1 Renal Section, Macrophage
dysfunction is considered an important contributory factor for
increased propensity of infections in uremia. Because nitric oxide (NO)
is believed to be an effector molecule of macrophage cytotoxicity, we
propose that the dysfunction may be related to impaired NO synthesis.
To verify this hypothesis, we evaluated macrophage NO synthesis in the
presence of urea, a compound that accumulates in renal failure and is
believed by some to be a uremic toxin. Macrophages (RAW 264.7 cells)
were incubated with bacterial lipopolysaccharide to induce NO
synthesis, whereas the test groups had various concentrations of urea
in addition. NO synthesis was measured by assaying the supernatant for
nitrites and nitrates by chemiluminescence. We observed that urea
consistently produced a dose-dependent reversible inhibition of
inducible NO production in macrophages, whereas parathormone, another
toxin retained in uremia, had no such inhibitory effects. Further
studies revealed that mRNA for inducible NO synthase was not inhibited
by urea. We thus conclude that urea inhibits inducible NO synthesis in macrophages by a posttranscriptional mechanism and that this may be
important in macrophage dysfunction of uremia.
inducible nitric oxide synthesis; macrophage dysfunction; uremia
INFECTIONS CONTINUE TO BE an important cause of
morbidity and mortality in patients with end stage renal disease.
Although several immunologic abnormalities have been described in
uremia, the precise mechanisms responsible for the impaired host
defenses remain unclear. Macrophages constitute the first line of host defenses in conferring immunity against infections in humans. Macrophages have been shown to be dysfunctional in uremia, and impaired
function of macrophage Fc Cell cultures.
Murine peritoneum-derived macrophages (RAW 264.7 cell line) were
obtained from the American Type Culture Collection, and
passages 5-10
were grown in RPMI 1640 (GIBCO BRL, Life Technologies, Grand Island,
NY) enriched with fetal calf serum (GIBCO BRL). Medium was customized
to contain L-glutamine but no
nitrate to avoid interference with the nitrate assay. Cells were
incubated at 1 × 105
cells/well in 96-well plates in a 5%
CO2 incubator at 37°C. NO synthesis was induced by bacterial lipopolysaccharide (LPS) derived from Escherichia
coli serotype 026:B6 (Sigma Chemical,
St. Louis, MO) at a concentration of 1 µg/ml in each well.
Chemicals.
L-Arginine, murine parathyroid
hormone (PTH), and urea were purchased from Sigma. Stock
solutions of E.
coli LPS serotype 026:B6 were stored
at 4°C until needed. Endotoxin-tested fetal calf serum purchased
from GIBCO BRL was used in the preparation of culture medium.
Reconstituted culture medium was stored at 4°C until needed.
NO measurement.
The supernatant of medium from each well was analyzed for NO production
by chemiluminescence, using a Sievers Instruments NO analyzer (Boulder,
CO). The NO produced by the cells is quickly converted to its stable
end products, namely nitrite and nitrate. Nitrite-nitrate content is
reduced to NO with a vanadium reducing mixture in a reaction tube
on-line with the analyzer. The NO is propelled with nitrogen gas into
another reaction chamber in the NO analyzer, where NO reacts with ozone
to form NO2. The luminescence associated with this reaction is read in millivolts and recorded as a
deflection on a data recorder. The area under the curve reflects the NO
content in the sample. This is read initially as a value in
luminescence units, which when divided by the slope of a standard curve
gives the precise NO content. The standard curve was made in culture medium spiked with nitrate. The NO activity of the untreated
medium was subtracted from all the measurements to eliminate the
baseline NO content.
mRNA extraction and reverse transcription-polymerase chain
reaction.
Total cellular RNA was extracted using the guanidinium thiocyanate
method (3). After aspiration of the supernatant, the pellet
of cells was washed with phosphate-buffered saline and resuspended at a
concentration of 1 ml
2,3,4,6-tetra-O-acetyl- Northern blot analysis.
RAW 264.7 cells were grown in large 150 cm2 flasks to the point of
confluence and further incubated with fresh medium. The test groups had
either LPS alone or LPS and urea, whereas the control group had
neither. The incubations were carried out for 18 h at 37°C in a
CO2 incubator. At the end of the
incubation, the supernatant was collected separately for NO analysis
and the cells were centrifuged into a pellet for mRNA extraction. Total cellular RNA was extracted from RAW 264.7 cells in guanidine
thiocyanate. Proteins were denatured and eliminated by
phenol-chloroform-isoamyl alcohol extraction. The total RNA was
precipitated using isopropanol. Contaminating DNA was removed by
digestion with ribonuclease-free deoxyribonuclease (Promega, Madison,
WI). Approximately 50 µg of total RNA was electrophoresed through a
1% agarose-formaldehyde gel, transferred to GeneScreen nylon membrane
(DuPont-NEN, Boston, MA), and vacuum dried at 80°C for 2 h,
followed by ultraviolet cross-linking (under conditions recommended by
manufacturer). iNOS mouse macrophage cDNA probe (Cayman Chemical, Ann
Arbor, MI) was labeled with 32P,
using the random prime kit (Boehringer Mannheim) to a specific activity
of ~2 × 108
counts · min Data analysis.
All data are expressed as means ± SE. The data were analyzed using
commercial computer software (SlideWrite Plus 2.0 for Windows, Advanced
Graphics Software) for statistical significance, means, and SE.
Comparisons between groups of samples were performed by independent
t-test or repeated measures analysis
of variance as appropriate. Specific comparisons between the means were
done with Student-Newman-Keuls multiple range test. A value of
P < 0.05 was considered
statistically significant. The slope of the standard curves, the
intercept, and the r values for
nitrite-nitrate measurements were calculated by linear regression
analysis, and all illustrations were made with SlideWrite Plus 2.0 for
Windows. In some cases, the means of several experiments were used to
illustrate the observations.
Previous work demonstrated that NO produced by activated macrophages
may be an effector molecule in mediating the cytotoxicity (5, 6a).
Macrophages are believed to be functionally defective in renal failure
(18). The nature of this functional alteration is not completely clear,
and thus it is reasonable to hypothesize that the defect may relate to
the ability of macrophages to produce NO in the uremic
environment. To further explore this hypothesis, we evaluated the
effects of urea, a metabolite that accumulates in renal failure, on the
NO production in RAW 264.7 cells.
NO production in activated macrophages.
Initially, we evaluated the ability of RAW 264.7 cells to synthesize NO
by induction with bacterial endotoxin. RAW 264.7 cells were incubated
at 1 × 105 cells/well in
96-well plates. After a 2-h incubation, the nonadherent cells were
removed by aspiration of the supernatant and fresh medium was added.
Bacterial LPS derived from the E.
coli of the serotype 026:B6 was added
to the test group to yield a final concentration of 1 µg/ml. The incubation was continued at 37°C for 18 h, after which the supernatants were collected for measurement of NO
production. The NO activity of the untreated medium was subtracted from
all the measurements to eliminate the baseline NO content. The NO content (as reflected by the nitrite and nitrate content in the medium)
in the control group (n = 8) was 21.8 ± 2.1 µM, whereas the activated cells produced 93.2 ± 3.6 µM (P < 0.001). The magnitude of
LPS induction of NO synthesis by macrophages was dose dependent. Although we found significant and progressively greater induction of NO
synthesis with concentrations of 0.1, 1, and 10 µg/ml (Fig. 1), the cytopathic effects of
LPS were distinctly prominent at a concentration of 10 µg/ml. We thus
used an LPS concentration of 1 µg/ml in all our experiments. We also
noted that the induction of NO synthesis was noticeable as early as
after 6 h of incubation, with maximal activity seen after 18 h.
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
receptors was recently described in end stage renal disease (18).
However, the impaired cytotoxicity of macrophages in uremia remains to
be explained. Macrophages are now well known to produce nitric oxide
(NO), and there is evidence to believe that NO may be an effector
molecule of several functions of macrophages, especially cytotoxicity
(6a, 7). It is thus possible that macrophage dysfunction in uremia may
be related to defective NO production. We, therefore, studied the NO
production from macrophages and the effect of some of the known uremic
toxins such as urea. The results indicated that the urea inhibited
synthesis of inducible NO synthase (iNOS) in activated macrophages. We
further attempted to define the mechanisms of such an inhibition.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-D-glucopyranosyl isothiocyanate (GITC)
buffer/107 cells. The lysate was
passed through a pipette several times before addition of 0.1 ml of 2 M
sodium acetate at pH 4.0, 1 ml of water-saturated phenol, and 0.2 ml
49:1 chloroform-isoamyl alcohol. The lysate was extracted by gentle
mixing before incubation at 4°C for 15 min. The lysate was
centrifuged at 10,000 g for 20 min.
The aqueous phase was precipitated in an equal volume of 100%
isopropyl alcohol at
20°C for 30 min. The RNA was further centrifuged at 10,000 g for 10 min.
Once dissolved in 1/3 volume of GITC buffer, the RNA was precipitated
with 1 volume of 100% isopropyl alcohol for 30 min at
20°C.
The RNA pellet was cleaned using 75% ethanol in diethyl pyrocarbonate
(DEPC)-treated water. The RNA pellet was resuspended in DEPC-treated
water. RNA was quantitated by spectrophotometry at 260 nm. The purity
was assessed by measuring the 260 nm-to-280 nm ratio. For reverse
transcription-polymerase chain reaction (RT-PCR), the primers were
designed from the highly homologous region of the published mouse cDNA
sequences and chemically synthesized by Biosynthesis (Lewisville, TX).
The cDNA was then amplified with specific primers for 30 cycles with 1 min at 94°C, 1 min at 55°C, and 2 min at 72°C. The PCR
products were size fractionated on 1.2 agarose gel and detected by
ethidium bromide staining.
1 · µg
1.
The labeled probe was then hybridized to the membrane in 50% formamide, 10% dextran sulfate, 1% sodium dodecyl sulfate (SDS), and
5× SSPE (1× SSPE is 0.15 M NaCl, 0.01 M
NaHPO4, and 0.001 M EDTA, pH
7.4). The hybridization was done at 42°C for 24 h. The
membranes were washed once at room temperature for 15 min with 2×
SSPE. This was followed by a wash with 1× SSPE and 2% SDS at
65°C for 30 min, followed by a wash in 0.1× SSPE at room temperature for 15 min. Autoradiograms were prepared using Kodak XAR-5
film with intensifying screens at
80°C for 24 h.
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
View larger version (16K):
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Fig. 1.
Effects of bacterial lipopolysaccharide (LPS) on nitric oxide (NO)
synthesis by macrophages expressed as nitrite-nitrate content in
medium. Time- and dose-response relationship
(n = 8 in control and all test
groups). Values are means ± SE of 4 experiments.
# P < 0.05 vs. control.
Effects of urea on NO synthesis. We found that urea inhibited the NO production by activated RAW 264.7 macrophages. The inhibition was concentration dependent (Fig. 2) and was seen at concentrations as low as 10 mM, and maximal inhibition was seen at 100 mM concentration. Because a concentration of 50 mM corresponds to a urea nitrogen of ~150 mg/100 ml, concentrations seen in uremic humans, we continued further experiments at a urea concentration of 50 mM in the culture medium.
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Effects of urea on cell viability. To evaluate the effects of urea on cell viability, we incubated the macrophages in the presence of urea (50 mM), and we found that under such conditions these cells exhibited growth and proliferation comparable to control conditions (i.e., in the absence of urea in the medium). When cells were incubated in 75-cm2 flasks (n = 4) at a concentration of 106 cells/ml, the cell growth and proliferation at the end of 48 h of incubation were not significantly different between control conditions and in the presence of urea. The mean density of cell population as measured by a cell counter was 4.86 × 106 cells/ml in the control flask vs. 5.18 × 106 cells/ml in the flask containing urea in the medium. In addition, the time needed to attain confluence in the flask was similar under both conditions (4 days in both). Furthermore, the cell viability as measured with trypan blue staining was 97.5 vs. 98.1%, respectively, in the control and urea groups. These findings indicated clearly that urea did not affect the viability of the macrophages in culture.
Effects of PTH. To evaluate whether other known uremic toxins have an effect on NO production by macrophages, we examined the effects of PTH on NO production. PTH is incriminated in defective phagocytosis in uremia, but its effects on iNOS are unknown. We incubated RAW 264.7 cells with a rat PTH preparation at a concentration of 400 pg/ml, a concentration of PTH seen in rodent models of advanced renal failure. The control group had an NO content of 22.9 ± 2.3 µM, whereas the group incubated in the presence of PTH produced 24.3 ± 1.8 µM (n = 8; NS). Thus PTH had no independent effects on NO synthesis in RAW 264.7 cells. Furthermore, when RAW 264.7 cells were activated with LPS in the presence of PTH, they produced NO measuring 79.2 ± 3.36 µM (n = 8), indicating that PTH did not interfere with LPS-induced NO synthesis.
Effects of L-arginine supplementation. To understand the mechanism of urea-induced inhibition of NO synthesis in macrophages, we first evaluated the effects of L-arginine supplementation to assess whether this inhibition was at the level of substrate utilization. Because L-arginine is a precursor of NO, enrichment of the culture medium with L-arginine would overcome the inhibition of NO by urea. RAW 264.7 cells were incubated in RPMI 1640 medium saturated with additional L-arginine to yield a final concentration of 1 mM. LPS-induced NO synthesis was 51.2 ± 5.58 µM vs. a control value of 12.5 ± 2.02 µM (P < 0.01; n = 12). L-Arginine supplementation enhanced the NO synthesis by LPS to 69.8 ± 2.6 µM (P < 0.05). Addition of urea significantly inhibited LPS-induced NO synthesis (n = 12; 31.2 ± 2.27 vs. 51.2 ± 5.58 µM; P < 0.01). Supplementation of L-arginine did not prevent urea-induced inhibition of NO synthesis (33.24 ± 2.94 µM compared with 32.16 ± 2.27 µM without additional L-arginine; NS). Thus L-arginine supplementation did not overcome the urea-induced suppression of NO synthesis from activated macrophages (Fig. 4), suggesting that inhibition is not due to interference with substrate utilization.
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mRNA analysis and northern blot studies. Our studies evaluating the iNOS mRNA expression in macrophages using RT-PCR and Northern blot techniques indicate that urea did not interfere with the iNOS gene expression. Figure 5 shows a representative Northern blot analysis of RNA (n = 3) hybridized to species-specific 32P-labeled iNOS. The untreated cells contain no discernible iNOS mRNA; however, LPS treatment induced iNOS mRNA synthesis. This induction of mRNA was not diminished to any appreciable level by the addition of urea to LPS, whereas the NO production was inhibited by >95%. This indicates that the inhibitory effects of urea may involve a posttranscriptional step at or beyond translation. However, more studies are required to define the precise mechanism of this inhibition.
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DISCUSSION |
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The defects of host defense mechanisms in renal failure are multiple and involve alterations at various levels. Macrophage dysfunction appears to be a major site of impairment of host defenses. First, Hibbs et al. (6a, 7) showed that NO may be a mediator of macrophage-mediated cytotoxicity. More recently, Vallance et al. (22) described and Brenner and Yu (2) discussed the inhibition of NO synthesis due to retained endogenous factors in uremia. It is therefore conceivable that the dysfunction of macrophages in uremia may indeed be due to inhibition of NO synthesis. Our experiments were designed to examine this hypothesis.
Macrophages are one of the earliest known sources of NO synthesis (4).
NO is a labile free radical that acts as a paracrine and autocrine
agonist formed exclusively from
L-arginine by the action of the
enzyme NO synthase (15), of which there are at least two subtypes: the
constitutive subtype commonly expressed in endothelium and neurons,
which is calcium dependent, and the inducible subtype, as seen in the
macrophage and vascular smooth muscle, which is calcium independent (6)
and is induced by cytokines and endotoxins and regulated by
prostaglandins (9) and guanidines (14). Although NO was first
discovered to be the endothelium-derived relaxing factor (8),
subsequently many physiological roles have been ascribed to NO in
various tissues and organ systems, such as control of systemic blood
pressure, platelet aggregation, control of renal hemodynamics,
regulation of renal tubular transport, immune defenses, and neuronal
signaling in central and peripheral nervous systems (13, 20). In the area of immune defense mechanisms, NO, which is released by macrophages on induction with bacterial endotoxins, -interferon, or tumor necrosis factor, is believed to function as the effector molecule mediating the cytotoxicity (7). The NO inhibits the iron-containing enzymes in the target cells, including several enzymes involved in
mitochondrial respiration (10). In addition, NO also inhibits DNA
synthesis in the target cells. Thus NO may mediate the
macrophage-dependent killing of bacteria, protozoa, and fungi. Thus,
although the increased susceptibility to infections that occurs in
uremic syndrome has been largely attributed to lymphocytopenia and
leukocyte dysfunction, impairment of NO-mediated macrophage
cytotoxicity may play an important role in the immune dysfunction in
uremia.
Our preliminary experiments demonstrated that incubation of RAW 264.7 cells in uremic plasma inhibited iNOS production in these cells. However, these findings are similar to those of Arese et al. (1), who showed that incubation of J774 murine macrophage cell line in uremic human plasma caused inhibition of iNOS synthesis. We then proceeded to examine the specific factors in uremia that could influence iNOS production in macrophages.
Figure 1 shows that NO production can be induced in the activated macrophages by bacterial LPS derived from E. coli. Our results indicated that the induction caused a significant increase in NO content from a control value of 21.8 ± 2.1 to 93.2 ± 3.6 µM and that the maximal increase was seen at 18 h of incubation. It is also of interest that the basal nitrite-nitrate level is of considerable amount, and possibly there is some release of constitutive NO from macrophages, which is seen particularly with cells in late passages. This observation is consistent with the studies described earlier in RAW 264.7 cells (19). We also noticed that the baseline NO generated from the control cells was a function of the passage number, i.e., the higher the passage number the higher the NO production or constitutive NO synthesis. A similar observation was made by Schmidt et al. (19).
Our experiments showed that urea inhibited inducible NO production from macrophages in a dose-dependent fashion. The inhibition was observed in low concentrations of 10 mM but was maximal in concentrations of 50-100 mM (Fig. 2). It is interesting that significant inhibition was seen at a urea concentration of 50 mM, which corresponds to a blood urea nitrogen of ~150 mg/dl, which is a concentration that is seen in patients with advanced renal failure. Thus our observations are relevant to human or animal models of advanced renal failure, and the inhibition of NO synthesis by urea may have an important consequence. The fact that the inhibition of NO production was seen at 6 h and not earlier indicates that the mechanism may involve protein synthesis or gene expression. The maximal inhibition was seen at 18-24 h, which is again similar to the NO production from activated macrophages. These temporal correlations indicate that the inhibition of urea may be directly related to inhibition of NO synthase. We evaluated the effects of urea on NO production because there is renewed interest in urea as a potential cellular toxin. Urea is well known as a protein metabolite that accumulates in renal failure and is used conventionally to measure the severity of renal impairment. Because we wanted to examine the effects of renal failure, we thought that urea might be a reasonable candidate, since urea may have effects on cellular metabolism that have not been hitherto described. Notwithstanding the fact that urea was conventionally regarded as an innocuous metabolite that accumulated in renal failure, the recent evidence indicates that urea in fact may interfere with cellular metabolism and protein synthesis (17). In addition, recently several studies have demonstrated that urea may interfere with transport of electrolytes and also inhibit cellular and extracellular enzyme synthesis (12). So, we hypothesized that urea may also have an effect on NO synthesis in cells that are known to synthesize NO. In this context, it is interesting to note that Szabo et al. (21) showed recently that S-methylisothiourea was a potent and selective inhibitor of iNOS in a mouse macrophage cell line, and they further concluded that this compound may be beneficial in the rodent model of septic shock. It is possible that the urea part of this compound may play a role in this inhibition. We chose to examine the effects of urea on NO synthesis because urea has been shown to interfere with cellular transport mechanisms and synthesis of various intracellular enzymes (12, 17). However, we recognize the fact that many other factors retained in uremia may also potentially interfere with NO synthesis.
Our data suggest that urea had no effect on the basal or constitutive NO synthesis as reflected by our results (28.6 ± 2.1 µM vs. control 23.8 ± 2.4 µM after 18 h of incubation; n = 8; NS) These data lend further support to the conclusion that urea-induced effects on iNOS are quite specific. Our studies also demonstrate that urea-induced inhibition of NO synthesis is reversible as is evident from the observations that cells preincubated in the presence of urea retained their ability to be induced by LPS to synthesize NO when reincubated in a urea-free medium (Fig. 3). Thus urea specifically and reversibly inhibited the inducible NO synthesis in macrophages, and these observations may have important implications in the macrophage dysfunction of uremia.
The impact of renal failure on NO production in vivo is not well understood (11). There are conflicting data about the significance of NO in the pathophysiology of renal failure (16). It appears that the plasma levels may be elevated in patients with renal failure, especially in the face of fluctuations of blood pressure. However, most of this NO is derived from endothelial cells, and hence it is not clear how uremia affects the production of iNOS from macrophages. Our data show that urea, a metabolite that accumulates in the presence of renal failure, inhibited iNOS in RAW 264.7 cells (Fig. 2). We also explored other compounds that accumulate in renal failure in terms of their effects on NO production. PTH is known to accumulate in the presence of renal failure in humans and also in rodent models of renal failure. Interestingly, there are very few data on the effect of PTH on the iNOS in macrophages. We used various concentrations of PTH and found that there was no effect on NO production at any concentration. At 400 pg/ml, a concentration seen in rodent models of advanced renal failure, we did not find any effect on iNOS production in the RAW 264.7 cells. Our data indicate that PTH did not interfere with LPS-induced NO synthesis. Although iNOS was not analyzed by Northern blots, it is extremely unlikely that LPS-induced NO synthesis in PTH-treated cells occurred without iNOS mRNA synthesis. It is known that certain agents such as cyclohexamide inhibit NO synthesis beyond the phase of iNOS mRNA synthesis, but there is no substance described that facilitates an LPS-induced NO synthesis in the absence of the iNOS mRNA synthesis. Hence we did not feel it necessary to perform Northern blots in PTH experiments.
We thus concluded that the NO inhibition from macrophages in renal failure could be predominantly from the effects of urea. In this context, it is interesting to review the results of Vallance et al. (22), who found that NO synthesis is impaired in the uremic syndrome. They proposed that this inhibition is mediated by asymmetric dimethyl arginine, a compound that accumulates markedly in patients with end stage renal failure. So it is possible that there may be several compounds that accumulate in renal failure that inhibit the NO synthesis in vivo.
The fact that L-arginine supplementation, which normally enhances NO synthesis, failed to prevent the urea-induced inhibition of NO synthesis indicates that urea-induced inhibition could not be overcome by enhanced substrate availability (Fig. 4). Despite saturation of the cell system with maximum concentrations of L-arginine (1 mM), urea continued to inhibit NO synthesis, implying that the inhibition is not at the level of substrate utilization. If significant, uncontrolled arginase were present in the medium, the resultant conversion of arginine to urea would actually decrease and not increase NO synthesis with L-arginine supplementation. Our experimental data indeed showed that L-arginine significantly enhanced inducible NO synthesis (51.2 ± 5.58 µM with LPS vs. 69.8 ± 2.6 µM with supplemental arginine; P < 0.05), implying that, if any arginase was present, it was insufficient to affect NO synthesis. Additionally, because the same medium was used in all our experiments, arginase, if at all present in any amount, would affect all experimental groups equally, diminishing its significance. Furthermore, we found no evidence in the literature to postulate that urea stimulates arginase activity.
Our extensive studies examining the iNOS mRNA expression using the RT-PCR and Northern blot analysis indicated that the urea did not interfere with the synthesis of mRNA (Fig. 5), and hence we believe that the inhibition is at a posttranscriptional level. It is possible that urea inhibits translation of iNOS or inhibits a posttranslational step such as calmodulin binding of iNOS or binding with cofactors such as NADPH, tetrahydrobiopterin, or flavin mononucleotide. Further experiments would be necessary to clarify the details of this mechanism. It is interesting that, although urea inhibits many enzymes by impairing the cellular synthesis of these proteins, urea-induced inhibition of NO synthase may involve a step different from the other enzymes. The significance of this inhibition may be clinically important. If these results of in vitro studies are extrapolated into an in vivo situation, it is possible that, with increasing concentration of urea in advancing renal failure, there may be impairment of intrarenal production of NO. This effect could potentially influence the intrarenal hemodynamics in an unfavorable manner, leading to a progressive intrarenal vasoconstriction. With progressive loss of renal mass, elevation of blood urea may thus be an important factor contributing to decreased intrarenal NO synthesis in addition to renal depletion of arginine synthesis. The decreased NO synthesis may thus contribute to the progressive nature of renal disease. Thus one of the several adverse factors leading to relentless progression of renal failure may be impaired production or "dysregulation" of NO synthesis within the kidney, possibly resulting from retention of endogenous metabolites such as urea.
In conclusion, we believe that urea, a compound considered to be innocuous that accumulates in renal failure (23), may have potentially important implications in the pathogenesis of the uremic syndrome. Our experiments show that urea inhibits NO production by the macrophages by a posttranscriptional mechanism. This observation may have a clinical relevance in that urea may play a role in impaired macrophage function in renal failure by decreasing the NO production, since NO may be the effector molecule of a macrophage cytotoxicity (6a). In addition, these observations may have even more serious clinical implications in that urea may impair the production of NO from other known sources in the kidney, such as the mesangial cells or glomerular endothelium, and thus may contribute to the progression of renal failure.
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ACKNOWLEDGEMENTS |
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We sincerely acknowledge Dr. F. Paronetto, VA Medical Center, Bronx, NY, for allowing us to use equipment needed for these studies. We thank P. Usha for manuscript preparation.
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FOOTNOTES |
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Parts of these studies were presented at the 30th Annual Meeting of the American Society of Nephrology, San Antonio, TX, 1997.
These studies were partly supported by Sandoz Pharmaceuticals Research Grant PNH-710.
Address for reprint requests: S. S. Prabhakar, Renal Section, 9A-17, Bronx VA Medical Center, 130 W. Kingsbridge Rd., Bronx, NY 10468.
Received 24 March 1997; accepted in final form 5 August 1997.
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