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*
Department of Physiology & Biophysics, University of Nebraska College
of Medicine, Omaha, Nebraska
Department of Pediatrics, University of Nebraska College of Medicine,
Omaha, Nebraska
Department of Integrative Biology & Pharmacology, University of Texas
Medical School at Houston, Houston, Texas
Vascular Biology Center and Department of Pharmacology & Toxicology,
Medical College of Georgia, Augusta, Georgia.
Correspondence to Dr. Pamela K. Carmines, Department of Physiology & Biophysics, University of Nebraska College of Medicine, 984575 Nebraska Medical Center, Omaha, NE 68198-4575. Phone: 402-559-9343; Fax: 402-559-4438. E-mail: pcarmines{at}unmc.edu
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Studies focusing on the renal microvascular influence of NO in IDDM have yielded results that are more consistent. Renal microvessels from rats with streptozotocin (STZ)-induced IDDM exhibit suppressed agonist-induced endothelium-dependent relaxation (7), diminished NO-dependent basal tone (8), and loss of the modulatory influence of endogenous NO on agonist-induced constriction (4,9). Superoxide anion (O2.-) production has been implicated as one factor that opposes the effects of NO on the renal microvasculature of diabetic rats (7,8,9). Because O2.- decreases the half-life of NO, increased renal O2.- levels in IDDM should reduce the bioavailability of NO and curtail its tonic impact on arteriolar tone. Although accumulating evidence suggests that renal oxidative stress accompanies IDDM, O2.- production has not been compared in kidneys from normal and diabetic animals. Discrete aspects of the renal oxidant/antioxidant balance have been addressed in IDDM (10,11,12); however, these investigations have focused on alterations evident in severely hyperglycemic animals studied months after onset of the disease. The relative status of renal NO synthesis and its degradation by O2.- has not been explored during the hyperfiltration stage of IDDM.
The present study was designed to evaluate the hypothesis that the hyperfiltration stage of IDDM is accompanied by an imbalance between NO synthesis and O2.--dependent NO degradation in the kidney. Attention was focused on the renal cortex, wherein lies the arteriolar vasculature that controls glomerular capillary hydrostatic pressure. Our approach was to quantify parameters related to NO synthesis (NOS; NOS activity, NOS protein levels and localization, and nicotinamide adenine dinucleotide phosphate [NADPH] diaphorase staining) and O2.- accumulation (O2.- production and superoxide dismutase [SOD] activity) in renal cortex of normal and diabetic rats. Because NO and O2.- react rapidly to form peroxynitrite (ONOO-), the actions of which include nitration of tyrosine residues on proteins, renal cortical nitrotyrosine levels were used as an index of ONOO- production.
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Group 1
Two wk after injection with STZ or vehicle, rats were anesthetized with
pentobarbital sodium (50 mg/kg, intraperitoneally) and tracheotomized, to
facilitate spontaneous respiration. Both kidneys were flushed with cold
isotonic saline from the abdominal aorta and were removed quickly. Cortical
tissue from the right kidney was used for assay of NOS activity, and that from
the left kidney was used for assay of SOD activity.
NOS Assay. Total NOS activity in renal cortex was determined on the
basis of the rate of L-[3H]citrulline formation from
L-[3H]arginine, as described previously
(13). Briefly, the renal
cortex was weighed, minced, and homogenized in ice-cold buffer containing 10
mM HEPES, 320 mM sucrose, 0.1 mM ethylenediaminetetraacetate, 1 mM
dithiothreitol, 0.1 mM soybean trypsin inhibitor, 0.1 mM aprotinin, and 1 mM
phenylmethylsulfonyl fluoride (pH 7.4). After 20 min of treatment of the
homogenate with 0.2 vol/wt 200 mM CHAPS at approximately 4°C and
subsequent centrifugation at 10,000 x g, DOWEX (50WX8-400;
Sigma) was used to remove endogenous L-arginine from the supernatant
(14). A 20-µl aliquot of
the supernatant was added to 50 µl of incubation buffer (pH 7.2) composed
of 20 mM HEPES, 1 mM CaCl2, 1 mM MgCl2, 5 mM L-valine, 2
µM tetrahydrobiopterin (BH4), 1 mM ß-NADPH, 0.5 µM
calmodulin, 20 µM L-arginine, and 0.2 µM
L-[2,3,4,5-3H]arginine (12.6 µCi/ml assay buffer, 63 Ci/mM
specific activity; Amersham Life Science, Buckinghamshire, UK). Parallel
reactions were performed in the presence of 300 µM
N
-nitro-L-arginine (L-NNA), a NOS inhibitor
(15). Samples were incubated
for 30 min at 37°C, after which the reaction was terminated by DOWEX
treatment to remove L-arginine. L-[3H]citrulline that formed during
the reaction was quantified by liquid scintillation counting. Total NOS
activity of each background-corrected sample was determined as the difference
between the radioactivity with and without 300 µM L-NNA and was expressed
as picomoles of L-[3H]citrulline formed per hour per milligram of
protein. The conditions of this assay minimized any confounding impact of
endogenous L-arginine (removed by DOWEX treatment) or arginase activity
(inhibited by exogenous L-valine). We could not control for the potential
recycling of L-citrulline to L-arginine via endogenous argininosuccinate
synthase and argininosuccinate lyase. However, because the
Km for argininosuccinate lyase is 3.7 mM
(16), the fact that
L-NNA-dependent processes in renal cortical homogenates yield nanomolar
concentrations of L-citrulline in the presence of micromolar concentrations of
L-arginine under these conditions makes it unlikely that the recycling
reaction significantly influences this widely accepted assay for NOS
activity.
Superoxide Dismutase Assay. Renal cortical tissue was weighed, minced, and homogenized in ice-cold 250 mM sucrose. The homogenate was centrifuged (10 min at 8500 x g, 4°C), and the supernatant was stored at -70°C until assay for SOD activity, as described previously (13). Briefly, the thawed sample was subjected to ethanol:chloroform extraction, mixed, and centrifuged (10 min at 3000 x g, 4°C). A commercially available kit (BIOXYTECH SOD-525; OXIS International, Portland, OR) was used to measure SOD activity in the aqueous upper layer of the extract.
Group 2
Two wk after injection with STZ or vehicle, rats were anesthetized with
pentobarbital sodium (50 mg/kg, intraperitoneally). The right renal artery and
vein were ligated, and the kidney was removed quickly and weighed, and
cortical samples were frozen in liquid N2. These samples were
stored at -70°C until protein isolation and Western blot analysis (NOS-1,
NOS-2, NOS-3, and nitrotyrosine). The distal aorta was cannulated and a 1-ml
blood sample was collected into a heparinized syringe. Plasma from this sample
was stored at -20°C until measurement of NOX concentration. The
left kidney was flushed with saline, perfused with 40 to 50 ml of 4%
paraformaldehyde in phosphate buffer (pH 7.4), and removed from the rat. After
overnight postifixation in paraformaldehyde at 20°C, the kidney was
maintained in 20% sucrose at 4°C until processing for NADPH-diaphorase
staining.
Protein Isolation. Tissue lysates were prepared by standard homogenization techniques. Briefly, frozen cortical tissue was pulverized to a powder and solubilized in homogenization buffer (vol/wt ratio of 10; 50 mM Tris-HCl [pH 7.4], 10 mM ethylenediaminetetraacetate, 0.1% 2-mercaptoethanol) in the presence of protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 2 µM leupeptin, 1 µM pepstatin A, and 0.1% aprotinin). The preparation was homogenized for 20 strokes in a Dounce glass/Teflon homogenizer on ice. Treatment with CHAPS (20 mM, 20 min, 4°C) was used to separate further the homogenate into a detergent-soluble fraction and detergent-insoluble fraction. The supernatant collected after centrifugation (10,000 x g, 45 min, 4°C) was considered the "soluble fraction." The pellet was resolubilized in half the original homogenization buffer volume with 20 mM CHAPS (20 min at 4°C). The resolubilized pellet was then centrifuged at 100,000 x g (30 min at 4°C); this is considered the "resolubilized pellet fraction." The resulting detergent-insoluble pellet was rehomogenized in half the original homogenization buffer volume and stored at -80°C until analysis. For detection of NOS-1 and NOS-3, the soluble fraction and resolubilized pellet fraction were purified further by 2',5'-adenosine diphosphate-Sepharose column chromatography as described previously (17).
Western Blot Analysis. Proteins were separated on 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose by wet blotting (Bio-Rad Laboratories, Hercules, CA) for 45 min. The blots were allowed to air-dry for 30 min and blocked with 5% nonfat dry milk diluted in Tris-buffered saline (blocking buffer) for 1 h at room temperature. The blots were incubated with the primary antibody diluted in blocking buffer overnight at 4°C, followed by two washes with blocking buffer at room temperature. The blots then were incubated with the secondary antibody (horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit antibody; Amersham) for 1 h at room temperature, followed by four washes with Tris-buffered saline. The specific bands were detected by use of the enhanced chemiluminescence system (Amersham). Densitometry was used to semiquantify the bands with an Alpha Innotech Imaging System (San Leandro, CA). Primary antibodies used were anti-NOS-1 (1:1000, SA-227; Biomol Research Laboratories, Plymouth Meeting, PA), anti-NOS-2 (1:3000, SA-200; Biomol), anti-NOS-3 (1:1000, N30020: Transduction Laboratories, Lexington, KY), and nitrotyrosine-specific antibody (1:50 (18). The antinitrotyrosine mouse monoclonal antibody has high affinity for nitrotyrosine-containing proteins, is specific for nitrotyrosine-residues, and recognizes a variety of nitrated proteins with similar affinity (18). Molecular weights were estimated by use of rainbow (32 to 216 kD; Amersham) and enhanced chemiluminescence markers (32 to 97 kD; Amersham).
NADPH-Diaphorase Histochemistry. Histochemical localization of the catalytic activity of NOS was assessed by use of the NADPH-diaphorase reaction. This method has been exploited to localize in situ NOS activity in a variety of tissues, including kidney. Briefly, postifixed kidneys in 20% sucrose were blocked in the coronal plane and sectioned in a cryostat. The sections were collected in phosphate buffer (pH 7.4) containing 0.3% Triton X-100, 0.1 mg/ml nitro blue tetrazolium, and 1.0 mg/ml ß-NADPH. The sections were warmed to 37°C for 75 min, rinsed in phosphate buffer, mounted on chrome-alum coated slides, and dried, and coverslips were mounted directly with Permount (Fisher Scientific, Fairlawn, NJ). NADPH-diaphorase staining in sections from sham and STZ rats was quantified by computerized assessment of blue saturation density (19). Animal means were calculated on the basis of 20 to 30 measurements per rat (10 regions per slide; 2 to 3 slides per rat), and statistical analyses were performed by use of these animal means.
Group 3
Two wk after injection with STZ or vehicle, rats were anesthetized with
pentobarbital sodium (50 mg/kg, intraperitoneally). The abdominal aorta was
cannulated, allowing the kidneys to be flushed with heparinized saline. Each
kidney was excised and weighed. Two nearmedial slices were obtained from each
kidney with a Stadie-Riggs microtome (Thomas Scientific, Swedesboro, NJ). The
medullary portion of each slice was removed carefully and discarded. The
cortical slices thus obtained were bisected and used for measurement of
O2.- production.
O2.- Production Assay. O2.- production was measured on the basis of its ability to reduce ferricytochrome c, according to established methods (20). Renal cortical slices were incubated at 37°C in Hank's balanced salt solution containing 80 µM cytochrome c and either 5 mM glucose (right kidney) or 20 mM glucose (left kidney). To control for nonspecific reduction of ferricytochrome c, slices from each kidney were incubated in cytochrome c solution containing 500 U/ml SOD. As a positive control, slices from each kidney also were incubated in the presence of 250 µg/ml heat-aggregated bovine IgG, a known stimulant of renal O2.- production (20). The supernatant was removed at 5, 30, 60, or 90 min after initiating the cytochrome c incubation, centrifuged (10,000 x g) to remove any cellular debris, and absorbance was measured at 550 nm. The extinction coefficient of cytochrome c was assumed to be 2.1 x 104 M-1 cm-1. SOD-sensitive O2.- production was expressed as nanomoles of reduced cytochrome c per milligram of protein.
Group 4
Two wk after injection with STZ or vehicle, rats were decapitated and
tissues were harvested for studies relating to another project
(21). A 3-mm-thick midcoronal
section of the left kidney was reserved for immunohistochemistry.
NOS-1 and NOS-3 Immunohistochemistry. Tissue was immersed overnight in 10% buffered formalin, dehydrated, embedded in paraffin blocks, sectioned (4 µm) onto super-frost plus slides, and stained by the Labeled Streptavidin-Biotin method for rat (DAKO, Carpinteria, CA). Endogenous peroxidase was blocked by 3% H2O2 for 15 min, followed by washing successively in water and Tris-buffered saline. Sections were incubated in humidity chambers with diluted primary antibody or control solution (anti-NOS-1: Transduction N31020, 1:500, 3 h; anti-NOS-3: N30020, 1:800, 1 h). The slides were washed with Tris-buffered saline, followed by incubation with biotinylated horse universal secondary antibody according to the kit instructions for 10 min. Staining was detected with streptavidin peroxidase and diaminobenzidine according to the kit instructions. The sections were counterstained with hematoxylin, rinsed with water, dehydrated with increasing percentages of alcohol, and mounted with cytoseal 60. The stained sections were viewed with a Zeiss Axiophot microscope on bright field setting fitted with a digital camera (Spot 2; Diagnostic Instruments, Sterling Heights, MI) and scanned with Adobe Photoshop imaging software (Adobe Systems, San Jose, CA).
Creatinine, NOX, and Protein Assays
Protein concentrations were determined by a standard Bradford assay
(Bio-Rad). Creatinine concentration in urine samples was measured by use of a
picric acid-based microplate assay
(22). The
ozone-chemiluminescence method (Model 280 NO Analyzer; Sievers Instruments,
Boulder, CO) was used to measure NOX concentration in urine and
plasma (deproteinized by cold ethanol precipitation, according to the
manufacturer's instructions).
Statistical Analyses
Data were analyzed by ANOVA for repeated measures and Newman-Keuls tests,
or unpaired t tests, as appropriate. P < 0.05 was
considered significant. All data are reported as the mean ± SD.
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NOS Activity, NOS Protein Levels and Localization, and
NADPH-Diaphorase Staining
Total NOS activity in renal cortex of sham and STZ rats was measured as
L-NNA-sensitive L-[3H]citrulline formation from
L-[3H]arginine in the presence of 1 mM Ca2+. NOS
activity in cortical tissue from sham rats averaged 15.9 ± 2.0 pmol/h
per mg protein (n = 11) and was increased by 80% in STZ rats
(Figure 1A). Western
immunoblots were prepared to determine the relative contributions of each NOS
isoform to the increase in NOS activity evident in STZ kidneys
(Figure 2). NOS-1 protein was
detectable in renal cortex only after adenosine diphosphate purification of
the soluble fraction. Densitometry values suggested decreased cortical NOS-1
protein in STZ kidneys (372 ± 69; n = 4) compared with sham
kidneys (540 ± 133; n = 3), but this effect did not achieve
statistical significance. NOS-2 protein, located in the detergent-insoluble
pellet, averaged 359 ± 101 (n = 4) and 305 ± 108
(n = 3) in cortical samples from sham and STZ kidneys, respectively
(P > 0.05). NOS-3 was evident in the soluble fraction of renal
cortex but also did not differ between groups (sham, 603 ± 71
[n = 3]; STZ, 696 ± 166 [n = 4]).
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Immunohistochemical studies were performed to assess further NOS protein levels and localization (Figure 3). Intense NOS-1 staining was evident throughout the cytoplasm of macula densa cells. Cortical sections from Sham and STZ kidneys displayed similar intensity and distribution of NOS-1 staining (Figure 3, A and C). Preadsorption with the antigenic peptide prevented NOS-1 immunostaining (Figure 3E). NOS-3 staining was apparent in endothelial cells of all vascular structures, including glomerular capillaries, arterioles, and small arteries. As was the case for NOS-1, there was no apparent difference between sham and STZ kidneys with regard to the pattern and intensity of NOS-3 immunostaining (Figure 3, B and D). Sections incubated in the absence of the anti-NOS-3 antibody confirmed that no immunoreactive products were detected by the secondary antibody alone (Figure 3F).
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NADPH-diaphorase staining was used in a further attempt to identify site(s) of altered NOS activity in the cortex of STZ rats. NADPH-diaphorase staining was evident in cortical vascular and glomerular structures of sham and STZ kidneys. Macula densa staining was weak in both groups. Computer-generated saturation density values for glomerular staining averaged 194 ± 6 in sham rats and 189 ± 7 in STZ rats. Similarly, cortical microvessels failed to exhibit altered diaphorase staining between groups (sham, 197 ± 9; STZ, 203 ± 6). Thus, despite increased NOS activity measured by L-citrulline assay, the renal cortex of STZ rats exhibited no significant alterations NOS protein levels, NOS protein distribution, or NADPH-diaphorase staining.
Superoxide Anion Production and SOD Activity
Cumulative O2.- production by renal cortical slices
from normal rats (weight-matched to sham rats) is illustrated in
Figure 4A. During the 90-min
sample period, cortical tissue from these animals produced 1.58 ± 0.37
nmol O2.-/mg protein (n = 6) when incubated in
the presence of 5 mM glucose. Inclusion of aggregated IgG (250 µg/ml) in
the media evoked stimulation of O2.- production that
achieved statistical significance within 30 min. Because similar responses
were observed in tissue from sham rats, O2.- production
data from normal and sham rats were pooled for comparison with STZ responses.
As evident in Figure 4B, incubation of renal cortical tissue from sham/normal rats in 20 mM glucose
tended to increase O2.- production from 1.83 ±
0.35 to 3.79 ± 0.83 nmol/mg protein per 90 min (n = 11);
however, this effect did not achieve statistical significance (P =
0.098). Moreover, aggregated IgG failed to stimulate
O2.- production by sham/normal tissue in the presence of
20 mM glucose (3.16 ± 0.63 nmol/mg protein per 90 min; P =
0.494 versus sham/normal 20 mM glucose).
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When renal cortical tissue harvested from STZ rats was incubated in media containing 20 mM glucose (to simulate in vivo ambient glucose levels), cumulative O2.- production during the 90-min incubation period was more than double that measured in tissue from sham/normal rats studied in the normal glucose environment (P < 0.05 versus sham 5 mM glucose; Figure 4B). Enhanced O2.- production by STZ tissue remained evident after acute restoration of ambient glucose concentration to 5 mM (P < 0.05 versus sham 5 mM glucose). Under these conditions, O2.- production by renal cortex of STZ rats remained 1.5-fold higher than that observed in sham/normal tissue. Aggregated IgG failed to augment further the O2.- production by STZ tissue, regardless of whether the tissue was incubated in media containing 5 or 20 mM glucose (data not shown). In this setting of markedly increased O2.- production in STZ rats, renal cortical SOD activity was increased by 50% relative to sham rats (P < 0.001; n = 11 per group; Figure 1B).
Nitrotyrosine Levels
Diabetes-induced alterations in renal cortical protein tyrosine nitration
are summarized in Figure 5.
Western blot analysis revealed an approximately 70-kD nitrotyrosine-containing
protein in tissue from sham rats. The nitrated protein was evident in both the
detergent-soluble (supernatant) and detergent-insoluble (pellet) fractions of
the cortical homogenate. Immunostaining of this protein averaged 655 ±
340 densitometric units (supernatant + pellet; n = 3) in sham kidneys
and was increased threefold in STZ kidneys (2773 ± 553; n = 5;
P < 0.05). STZ kidneys also contained a faint
nitrotyrosine-positive protein with a molecular weight of approximately 40 kD.
These results were confirmed by use of a commercially available antibody
against nitrotyrosine (Cayman Chemical, Ann Arbor, MI; data not shown). Blots
run in the absence of the antinitrotyrosine antibody confirmed that no
immunoreactive products were detected by the secondary antibody alone.
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Conflicting evidence exists concerning whether IDDM increases (2,24,25) or decreases (26,27) renal NOS protein levels, with alterations in this parameter typically interpreted as indicative of alterations in NOS activity. The present study assessed NOS activity in renal cortical homogenates on the basis of L-NNA—sensitive L-citrulline production in the presence of exogenous substrate and required co-factors. The data indicate an increase in NOS activity in tissue harvested from male STZ rats. Although no specific attempt was made to measure Ca2+-independent NOS activity, the absence of NOS-2 in Western blot analyses of the soluble fractions from sham and STZ kidneys suggests that the measured increase in NOS activity in the post-CHAPS cortical homogenate represents primarily Ca2+-dependent activity (NOS-1 and/or NOS-3). In accord with this contention, Omer et al. (28) reported a 50% increase in Ca2+-dependent NOS activity measured in whole kidney homogenates from female rats studied 2 wk after STZ treatment. In contrast, Keynan et al. (26) reported that renal cortical Ca2+-dependent NOS activity is reduced 7 d after STZ treatment, raising the possibility that IDDM alters this parameter in a time-related manner.
The literature contains reports of increased NADPH-diaphorase staining in afferent arterioles and glomerular capillaries of rats studied 1 to 4 wk after induction of IDDM (24) and of reduced macula densa staining in rats studied 1 or 6 wk after STZ treatment (26,27). Although NOS activity was increased in cortical homogenates from diabetic rats in the present study, neither the distribution nor the intensity of NADPH-diaphorase staining differed between sham and STZ kidneys. It is possible that the NADPH-diaphorase staining method lacks the sensitivity required to distinguish reliably the modest alterations in NOS activity. However, we cannot discount the possibility that in vivo NOS activity is tempered by decreased substrate or co-factor availability in IDDM (29,30) and that this phenomenon explains why increased NADPH-diaphorase staining was not evident in kidneys from STZ rats.
The increase in cortical NOS activity measured in kidneys from STZ rats in the presence of exogenous substrate and required co-factors might be expected to mirror alterations in NOS protein levels. Accordingly, Western blot analysis was performed to identify the NOS isoform responsible for the increase in L-citrulline production. All three NOS isoforms were evident in purified samples of rat renal cortex. An unanticipated observation emanating from the Western blot analysis was that renal cortical NOS-2 immunoreactive protein demonstrated a lower molecular weight than mouse macrophage NOS-2 immunoreactive protein (Figure 2). NOS-2 has been shown to bind calmodulin tightly, even under the denaturing conditions of sodium dodecyl sulfate—polyacrylamide gel electrophoresis analysis (31). We speculate that the lower molecular weight of renal NOS-2 may result from release of bound calmodulin by the detergent treatment used in the present study, although further studies are needed to address this possibility. In any case, Western blot analysis failed to reveal a change in any NOS isoform in cortical tissue from STZ rats, relative to tissue from sham rats. Directionally opposite alterations in NOS protein levels might occur within discrete cortical structures, a situation that could have substantial functional relevance without altering NOS protein levels evident by Western blot. However, immunohistochemical assessment revealed no apparent difference between sham and STZ rats regarding the staining intensity or distribution of NOS-1 or NOS-3 within the renal cortex. Hence, although no ideal indicator of in situ NO production is available, our accumulated measures of NO synthesis suggest that normal or increased NO production occurs in the renal cortex of STZ rats in the absence of any alteration in NOS protein levels or localization. In addition to the widely recognized Ca2+ dependence of NOS-1 and NOS-3 activities, emerging evidence indicates that a variety of additional factors can evoke posttranslational alterations in NOS activity, e.g., Hsp-90 (32), Akt-kinase (33,34), AMP-activated kinase (35). Further studies are required to address the potential involvement of these factors in the increased renal cortical NOS activity evident during diabetic hyperfiltration, although it is at least apparent that the factor and/or its impact on enzyme activity is retained in the tissue homogenate utilized in the L-citrulline assay.
Numerous studies have provided indirect evidence that O2.- production is accelerated in IDDM, primarily on the basis of measured increases in renal thiobarbituric acid reactive substances or antioxidant enzyme activity (10,11,12). The present study confirms these observations by documenting increased renal cortical SOD activity in STZ rats studied 2 wk after onset of IDDM. The suggestion of renal cortical oxidative stress was validated further by measurement of renal O2.- production in moderately hyperglycemic STZ rats. These data reveal a marked increase in O2.- production by renal cortical slices harvested during the hyperfiltration stage of IDDM. Although attempts to localize O2.- production within the various renal cortical structures were beyond the scope of the present study, increased O2.- production was demonstrated recently in glomeruli isolated from rats with poorly controlled IDDM (36). Increased O2.- production in IDDM could involve any of a variety of mechanisms, some of which are triggered by elevated extracellular glucose levels. Although we were unable to document a significant acute effect of 20 mM glucose on O2.- production by normal renal cortex, chronic elevations in glucose concentration may be sufficient to drive elevated renal O2.- production in IDDM. Autoxidation of glucose, formation of advanced glycation end products, and activation of NADPH-oxidase have been suggested to provoke oxidative stress in IDDM. O2.- production in IDDM also may arise as a result of increased NOS-1 or NOS-3 activity during conditions of limited substrate or co-factor availability (37,38). Indeed, reduced L-arginine availability has been reported in IDDM (29), and we note that our O2.- production experiments were performed in media devoid of L-arginine. Such a shift in renal NOS action from NO production to O2.- production could underlie our observation of decreased NOX excretion in IDDM, although we note that urinary NOX excretion is a crude indicator of renal NO production (39).
The reaction of NO with O2.- (to form ONOO-) occurs six times faster than the Cu/Zn-SOD reaction with O2.- (40). Thus, the 50% increase in SOD activity detected in kidneys from STZ rats would not be expected to compensate for the twofold increase in O2.- production. Peroxynitrite is more stable than NO and exerts multiple cytotoxic effects, some of which involve formation of protein-associated nitrotyrosine. Tyrosine nitration occurs in a number of diseases associated with oxidative stress and increased NOS activity (40,41), and increased protein tyrosine nitration is viewed widely as an indicator of peroxynitrite formation. Although the vast majority of previous reports of nitrotyrosine formation associated with disease have relied on immunohistochemical methods (41), the present study used Western blot analysis to provide a semiquantitative measure of protein nitration in renal tissue. The data reveal a significant increase in renal cortical nitrotyrosine levels during the early stage of IDDM. The formation of protein nitrotyrosine residues in renal cortex during IDDM is not promiscuous but, rather, involves primarily an approximately 70-kD protein. A modest increase in nitration of an approximately 40-kD protein also is evident. Increased nitration of proteins with similar molecular weights also occurs in association with endotoxin-induced kidney injury (42) and in the renal cortex of the spontaneously hypertensive rat (43). Cytoskeletal proteins have been suggested as prominent targets of tyrosine nitration in pathophysiologic states, as have a variety of enzymes involved in signal transduction (41). For example, nitration of intramitochondrial Mn-SOD results in a decline in its enzymatic activity during chronic rejection of renal allografts (44), although our results do not likely reflect increased nitration of Mn-SOD, because its molecular weight is <30 kD. An important goal of future studies will be to identify and localize the renal cortical protein(s) nitrated during the hyperfiltration stage of IDDM. Regardless of the identity of the nitrated protein, the trapping of nitro groups on protein tyrosine residues may contribute to the decline in NOX excretion observed in STZ rats in the present study. Thuraisingham et al. (45) recently provided immunohistochemical evidence of increased proximal tubular nitrotyrosine staining in biopsies obtained from patients with established diabetic nephropathy. Our observations indicate that renal nitrotyrosine levels are increased very early in the course of IDDM, before the onset of proteinuria.
Pieper (46) argued that disease duration is a critical determinant of the impact of IDDM on endothelium-dependent relaxation, and Craven et al. (47) suggested that IDDM provokes a period of increased renal NO synthesis followed by a period of diminished NO action. The results of the present study indicate that renal cortical oxidant stress and resulting ONOO- formation (which would diminish NO action as well as cause tissue damage) is evident during the period of normal or increased NO synthesis that occurs early in the course of IDDM. The functional consequences of these events are evidenced by our observation that the suppressed arteriolar constrictor response to NOS inhibition is restored by acute exposure of STZ kidneys to exogenous SOD (8) and our recent report that SOD treatment unmasks the normal modulatory impact of endogenous NO on afferent arteriolar constrictor responses to angiotensin II in this same model (9). Both of these observations indicate that O2.- production curbs the functional impact of NO on the renal microvasculature in IDDM, presumably via ONOO- formation. The reduced bioavailability of NO should exert a constrictor influence on the renal vasculature, which seems paradoxical in the setting of hyperfiltration. In light of the pleiotropic determinants of renal arteriolar tone, we surmise that the persistence of diabetic hyperfiltration under these conditions must reflect an overriding impact of other (NO-independent) vasodilator influences. Indeed, previous studies from our laboratory (48,49) suggest that decreased functional expression of voltage-gated Ca2+ channels and/or a increased K+ conductance in preglomerular microvascular smooth muscle could engender diabetic hyperfiltration in the face of the reduced NO bioavailability that accompanies IDDM.
On the basis of our accumulated observations, we propose that the following scenario may accompany the early stage of IDDM in the rat. Normal or increased renal cortical NO synthesis in the setting of accelerated O2.- production results in rapid ONOO- formation, with at least two consequences. First, rapid ONOO- production establishes an imbalance between NO synthesis and degradation, promoting a decline in the bioavailability of NO that, in turn, diminishes the impact of endogenous NO on renal function. A second result of ONOO- production is the formation of nitrotyrosine residues on specific proteins, which also may engender specific functional consequences by interfering with tyrosine phosphorylation (50). Further experimental scrutiny is required to establish the validity of each aspect of this postulate.
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References |
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-Nitro-L-arginine: A potent inhibitor of
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 T. D. Bell, G. F. DiBona, R. Biemiller, and M. W. Brands Continuously measured renal blood flow does not increase in diabetes if nitric oxide synthesis is blocked Am J Physiol Renal Physiol, November 1, 2008; 295(5): F1449 - F1456. [Abstract] [Full Text] [PDF]
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 B. Hohenstein, C. P.M. Hugo, B. Hausknecht, K. P. Boehmer, R. H. Riess, and R. E. Schmieder Analysis of NO-synthase expression and clinical risk factors in human diabetic nephropathy Nephrol. Dial. Transplant., April 1, 2008; 23(4): 1346 - 1354. [Abstract] [Full Text] [PDF]
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H. Shi, D. Patschan, T. Epstein, M. S. Goligorsky, and J. Winaver Delayed recovery of renal regional blood flow in diabetic mice subjected to acute ischemic kidney injury Am J Physiol Renal Physiol, November 1, 2007; 293(5): F1512 - F1517. [Abstract] [Full Text] [PDF]
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 P. Pacher, J. S. Beckman, and L. Liaudet Nitric Oxide and Peroxynitrite in Health and Disease Physiol Rev, January 1, 2007; 87(1): 315 - 424. [Abstract] [Full Text] [PDF]
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T. D. Bell, G. F. DiBona, Y. Wang, and M. W. Brands Mechanisms for Renal Blood Flow Control Early in Diabetes as Revealed by Chronic Flow Measurement and Transfer Function Analysis J. Am. Soc. Nephrol., August 1, 2006; 17(8): 2184 - 2192. [Abstract] [Full Text] [PDF]
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 R. Komers, W. E. Schutzer, J. F. Reed, J. N. Lindsley, T. T. Oyama, D. C. Buck, S. L. Mader, and S. Anderson Altered Endothelial Nitric Oxide Synthase Targeting and Conformation and Caveolin-1 Expression in the Diabetic Kidney Diabetes, June 1, 2006; 55(6): 1651 - 1659. [Abstract] [Full Text] [PDF]
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F. Nakhoul, Z. Abassi, M. Morgan, S. Sussan, and N. Mirsky Inhibition of Diabetic Nephropathy in Rats by an Oral Antidiabetic Material Extracted from Yeast J. Am. Soc. Nephrol., April 1, 2006; 17(4_suppl_2): S127 - S131. [Abstract] [Full Text] [PDF]
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Y. Chen, P. S. Gill, and W. J. Welch Oxygen availability limits renal NADPH-dependent superoxide production Am J Physiol Renal Physiol, October 1, 2005; 289(4): F749 - F753. [Abstract] [Full Text] [PDF]
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D. L. Lee, J. M. Sasser, J. L. Hobbs, A. Boriskie, D. M. Pollock, P. K. Carmines, and J. S. Pollock Posttranslational regulation of NO synthase activity in the renal medulla of diabetic rats Am J Physiol Renal Physiol, January 1, 2005; 288(1): F82 - F90. [Abstract] [Full Text] [PDF]
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 B. F. Schrijvers, A. S. De Vriese, and A. Flyvbjerg From Hyperglycemia to Diabetic Kidney Disease: The Role of Metabolic, Hemodynamic, Intracellular Factors and Growth Factors/Cytokines Endocr. Rev., December 1, 2004; 25(6): 971 - 1010. [Abstract] [Full Text] [PDF]
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,
sham; 
, streptozotocin (STZ). Data are presented as mean ± SEM.
*, P < 0.05 versus sham; **,
P < 0.001 versus sham.
, murine macrophage;
BAE, bovine aortic endothelium.
) of 250 µg/ml
aggregated IgG. 