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*
Renal Unit, Fremantle Hospital, Fremantle, Australia.
Department of Pharmacology, University of Western Australia, Perth,
Australia.
Department of Renal Medicine, University of Sydney at Westmead Hospital,
Westmead, Sydney, Australia.
Correspondence to Dr. G. K. Rangan, Renal Unit (F Block), Fremantle Hospital, P.O. Box 480 (Alma Street), Fremantle, W.A., Australia 6959. Phone: 618-9431-3600; Fax: 618-9431-3619; E-mail: gkr{at}cyllene.uwa.edu.au
 |
Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B (NF-B). This study investigated the effect of continuous oral
administration of an NO donor (molsidomine [Mol]), NO precursor (L-arginine
[L-arg]), or selective inhibitors of inducible NO synthase (iNOS;
aminoguanidine [AG], L-N6-(1-iminoethyl)lysine [L-NIL]) on
the progression of tubulointerstitial inflammation and NF-B activation
in a non-immune model of chronic glomerular disease (Adriamycin nephropathy
[AN]), from day 8 until day 30 after disease induction. On day 30, rats with
AN had heavy proteinuria, reduced creatinine clearance, and tubulointerstitial
disease. Treatment with both AG and L-NIL exacerbated the progression of AN as
evidenced by (1) increased renal cortical malondialdehyde;
(2) reduced creatinine clearance; and (3) increased tubular
atrophy, interstitial volume, and monocyte infiltration. Unexpectedly, Mol
also increased renal malondialdehyde and worsened tubular injury, whereas
L-arg had no effect. The increase in renal cortical NF-B activation in
AN was not altered by AG, L-NIL, or Mol, but the mRNA expression of monocyte
chemoattractant protein-1, interleukin-10, and osteopontin were elevated in
these groups. Nitrite release from kidney slices reduced in AN. Treatment with
Mol restored renal nitrite release to normal, whereas neither L-arg nor the
NOS inhibitors had an effect. It is concluded that endogenous iNOS-derived NO
has a protective role against tubulointerstitial injury and cytokine
production in AN. However, the pro-oxidant activity of NO donors may limit
their potential benefit in proteinuric renal disease. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B (NF-B)
(3). The latter are a family of
inducible transcription factors that regulate the expression of multiple genes
involved in inflammation (4).
In vitro studies show that NO may either suppress or promote
NF-B activation, depending on the cell type and stimulant. NO has an
inhibitory effect on cytokine and lipopolysaccharide-induced NF-B
activation in macrophages and endothelial, vascular smooth muscle, and
mesangial cells
(5,6,7,8).
In contrast, NO promotes NF-B activation in lymphocytes and
neuronal-derived and malignant cell lines
(9,10,11).
These results could explain the divergent effects of NO on NF-B
activation in vivo
(12,13).
Tubular atrophy, interstitial inflammation, and heavy proteinuria are
important determinants of kidney function and prognosis in almost all types of
progressive renal diseases
(14). Animal models of
nonimmune chronic proteinuric renal disease (remnant kidney, protein overload,
and Adriamycin nephropathy [AN]) are associated with increased NF-B
activation and tubulointerstitial injury
(15,16,17).
Little is known about the effect of modulating NO on NF-B activation in
these models. Available evidence in nonproteinuric experimental renal disease
suggests that NO inhibits NF-B activation
(18,19).
For example, L-arg, a physiologic precursor of NO, reduced NF-B
activation and tubulointerstitial disease in rats with ureteral obstruction
(18). Moreover, acute
administration of L-arg reduced induction of regulated upon activation, normal
T cell expressed and secreted (RANTES; an NF-B—dependent
chemokine) in rats with endotoxin-induced glomerular injury
(19). However, NO also has
proinflammatory effects (2),
which could potentially limit the ability of NO-based therapies to attenuate
NF-B activation in vivo
(2).
In the present study, we hypothesized that the continuous oral
administration of an NO precursor/donor would reduce chronic NF-B
activation and tubulointerstitial injury in non-immune proteinuric renal
disease, whereas inhibitors of NOS would exacerbate these parameters. To test
this hypothesis, rats with established AN received an NO precursor (L-arg),
long-acting synthetic NO donor
(N-ethoxycarbonyl-3-morpho-linosydnonimine [molsidomine (Mol)]), or
selective inhibitors of inducible NO synthase (iNOS; aminoguanidine [AG] or
L-N6-(1-iminoethyl)lysine [L-NIL]) in their drinking
water.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All drugs were purchased from Sigma-Aldrich (Sydney, Australia) and commenced from day 8 to exclude the confounding possibility that they might alter glomerular injury and proteinuria. The doses were based on those of previous studies that showed modulation of the urinary excretion of NO metabolites, iNOS expression, and tubulointerstitial disease and no affect on mean arterial BP (18,20,21,22,23,24).
Renal Cortical Malondialdehyde
Lipid peroxidation was assessed by measurement of malondialdehyde (MDA) in
renal cortical homogenates as described previously
(17).
Renal Function
Analysis of biochemical parameters and calculation of CrCl was performed as
described previously (17).
Renal Histology
Light Microscopy. A midcoronal slice of the right kidney was
immersion-fixed in 10% neutral buffered formalin for 24 h. An arbitrary
coronal section, 3 µ in thickness, from each animal was stained with
periodic acid-Schiff (PAS) and used for morphometric analysis. A uniformly
random cluster method (25) was
used to determine the microscopic fields for morphometric evaluation. Five
points were marked circumferentially around the coronal section with a
fine-point marking pen. A midcortical field (x400), adjacent to each pen
mark, which contained tubular structures only, was evaluated for morphometric
measurements. The cross-sectional diameter and cell height of all tubules
present in the selected cortical field, as well as interstitial volume, were
measured by line and area computer morphometric measurements respectively, as
described previously using digitized images and image analysis software
(Optimas version 5.2; Optimas Corp., Seattle, WA)
(17). Morphometric analysis
was restricted to tubules and interstitium, as there are minimal quantifiable
glomerular alterations visible by light microscopy in AN at day 30
(17).
ED-1 Infiltration. Interstitial monocyte infiltration was assessed by ED-1 immunohistochemistry in frozen sections as described previously (17). The number of ED-1-positive interstitial cells was quantified in 10 nonoverlapping cortical fields (x400, measuring 0.075 mm2 each).
Electrophoretic Mobility Shift Assay and Immunohistochemical Staining
for NF-B
Nuclear protein extraction and electrophoretic mobility shift assay in
renal cortical homogenates were performed as described previously
(17). Autoradiographs from all
groups were exposed simultaneously using the same film and intensifying
screen. The density of each band was determined by densitometry (Molecular
Dynamics, Sunnyvale, CA).
For immunostaining of the NF-B1 (p50) subunit, paraffin sections
were dewaxed in Histo-clear (National Diagnostics, Atlanta, GA) and rehydrated
in graded alcohols. The tissue sections then were incubated sequentially in 3%
hydrogen peroxide (10 min), rabbit serum (1:5 diluted in Background Buster
[NB306; Accurate Chemicals, Westbury, NY]) for 30 min, goat polyclonal
anti—NF-B1 (p50; 1:1000 diluted in 1% bovine serum
albumin/phosphate-buffered saline [SC-114X; Santa Cruz Biotechnology, Santa
Cruz, CA] a gift from Dr. Couser, University of Washington, Seattle, WA) for
16 h at 4°C, biotinylated rabbit anti-goat IgG (1:1000; BA-5000 [Vector
Laboratories, Burlingame, CA]) for 30 min and then Vectastain Elite ABC
reagent (Vector Laboratories) for 20 min. The antigen was visualized with
3,3'-diaminobenzidine and counterstained with methyl green. As a
negative control, the primary antibody was replaced with normal goat
serum.
Reverse Transcription-PCR
The renal cortical mRNA expression of NF-B—dependent (tumor
necrosis factor-
[TNF-], monocyte chemoattractant protein-1
[MCP-1], interleukin-10 [IL-10]) and —independent (osteopontin)
cytokines was determined by semiquantitative reverse transcription-PCR
(26). Briefly, 1 µg of
total RNA, isolated from 100 mg of renal cortex (RNAzol, Teltest), was reverse
transcribed to yield cDNA using techniques previously described in detail
(26). The PCR samples were
incubated at 94°C for 3 min and then cycled 24 to 38 cycles through
denaturation/annealing at 94°C for 30 s and extension at 60 to 68°C
for 90 s. A final extension step was performed at 72°C for 5 min. Twenty
percent of the PCR product was separated on a 1.6% agarose gel containing
ethidium bromide (0.5 µg/ml). The bands were photographed under ultraviolet
illumination using positive/negative film (Type 665; Polaroid, Cambridge, MA).
Cytokine gene expression was the ratio of the volume density of the cytokine
to that of the house-keeping gene glyceraldehyde-3-phosphate dehydrogenase as
determined by densitometry of the negatives.
Urinary Nitrite Excretion and NOS Activity in Kidney Slices
Urine samples were deproteinized by equal volumes of 0.3 M sodium hydroxide
and 5% zinc sulfate and centrifugation at 6400 x g for 20 min.
The supernatants were added in duplicate to 96-well plates and reacted with 1%
sulfanilamide in 5% phosphoric acid and 0.1%
N-1-napthylethylenediamine dihydrochloride. Nitrite levels were
assessed by measurement of absorbance (550 nm) using a microplate reader
(Model 550; Bio-Rad laboratories, Hercules, CA)
(27). Serial dilutions of 0.1
M sodium nitrite were the external standard.
Renal NOS activity was assessed by nitrite release from kidney slices as described previously (27). Briefly, a midcoronal slice of kidney was immersion-fixed in 2% paraformaldehyde lysine periodate for 24 h. Five sections, 50 µ in thickness, were incubated with 10 mM L-arg in a buffer containing 25 mM HEPES, 140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, and 5 mM glucose at 37°C for 24 h. Nitrite release in supernatants was expressed relative to milligrams of protein.
Statistical Analyses
Data are expressed as mean ± SEM. Comparisons between experimental
groups were performed using the independent t test and Mann-Whitney
U test for parametric and nonparametric data, respectively.
P < 0.05 indicated a significant difference among groups.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Renal Function
By design, proteinuria, SCr, and CrCl were similar among groups
with AN at baseline (day 6; Table
2 and CrCl: AN+vehicle, 1.50 ± 0.11; AN+L-arg, 1.45
± 0.19; AN+Mol, 1.46 ± 0.04; AN+AG, 1.43 ± 0.18;
AN+L-NIL, 1.45 ± 0.13 ml/min; P = NS). The progression of
proteinuria was not altered by treatment with any of the NO modulating drugs
on neither day 20 (data not shown) or day 30
(Table 2). CrCl was decreased
on day 30 in rats with AN (P = 0.08 versus normal control
group) (Figure 1). Treatment
with both iNOS inhibitors and, unexpectedly, Mol worsened the decline in renal
function in AN on day 30, as assessed by SCr, SUr and
CrCl (Table 2 and
Figure 1). Urinary excretion of
urea was reduced in AN on day 30 and increased to normal only by L-arg
administration.
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Renal Cortical MDA
Renal cortical MDA was increased in AN
(Figure 2). L-arg had no effect
on renal MDA production, whereas Mol, AG, and L-NIL increased MDA
production.
|
Renal Histology
Rats with AN developed prominent cortical tubulointerstitial disease
characterized by tubular atrophy, interstitial volume expansion, and
interstitial cell infiltration (Figure
3). The glomerular changes were characterized by a mild increase
in mesangial matrix only. Treatment with Mol, AG, or L-NIL increased the
extent and severity of tubulointerstitial injury
(Figure 3). This was not
accompanied by an increase in glomerular mesangial matrix or microvascular
thrombosis (data not shown). Wet kidney weight was increased in groups that
were treated with AG and L-NIL (Table
3). By quantitative morphometric analysis, cortical tubule cell
height and interstitial volume were worsened by Mol and the iNOS inhibitors
(Figure 3 and
Table 3). In contrast, L-arg
had no effect on the progression of the histologic changes in AN.
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Interstitial monocyte infiltration was increased approximately fivefold in AN. Treatment with both iNOS inhibitors increased interstitial ED-1 infiltration in AN, whereas L-arg and Mol had no significant effect (Table 3).
Renal Cortical NF-B Activation
NF-B DNA binding activity was dramatically increased in rats with
AN, whereas it was barely detectable in the control group (Figures
4A and
5A). The specificity of the
retarded band was confirmed by competition studies
(Figure 4B). Supershift
analysis demonstrated that NF-B1 (p50) was the predominant protein
present in the retarded band (Figure
4C). The induction of renal cortical NF-B DNA binding
activity in AN was not affected by any of the drugs
(Figure 5B).
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Immunostaining was performed to localize the cellular source of
NF-B1. In control rats, occasional cortical tubules stained for
NF-B1 (Figure 6, A and
C). In contrast, in AN, there was a dramatic increase in nuclear
staining for NF-B1, and this was localized predominantly to tubular
epithelial and interstitial cells (Figure
6, B and D). The pattern of NF-B1 staining in AN was not
altered by treatment with any of the NO-modulating drugs.
|
Renal Cortical mRNA Expression of MCP-1, TNF-, IL-10, and
Osteopontin
Renal cortical mRNA expression of MCP-1, TNF-, IL-10, and
osteopontin were increased in AN (Table
4). Administration of L-NIL, AG, or Mol increased MCP-1, IL-10,
and osteopontin mRNA in AN, compared with the vehicle alone. It is interesting
that L-arg increased IL-10 and reduced TNF- without any change in MCP-1
and osteopontin compared with vehicle-treated rats with AN.
|
Urinary Nitrite Excretion and NOS Activity in Kidney Slices
On day 30, urinary nitrite excretion was not altered in AN
(Figure 7A). Urinary nitrite
excretion was increased by Mol but was unaffected by the other treatments. NOS
activity, as determined by nitrite release from kidney slices, was reduced in
rats with AN and normalized by Mol (Figure
7B). In contrast, NOS activity remained reduced in kidney slices
from groups that were treated with either L-arg or NOS inhibitors.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B-dependent and —independent cytokine genes. However,
surprisingly and contrary to our original hypothesis, treatment with the NO
donor Mol also increased renal MDA and worsened tubular injury in AN, whereas
the NO precursor L-arg was not effective. These data suggest that whereas
endogenous iNOS-derived NO has a protective role in proteinuric
tubulointerstitial injury, excess exogenous NO has adverse effects in the same
situation. Among its multiple and divergent functions, NO has several anti-inflammatory properties. Apart from maintaining perfusion of the renal microcirculation (28), NO inhibits leukocyte and platelet endothelial adhesion (29), suppresses T-cell and monocyte proliferation (30,31), and preserves integrity of the vascular endothelial permeability barrier (32). NO also is a physiologically important scavenger of superoxide radicals, being more potent than superoxide dismutase (33). For examination of the role of endogenous NO in AN, rats were treated long term with selective inhibitors of iNOS. Because AG has NO-independent actions (34), we also included a group that was treated with L-NIL, an amino acid—based selective inhibitor of iNOS (24). Both inhibitors increased lipid peroxidation and interstitial monocyte infiltration, suggesting that endogenous iNOS-derived NO has antioxidant and anti-inflammatory properties in AN. Our results suggest that the increase in interstitial monocyte infiltration by NOS inhibition in AN may be due partly to an increase in gene expression of MCP-1 and osteopontin. In addition, preliminary data in AN show that this effect may be due to a reduction in monocyte apoptosis (35).
In contrast to these findings, Mol worsened tubulointerstitial injury in AN, despite increasing urinary nitrite excretion and normalizing renal NOS activity. Mol is a long-acting NO donor (36) reported to improve significantly the survival of proteinuric rats with remnant kidney disease (22). In vivo, Mol is absorbed efficiently from the gastrointestinal tract and converted enzymatically in the liver to yield the active metabolite 3-morpholino-sydnonimine-hydrochloride (SIN-1) (36). SIN-1 is decomposed spontaneously into NO and superoxide anion (37,38). The latter can combine rapidly to form the potent free radical peroxynitrite (ONOO-) and cause tissue injury (2). Mol significantly increased renal cortical lipid peroxidation in AN, suggesting that this mechanism could explain the results of our study. Although in vitro data suggest that other NO donors may not lead to spontaneous ONOO- generation as readily as Mol (38), it is likely that these compounds also would behave as pro-oxidants in proteinuric renal disease. This is because the elevated levels of renal cortical superoxide anions in AN (39) remain a potential endogenous stimulus for ONOO- formation in the presence of excess exogenous NO. Additional studies are needed to test this hypothesis and to evaluate whether combination therapy with hydroxyl radical scavengers (40), flavonoids (37), and/or ONOO- scavengers (e.g., uric acid) (38) could attenuate the deleterious renal consequences associated with NO donors.
Stimulated by previous observations, we investigated whether NO modulates
chronic NF-B activation in AN. The confounding pro-oxidant effects
associated with Mol, however, do not permit us to address this hypothesis
adequately in the present study. Although the increase in renal cortical
NF-B activation in AN was not altered by either Mol or NOS inhibitors,
the mRNA expression of NF-B—dependent cytokines (MCP-1, IL-10)
was elevated compared with vehicle. The discrepancy between the DNA-binding
activity and gene expression data may be due to the specificity of the
activated NF-B dimers in vivo
(41), insensitivity of the
electrophoretic mobility shift assays to detect small increases in nuclear
protein translocation, and/or induction of other transcription
factors/coactivators
(16,41).
At variance with previous experimental data
(18,20,21),
L-arg did not significantly attenuate tubulointerstitial disease in AN,
despite using a standard method of administration. The same results were
obtained when L-arg hydrochloride was administered by daily intraperitoneal
injections to rats with AN (250 mg/kg twice a day from days 7 to 30;
n = 8; Rangan GK, Wang Y, Harris DCH, unpublished observation).
Interestingly, we found that L-arg was associated with an increase in renal
cortical IL-10 mRNA and reduction in TNF-, without altering the mRNA
expression of monocyte chemoattractants (MCP-1, osteopontin). The latter could
explain the ineffectiveness of L-arg in AN. Furthermore, unlike Mol, L-arg
increased urinary excretion of urea but had no effect on urinary nitrite
excretion, suggesting an effect independent of NO
(1,2).
These results are not surprising, considering the multiple pathways involved
in L-arg metabolism in vivo
(1,2).
In addition, as NOS activity may be reduced in AN, it seems unlikely that
providing more substrate could directly increase local NO generation. Similar
reasons were proposed recently to explain the failure of chronic L-arg therapy
to increase urinary nitrite excretion in patients with progressive renal
disease (42).
Consistent with previous data, we found that urinary nitrite excretion was
not altered in AN (43).
Nitrite release from kidney slices was reduced, however. As in the remnant
kidney model (1), the deficit
in renal NO production in AN therefore could be due to suppression of iNOS
expression mediated by tubular dysfunction and damage
(1); elevation of specific
cytokines (osteopontin, endothelin-1, transforming growth factor-ß1,
IL-4, and IL-10)
(1,44);
and/or the effects of proteinuria and hyperlipidemia
(45). In addition, the
concomitant increase in NF-B activation in both AN and the remnant
kidney model
(15,17)
support previous in vitro observations that suggest that induction of
iNOS in tubules does not correlate with NF-B DNA-binding activity
(46).
In conclusion, the data presented in this article demonstrate three
important findings: (1) iNOS-derived NO plays a protective role in
mediating tubulointerstitial injury and NF-B activation, possibly
involving effects that suppress monocyte accumulation and production of
reactive oxygen species; (2) administration of Mol was associated
with renal pro-oxidant effects that may have negated its ability to suppress
NF-B activation; and (3) the NO precursor L-arg did not
increase urinary nitrite excretion and is not universally protective in
experimental proteinuric renal disease. Given that NO donors are potential
therapeutic agents in chronic renal disease
(1,2),
further experimental studies need to address whether the pro-oxidant effects
of Mol and (possibly other NO donors) can be prevented by combination therapy
with antioxidant compounds and/or ONOO- scavengers
(37,38,40).
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Acknowledgments |
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