mRNA, on the partitioning and oxidation of NO, and on the biological response to NO in rat glomerular mesangial cells. We6 and others28 have recently shown that these cells possess an iNOS that can be induced by LPS and cytokines and is similar to that found in macrophages and vascular smooth muscle cells. The present study demonstrates that although NO in the headspace and NO2-+NO3- in the media are altered by hypoxia, the induction of iNOS mRNA and the intracellular cGMP levels are preserved even during severe hypoxia.
There are many steps in the L-arginine–NO pathway
that must occur for LPS to elicit an effective increase in NO
production and biological activity (Fig 1). First, the NOS gene must be
transcribed to mRNA, an important regulatory step in the case of iNOS.
Using Northern hybridization with a probe cloned from mouse
macrophages, we have been unable to detect iNOS mRNA in unstimulated
mesangial cells. However, within 4 hours of incubation with LPS, iNOS
mRNA is detectable, and by 24 hours, an eightfold increase in this mRNA
is observed.6 In the present study, similar levels of
iNOS mRNA were seen after 24 hours of incubation with LPS, despite
exposure to different oxygen tensions (Fig 6). Therefore, we conclude
that hypoxia does not affect the transcription of gene to mRNA
for iNOS.
The second point in the L-arginine–NO pathway that we
explored was the direct measurement of headspace NO by
chemiluminescence. This method is quite sensitive and can detect NO in
the headspace of mesangial cells after as little as 6 hours of
incubation with LPS.6 By sampling from the gas above the
mesangial cells and media, we measure only that proportion of the total
NO that manages to escape the cell and that has avoided oxidation in
the media. This is a small fraction of the total NO released from the
cell, as demonstrated by the finding that media
NO2-+ NO3- levels are
200- to 300-fold greater than headspace NO levels (Fig 2). Furthermore,
the headspace NO content is not solely a function of the rate of NO
synthesis but also its rate and degree of decomposition. Moderate
hypoxia, compared with normoxia, increased headspace NO,
whereas severe hypoxia markedly reduced NO to a level slightly
lower but not significantly different from that of normoxia (Fig 3A).
This biphasic change in headspace NO can be explained by alterations in
both its partitioning and decomposition. During normoxia, headspace NO
is low because most of it either decomposes while in the media or
rapidly after exposure to the oxygen in the headspace. Our results
suggest that this partitioning and decomposition can be reduced by
limiting the oxygen in the system. The cell-free in vitro studies with
authentic NO confirm that hypoxia favors the survival of NO, so
that it may escape the media and enter the headspace (Fig 4). Thus,
under conditions of moderate hypoxia, more NO will appear in
the gas phase from a fixed amount of NO, whether that NO is generated
by cells or added to the aqueous phase as "authentic" NO gas in
solution. Furthermore, as a result of the preservation of NO in
hypoxia, less
NO2-+NO3- is
detectable in the media (Fig 3B). We conclude that although headspace
NO is a very sensitive marker of iNOS function, it is a tiny portion of
the total NO synthesized and is susceptible to changes in partitioning
with variation in ambient PO2. Consequently,
headspace NO should probably not be used in isolation to monitor NO
synthesis.
The total amount of the two major NO oxidation products,
NO2- and NO3-,
were found to progressively decrease with increasing severity of
hypoxia (Fig 3B). Because, in the presence of
O2, NO rapidly oxidizes to form these products and
because these products are stable enough to accumulate during the 24
hours of incubation, the total
NO2-+NO3- accounts
for the majority of NO produced during the incubation period. Thus, the
finding that severe hypoxia reduced the total
NO2-+NO3- in the
media as well as the NO in the headspace suggests that iNOS activity is
reduced in severe hypoxia. This is consistent with a previous
report that severe hypoxia
(PO2, 28 mm Hg), compared with
normoxia (PO2, 130 mm Hg), markedly
inhibits the activity of isolated bovine cerebellar cNOS16
and the recent findings of McQuillan et al29 demonstrating
a decrease in steady state mRNA for cNOS from human vascular
endothelial cells after 24 to 48 hours of oxygen levels of 20 mm Hg.
In addition, the latter authors also found evidence for decreased NO
generation by these endothelial cells, in that reporter smooth muscle
cells showed little change in cGMP after coincubation with endothelial
cells exposed to hypoxia.
In contrast, we have found that intracellular cGMP levels after incubation with LPS are not different in mesangial cells during normoxia, moderate hypoxia, and severe hypoxia. Rat glomerular mesangial cells possess not only the ability to synthesize NO but also directly respond to it with changes in intracellular cGMP, increases in cell surface area (ie, relaxation), and inhibition of proliferation.17 18 30 We have previously shown that LPS-induced changes in cGMP are mainly dependent on NO by demonstrating significant inhibition of these changes with the NO synthesis inhibitor L-NAME.6 In the present study, the inhibitory effect of L-NAME on cGMP levels after incubation with LPS was similar in normoxia and severe hypoxia. NO interacts with cytosolic guanylate cyclase in these cells, resulting in accumulation of cGMP.31 32 cGMP accounts for most of the physiological effects of NO through its ability to stimulate kinases,33 34 activate potassium channels in vascular smooth muscle,35 36 and lower cytosolic calcium.34 37 It is possible that the cGMP is preserved in hypoxia because the amount of NO produced during hypoxia, even if slightly reduced, exceeds some "threshold" for activation of guanylate cyclase. Alternatively, hypoxia, by enhancing the survival of NO, could yield the same number of NO molecules surviving to activate guanylate cyclase from a smaller amount of NO synthesis. An additional consideration is that the intracellular location of both the iNOS and guanylate cyclase in mesangial cells protects this pathway from changes in oxygen tension in the headspace or media during the time of our incubations. Finally, and most likely, is the conclusion that the actual amount of NO produced from mesangial cell iNOS is not altered by hypoxia, since the cGMP response and mRNA induction are not different. Therefore, the alterations in NO in the headspace and NO2-+ NO3- in the media are mainly due to changes in partitioning and decomposition. Thus, it would seem that the function of cNOS (at severe hypoxia in some cell types) is suppressed by hypoxia,16 29 whereas iNOS is preserved, suggesting differing roles for these isoforms in the pathophysiological effect of hypoxia.
An additional finding in our studies is that in hypoxic environments,
the ratio of the decomposition products NO2-
and NO3- is altered. In normoxia, 65% of
the total NO2-+NO3-
is accounted for by NO3-, whereas in
severe hypoxia 90% is NO3- and <10%
is NO2- (Fig 5). These findings suggest that
NO is oxidized in a different manner in hypoxia than in
normoxia. It is possible, though unproved, that hypoxia
promotes NO3- formation by increasing the
availability of the forms of hemoglobin and cytochromes, which are
needed to convert NO2- to
NO3-.38 An alternative
explanation is that hypoxia favors the conversion of NO to
peroxynitrite, which is then converted to
NO3-, without the intermediate
formation of NO2-.39 On the basis
of these findings, we conclude that an additional effect of
physiological levels of hypoxia on the
L-arginine–NO pathway is to alter the oxidation pathways
for NO. These effects could have implications for the duration of
action and the bioavailability of NO in biological systems.
Furthermore, it suggests that conclusions regarding alterations in the
L-arginine–NO pathway in different pathophysiological
states require careful examination and measurement of more than one
point in this pathway.
We have previously demonstrated that glomerular mesangial cells in culture produce NO via an LPS-stimulated iNOS6 and have evidence that the kidney is an important producer of NO in vivo.24 Furthermore, iNOS is found in the juxtaglomerular apparatus,40 arcuate and interlobular arteries, glomeruli, and many parts of the nephron, especially the medullary thick ascending limb and inner medullary connecting duct.41 NO is important in maintaining basal renal vascular tone, glomerular filtration rate,42 and sodium balance, the latter by its natriuretic effect.43 Thus, NO produced by glomerular mesangial cell iNOS could have a number of important actions within the glomerulus and, perhaps by entering the glomerular filtrate, could affect tubular function as well.
Chronic hypoxia can affect vascular remodeling, vessel tone, and function in many organs, including the kidney. Abnormalities in the kidney due to long-term hypoxia include glomerular hypertrophy and focal glomerulosclerosis, although the frequency of clinical renal dysfunction due to chronic hypoxia is unknown. Furthermore, acute hypoxia due to renal hypoperfusion or ischemia is a frequent cause of acute renal failure. Recent studies demonstrated that calcium-independent NO activity was increased in isolated proximal tubules after 15 minutes of severe hypoxia and increased further 35 minutes after reoxygenation.44 Inhibition of the NO with L-NAME reduced the hypoxia/reoxygenation injury in these cells. Thus, given the work of others along with the findings of the present study, it is quite possible that NO is important in the pathophysiological effects of hypoxia on the kidney.
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Acknowledgments |
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This study was supported by research funds from the Department of Veterans Affairs and by National Institutes of Health grant HL-45735 (Dr Archer). The authors gratefully acknowledge Daniel P. Nelson and Nancy Cowan for their technical contributions to these studies.
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Footnotes |
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Reprint requests to Stephen Archer, MD, Cardiology Section (111C), VA Medical Center, 1 Veterans Dr, Minneapolis, MN 55417.
Received September 14, 1994; accepted March 13, 1995.
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References |
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