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Physiol. Genomics 28: 158-167, 2007. First published October 17, 2006; doi:10.1152/physiolgenomics.00077.2006
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Received 2 May 2006; accepted in final form 16 October 2006.
Physiological Genomics 28:158-167 (2007)
1094-8341/07 $8.00 © 2007 American Physiological Society

Translational Physiology

Transcriptome-based identification of pro- and antioxidative gene expression in kidney cortex of nitric oxide-depleted rats

Sebastiaan Wesseling1, Jaap A. Joles1, Harry van Goor3, Hans A. Bluyssen1, Patrick Kemmeren2, Frank C. Holstege2, Hein A. Koomans1 and Branko Braam1,4

1 Nephrology and Hypertension, Netherlands
2 Genomics Laboratory, University Medical Center Utrecht, Netherlands
3 Pathology, University of Groningen, Netherlands
4 Nephrology and Immunology, University of Alberta, Canada


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Nitric oxide (NO) depletion in rats induces severe endothelial dysfunction within 4 days. Subsequently, hypertension and renal injury develop, which are ameliorated by {alpha}-tocopherol (VitE) cotreatment. The hypothesis of the present study was that NO synthase (NOS) inhibition induces a renal cortical antioxidative transcriptional response and invokes pro-oxidative and proinflammatory gene expression due to elimination of dampening effects of NO and enhanced oxidative stress. Male Sprague-Dawley rats received NOS inhibitor N{omega}-nitro-L-arginine (L-NNA, 500 mg/l water) for 4 (4d-LNNA), 21 (21d-LNNA), or 21 days with VitE in chow (0.7 g/kg body wt/day). Renal cortical RNA was applied to oligonucleotide rat arrays. In 4d-LNNA, 21d-LNNA, and 21d-LNNA+VitE, 120, 320, and 184 genes were differentially expressed, respectively. Genes related to glutathione and bilirubin synthesis were suppressed during 4d and 21d-LNNA and not corrected by VitE. Proteinuria, tubulointerstitial macrophages, and heme-oxygenase-1 (HO-1) expression were strongly correlated. Remarkably, pro-oxidative genes were not induced. Inflammation- and injury-related genes, including kidney injury molecule-1 and osteopontin, were unchanged at day 4, induced at 21d, and partly corrected by VitE. Superimposing HO-1 inhibition on NOS inhibition had no impact on the development of hypertension. To summarize, renal expression of genes involved in synthesis of the antioxidants glutathione and bilirubin seemed directly NO dependent, but there were no direct effects of NO depletion on pro-oxidant systems. This indicates that renal transcriptional regulation of two defense systems, glutathione and bilirubin syntheses, seems to depend upon adequate NO synthesis. Interaction between NO synthesis and heme degradation pathways for blood pressure regulation was not found.

nitric oxide synthase inhibition; bilirubin; microarray; {alpha}-tocopherol; proteinuria; hypertension


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
THE PATHOGENESIS OF THE HYPERTENSION and renal damage due to nitric oxide (NO) deficiency is complex and only partially understood (44, 45). In general, a disturbed redox balance is considered to be inherent to all phases of renal injury (15). Redox regulation involves the delicate balance between pro- and antioxidative forces. NO depletion affects this balance and causes oxidative stress (2). A comprehensive analysis of renal gene expression of systems that are involved in production and disposition of reactive oxygen species during NO deficiency is not available. These systems include the superoxide dismutase family, catalase, enzymes responsible for glutathione synthesis, as well as the degradation pathway of heme that results in formation of the dilator CO and the potent scavenger bilirubin (36).

Variable changes in expression and activity of antioxidant enzymes have been reported in renal tissue during NO synthase (NOS) inhibition with N{omega}nitro-L-arginine methyl ester (L-NAME) (11, 20). However, one study was performed in spontaneously hypertensive rats (20), and measurements in both studies were performed at a relatively late stage when profound hypertension and renal injury were apparent. At this stage the interplay between disturbed redox balance, inflammation and interstitial fibrosis complicate dissection of the direct effect of loss of NO. Acute application of SOD mimetics and inhibitors during NOS inhibition suggest increased superoxide formation (22, 23). Similarly, acute inhibition of heme oxygenase during NOS inhibition suggest protection by the degradation of heme to CO and bilirubin (33), but such studies do not allow identification of adaptive changes.

The hypothesis of the present study was that NO synthesis inhibition induces a renal cortical antioxidative transcriptional response and invokes pro-oxidative and proinflammatory gene expression due to elimination of dampening effects of NO on the one hand and enhanced oxidative stress on the other. In previous studies, we have applied genomics approaches to identify cellular transcriptional consequences of NO donors (7) and NO synthesis inhibition (6). In analogy, we now applied this technique to investigate transcriptomes of kidneys of rats with NO deficiency induced by the potent NOS inhibitor N{omega}-nitro-L-arginine (L-NNA) (2, 42). Transcriptional responses in renal cortex were studied under three different circumstances; first preceding severe hypertension and renal damage, i.e., after 4 days (4d) of L-NNA, second when hypertension and renal damage are present after 21d of L-NNA, and finally after 21d of L-NNA with vitamin E to dissect which part of the complex response at this late stage was driven by increased oxidative stress.

Two important transcriptional pathways were identified and prompted us to perform follow-up experiments. Since several genes related to glutathione (GSH) synthesis were already suppressed after 4d of L-NNA, renal GSH content was measured at this early time point. Furthermore, because heme oxygenase-1 (HO-1) gene expression was strongly enhanced in the chronic phase of NO suppression only, i.e., after 21d, we studied whether this was related to proteinuria and inflammation by assessing renal HO-1 expression and macrophage influx (ED-1 staining).


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Experimental design.
Male Sprague-Dawley rats (8–12 wk; Harlan-Olac, Blackthorn, Bicester, Oxon, UK) were treated with L-NNA for 4 (4d-LNNA; n = 5, 500 mg L-NNA/l) or 21 days (21d-LNNA; n = 5, 500 mg L-NNA/l) in the drinking water. A subgroup of 21d-LNNA was cotreated with vitamin E (21d-LNNA+VitE; n = 5, 0.7 g/kg body wt per day) in finely ground chow. Control rats (Controls, n = 12) received standard diet. The chow contained 100 mmol/kg sodium and 82 mg/kg VitE (RMH-TM; Hope Farms, Woerden, Netherlands). At the end of the experiment the rats were anesthetized with pentobarbital, an aortic blood sample was drawn, the kidneys were excised, and parts of the kidney were snap frozen and stored at –80°C or stored in 4% formaldehyde for paraffin embedding and histochemical analysis. Systolic blood pressure, proteinuria, plasma creatinine, and aortic NO-dependent relaxation were measured as described previously (2, 3).

In a second experiment rats, similarly treated with L-NNA for 4d (n = 4) and aged-matched controls (n = 4) were killed, and the kidneys, liver, heart, and blood were collected for GSH measurements. Care was taken to collect and homogenize the organs in 5% sulfosalicyclic acid (wt/vol in water) to rapidly inactivate {gamma}-glutamyltranspeptidase, the enzyme responsible for the first step in the degradation of GSH (32).

In a third experiment HO-1 was inhibited with Sn(IV) protoporphyrin IX dichloride (SnPP; Frontier Scientific, Carnforth, UK) in NO-depleted rats. First, rats were treated with L-NNA (100 mg LNNA/l) and/or SnPP for 21d (21d-LNNA, 21d-LNNA+SnPP or 21d-SnPP, respectively). Second, a subgroup of rats treated with same dose of LNNA for 28d (28d-LNNA) were concomitantly treated with SnPP starting at day 14 (28d-LNNA+14d-SnPP). Rats were also treated with SnPP for 28d. All groups in the third experiment, including two untreated groups (Control), contained five rats. SnPP was dissolved in saline (pH 7.4) and subcutaneously injected at dose of 50 µmol SnPP/kg body weight/wk.

During the experiments sentinel rats were regularly monitored for infection by nematodes and pathogenic bacteria, as well as antibodies to rodent viral pathogens. The Animal Ethical Committee of the University of Utrecht approved the protocol.

Total RNA isolation, microarray procedures, and analysis.
Details of the procedures are available in the web appendix (www.nephrogenomics.net/data/appendices/Rat-LNNA-2006). In short, total RNA was extracted (TRIzol) from renal cortex cryostatically sliced off frozen kidney, dissolved in distilled H2O, and stored at –80°C. For microarray analysis, total RNA was pooled per group in equal amounts per subject: control (n = 12), 4d-LNNA (n = 5), 21d-LNNA (n = 5), and 21d-LNNA+VitE (n = 4), reverse transcribed (RT) with allyl-dUTP incorporation and labeled with Cy3 or Cy5 dyes (Amersham Biosciences, Piscataway, NJ). Samples were hybridized to rat 7.5k Oligo Chips manufactured in the Genomics Laboratory, containing Rat Genome Array-Ready Oligo Set version 1.1 of Qiagen-Operon (40), spotted in duplicate. Samples of treated rats were compared with controls and a dye switch procedure was applied (21). Control samples were also compared with each other to allow elimination of unreliable spots. After being washed, slides were stored in the dark, until scanning using the ScanArray 4000XL (BioDiscovery, El Segundo, CA). Images were quantified using Imagene Software (BioDiscovery), and data were normalized as described previously (21). Duplicates of the genes were averaged. Ratios of ≥0.7 or ≤–0.7 were considered significant. Microarray data in MIAME format were submitted at European Bioinformatics Institute under accession number E-UMCU-18 for experiments and A-UMCU-5 for arrays.

Semiquantitative and real-time quantitative PCR.
For semiquantitative PCR total RNA was pooled per treatment group in equal amounts per subject. For real-time PCR, total RNA from individual samples was used (at least n = 3 per group in the LNNA experiment). Real-time PCR of renal HO-1 was performed on 42 samples: 19 Sprague-Dawley rats (8 control and 11 LNNA) and 23 FHH rats. RT was performed in batches of 5 µg of total RNA. Laboratory details on RT-PCR and the primer conditions are available in web appendix table 1. PCR samples were run on a 2% agarose gel containing ethidium bromide (17 µl/l Agarose; MP Biomedicals, Irvine, CA).


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  		Table 1. Systolic blood pressure, proteinuria, plasma creatinine, and acetylcholine-mediated endothelial relaxation (aorta)

 
TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA) were used for real-time quantitative PCR, and the protocol was performed as recommended by the supplier. In short, cDNA (50 ng of starting material total RNA) was mixed with Taqman Universal PCR Master Mix (with AmpErase UNG) and TaqMan Gene Expression Assay in end volume of 25 µl. Quantitative PCR was performed in ABI 7700 Single Reporter. The gene threshold was determined by SDS 1.91 (Applied Biosystems). Genes studied were HO-1 (Rn00561387), NADPH oxidase 4 (NOX4; Rn00585380), biliverdin reductase A (BVRA; Rn00587773), and 18S (18S; Hs99999901). Calculation of the ratios is explained in the web appendix (www.nephrogenomics.net/data/appendices/Rat-LNNA-2006).

Biochemical measurements.
Stable metabolites of NO, NO2 and NO3 (NOx), and lipid peroxides [thiobarbituric acid-reactive substances (TBARS)] in plasma and urine were measured as described previously (1). GSH in organ homogenates was measured as described previously (10). Oxidized glutathione (GSSG) was first converted into reduced GSH by adding NADPH. GSSG alone was measured by removing GSH first with N-ethylmaleidimide. Subtraction of GSSG from total GSH plus GSSG, resulted in GSH values.

Histology: osteopontin and macrophages (ED-1).
Paraffin sections were deparaffined, placed in 10 mmol/l citric buffer (pH 6.0), and subjected to heat-induced antigen retrieval by using a microwave at 400 W for 15 min. After endogenous peroxidase blockade, sections were incubated with osteopontin antibody (1:2,000, kindly provided by Dr. Sharon Ricardo, Monash University, Clayton, Victoria, Australia) for 1 h. Secondary peroxidase-labeled rabbit anti-mouse antibodies (1:50; Dakopatts, Glosstrup, Denmark) were applied, followed by tertiary peroxidase-labeled goat anti-rabbit antibodies (1:100; Dakopatts). Peroxidase activity was developed with 3,3'-diaminobenzidine tetrachloride. Sections were counterstained with Mayer's hematoxylin, dehydrated, and mounted. All rats were analyzed. Per rat, eight fields of the renal cortex were randomly selected, and the percentage of positive area was determined with Optimas (MediaCybernetics, Gleichen, Germany) in a blinded fashion.

To visualize monocytes/macrophages, ED-1 mouse monoclonal antibody (kindly provided by E. Döpp, Dept. of Cell Biology, Free University, Amsterdam, the Netherlands) was used. Paraffin sections of kidney were pretreated by autoclaving the sections with 10 mM citrate buffer (pH 6). After rapid cooling, the slides were incubated with ED-1 antibody (dilution 1:2,000 in PBS containing 1% BSA and 0.4% sodium azide) at room temperature for 1 h. Bound antibody was detected using a secondary antibody rabbit anti-mouse horseradish peroxidase (HRP) and tertiary antibody swine anti-rabbit HRP, both incubated for 30 min at room temperature and diluted 1:100 in PBS containing 5% normal rat serum. Both antibodies were obtained from Dako.

The number of ED-1-antigen-positive monocytes/macrophages was counted in 20 tubulointerstitial fields of 0.245 mm2 using x200 magnification. An average score per field was calculated.

Gene expression analysis and statistics.
Results are expressed as means ± SE. Data were compared using one-way analysis of variance where appropriate followed by a Student-Newman-Keuls post hoc test. Correlations between proteinuria, macrophage influx, and renal HO-1 expression were calculated using linear regression analysis. P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Endothelial function, blood pressure, and renal function during chronic L-NNA.
NOS inhibition induced severe aortic endothelial dysfunction in all treated groups (Table 1). Long-term NO depletion caused hypertension (P < 0.01 vs. Control), which was ameliorated by VitE. Proteinuria was severe at 21d-LNNA (P < 0.01 vs. Control) and ameliorated by vitamin E (P < 0.05 vs. 21d-LNNA). Plasma creatinine was elevated in 21d-LNNA (P < 0.01 vs. Control). This was prevented by vitamin E (P ≤ 0.05 vs. 21d-LNNA).

Regulated genes during L-NNA.
The self-vs.-self microarray showed 47 genes with absolute log2 ratios of >0.7; these genes were excluded from further data analysis. The numbers of regulated genes in 4d-LNNA, 21d-LNNA, and 21d-LNNA+VitE were 120, 320, and 184, respectively. Figure 1A shows that coregulation of genes was most prominent between 21d-LNNA and 21d-LNNA+VitE. The distribution of expression ratios was symmetrical in all groups (Fig. 1B). The full dataset is available online (www.nephrogenomics.net/data/appendices/Rat-LNNA-2006).


Figure 1
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  		Fig. 1. All regulated genes were presented in an overlap model to show the number of shared regulated genes between treatment groups (A). The distribution of log2 ratio for all genes was binned per 0.05 log2 ratio. The number of genes per bin close to 0 (indicating no change) was greatest in control (B). L-NNA, N{omega}-nitro-L-arginine; VitE, vitamin E; d, day.

 
Transcriptional responses of redox-related genes to NO synthesis inhibition.
In the antioxidative systems almost all genes related to synthesis of GSH were decreased in LNNA groups (Table 2; RT-PCR data are shown in GoGoFig. 4). Genes coding for both components of {gamma}-glutamylcysteine ligase [catalytic (GCLc) and modifier (GCLm) subunits], the rate-limiting enzyme of GSH synthesis, were persistently decreased and not affected by vitamin E. Similarly, BVRA was suppressed in all 21d-LNNA-treated rats. This decrease was found to be significant with quantitative PCR but also in 4d-LNNA-treated rats (Fig. 2). Hypoxia-inducible factor 3-alpha (HIF-3{alpha}), a negative regulator of the hypoxia-inducible response, was downregulated by NO depletion after 4d, but not after 21d. Catalase (CAT) was suppressed in 21d-LNNA only. HO-1, ceruloplasmin (Cp), and all isoforms of metallothionein (MTs) were induced in 21d-LNNA, and only MT-1 and Cp were normalized by vitamin E. Quantitative PCR confirmed the increased HO-1 at 21d (n = 5), but in contrast to microarray, vitamin E appears to blunt this response (n = 5, Fig. 2).


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  		Table 2. Anti- and pro-oxidative genes regulated (ratio ≥ 0.7 or ≤ –0.7) after 21 days of L-NNA treatment, and their ratios in the other groups

 

Figure 2
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  		Fig. 2. Real-time quantitative PCR was applied on heme oxygenase-1 (HO-1), biliverdin reductase A (BVRA), NADPH oxidase 4 (NOX4), and 18S. At least 3 individuals per treatment group were used in duplicate per gene. Duplicates were averaged and corrected by 18S as reference gene per individual. Subsequently all corrected expressions per gene within a treatment group were compared with averaged Control gene expression and finally averaged. #P < 0.005, ##P < 0.001 vs. Control; {dagger}P < 0.005 vs. Control and P < 0.001 vs. 21d-LNNA; {dagger}{dagger}P < 0.001 vs. 21d-LNNA.

 

Figure 3
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  		Fig. 3. Histology was performed on renal cortex for localization of osteopontin (magnification x200). A: 21d-LNNA; B: 21d-LNNA+VitE. #P < 0.001 vs. control and 4d-LNNA; ##P < 0.05 vs. 21d-LNNA and P = 0.05 vs. control.

 

Figure 4
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  		Fig. 4. RT-PCR was applied to confirm expression changes of catalase, ceruloplasmin, fibrinogen-ß, catalytic subunit of glutamate-cysteine ligase, glutathione synthetase, kidney injury molecule-1, metallothienin-1, osteopontin, tissue inhibitor of metalloproteinase-1, and vimentin. Ratios of regulated genes are indicated in bold type.

 
In the pro-oxidative systems the overall tendency was downward regulation, although only flavin-containing mono-oxygenase 3 (FMO3) was persistently significantly suppressed. Two other FMO isoforms were also suppressed at 21d-LNNA, while only FMO1 gene expression was normalized by vitamin E. Kidney-specific NADPH oxidase (NOX4) was significantly downregulated at 21d-LNNA, and this was prevented by vitamin E. Quantitative PCR confirmed these changes in NOX4 (Fig. 2).

Transcriptional responses of genes related to inflammation.
Remarkably few genes related to inflammation were persistently differentially expressed. Exceptions were one interferon-{alpha}-inducible protein and one serine/cysteine proteinase inhibitor, which were consistently increased. All other inflammatory genes that were up after 21d but not 4d of LNNA (arachidonate 12-lipoxygenase, chemokine ligands 9 and 12, caspase 12, interleukin 1ß, interleukin 3-regulated nuclear factor, tumor necrosis factor receptor 1{alpha}, CD12, CD44, CD53, tissue plasminogen activator, and EGR1) were fully corrected by vitamin E, pointing at a response to oxidative stress.

Transcriptional responses of genes related to damage.
Web appendix Table 2 lists a selection of the regulated genes related to damage. At 4 days, practically no genes related to damage were differentially regulated. Kidney injury molecule-1 (KIM-1) was the strongest induced gene in kidneys from 21d NO-depleted rats with or without vitamin E. Osteopontin was the next mostly strongly regulated gene in 21d NOS-inhibited rats. Immunohistochemistry was performed to determine the renal localization of osteopontin. Kidneys from control and 4d-LNNA-treated animals revealed almost no staining (figures not shown), whereas long-term NO depletion resulted in strong staining of osteopontin primarily in renal tubules (Fig. 3A). Vitamin E cotreatment ameliorated staining of osteopontin (Fig. 3B). The protein expression of osteopontin followed proteinuria. Two metalloproteinase-related genes meprin 1a and 1b were suppressed in long-term NO-depleted rats, irrespective of vitamin E cotreatment. Expression of epidermal growth factor (EGF) and the corresponding receptor-related gene (EGFR) were suppressed at 21d; this was normalized by vitamin E.

Confirmation of a selection of the differentially expressed genes by semiquantitative PCR.
Semiquantitative PCR was performed on the genes CAT, Cp, fibrinogen-ß, {gamma}-GCSc, GSH, KIM-1, MT1, osteopontin, tissue inhibitor of metalloproteinase (TIMP)-1, and vimentin (VIM) (Fig. 4). All gene expressions were confirmed, except MT1 and Cp, which, in contrast to microarray, were still induced during vitamin E cotreatment. There was a tendency toward decreased gene expression of CAT in all treatment groups. The genes that showed a clear induction at 21d-LNNA and less during vitamin E cotreatment were KIM-1, osteopontin, TIMP-1, and VIM.

Glutathione synthesis.
Following up on the finding that after only 4d of L-NNA treatment, gene expression of the regulatory and catalytic subunits of GCL, the rate-limiting enzyme of GSH synthesis, was already decreased in the kidney (Table 2), renal GSH content was assessed at this early time point. Interestingly, despite the fact that plasma levels and urinary excretion of nitrite plus nitrate (NOx) was clearly decreased confirming adequate NOS inhibition, there was no decrease in glutathione content in blood or any of the organs studied including the kidneys (Table 3). TBARS were not changed after 4d of LNNA in either plasma or urine.


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  		Table 3. Blood and organ glutathione content, proteinuria, plasma creatinine, NOx and TBARS, and urine NOx and TBARS after 4 days of L-NNA

 
Renal injury and HO-1.
In controls, proteinuria ranged from 10 to 34 mg/d. In 21d LNNA-treated rats proteinuria ranged from 11 to 396 mg/d. Rats treated with LNNA for 21d showed increased macrophage influx, while vitamin E cosupplementation reduced tubulointerstitial inflammation (Table 4). ED-1 staining correlated with proteinuria (Fig. 5A). Gene expression of HO-1 was strongly correlated with proteinuria and ED-1 staining (Fig. 5, B and C).


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  		Table 4. Proteinuria, tubulointerstitial macrophages (ED-1-positive cells), and renal cortical HO-1 gene expression

 

Figure 5
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  		Fig. 5. Relationship between renal tubulointerstitial ED-1-positive cells, proteinuria, and cortical HO-1 gene expression. ED-1-positive cells (field area 0.245 mm2) was compared with proteinuria (A), and HO-1 gene expression was compared with ED-1-positive cells (B) and proteinuria (C).

 
Functional study of HO-1 during NO depletion.
NO-depleted rats were hypertensive after 2 and 3 wk (P < 0.001 vs. Con) and proteinuric after 3 wk (P < 0.05 vs. Control and 21d-SnPP). These changes in SBP induced by NO depletion were not affected by superimposed treatment of HO-1 inhibition (Fig. 6, A and B). Similarly, NOS-inhibited rats displayed hypertension from 2 wk onward (P < 0.005 vs. Control and 28d-SnPP), and superimposing HO-1 inhibition starting at week 2 had no impact on the development of hypertension. Interestingly, while the NOS-inhibited rats were proteinuric at weeks 3 and 4, HO-1 inhibition starting at week 2 in NO-depleted rats had a protective effect (P < 0.05 28d-LNNA vs. all groups; Fig. 6, C and D). No changes occurred in rats treated with only SnPP for 21 or 28 days (Fig. 6, A–D).


Figure 6
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  		Fig. 6. Functional study of HO-1 inhibition in nitric oxide (NO)-depleted rats. A and B: systolic blood pressure (SBP) and proteinuria (UpV) in NOS and/or HO-1 inhibited rats. •, Control; {circ}, 21d-LNNA, {blacktriangledown}, 21d-LNNA+SnPP; {triangleup}, 21d-SnPP. *P < 0.001 vs. Control and 21d-SnPP; **P < 0.005 vs. Control and 21d-SnPP; {dagger}P < 0.05 vs. Control and 21d-SnPP. C and D: SBP and UpV in nitric oxide synthase (NOS) or HO-1 inhibited rats and NOS and subsequently HO-1 inhibited rats. •, Control, {circ}, 28d-LNNA, {blacktriangledown}, 21d-LNNA+14d-SnPP; {triangleup}, 21d-SnPP. #P < 0.005 vs. Control and 28d-SnPP; ##P < 0.05 vs. all groups.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Experimental NO depletion in rats is associated with severe hypertension, and with increased oxidative stress in kidney cortex (2, 3). The hypothesis of the present study was that NO synthesis inhibition induces a renal cortical antioxidative transcriptional response and invokes pro-oxidative and proinflammatory gene expression. NO depletion directly caused a complex disturbance in gene regulation of antioxidant systems, pointing at diminished GSH and bilirubin synthesis. After 21d of LNNA, expression of catalase was decreased, suggesting further loss of antioxidant defense. Catalase expression was partly corrected by vitamin E. Gene expression of the pro-oxidant NOX4 was suppressed after 21d. Hence, we report that depression of antioxidant defense genes, rather than enhanced expression of pro-oxidant genes, is an early response to NO depletion.

The decrease in expression after 4 days of NO synthesis inhibition of the regulatory and catalytic subunits of GCL, the rate-limiting enzyme of GSH synthesis, clearly preceding renal damage, extends observations that NO at physiological rates of production induces GSH synthesis from vascular smooth muscle cells (26) and endothelial cells (25) to the renal cortex. In other studies NO depletion resulted in decreased renal and cardiac GCL activity (5, 20). Polymorphisms of GCLm and GCLc are associated with lower plasma GSH levels, myocardial infarction (17, 27), and impairment of NO-mediated coronary vasomotor function (17, 28). These associations appear to be independent of traditional risk factors. Thus the GSH/NO axis is needed to counteract the effects of ROS constitutively produced under control circumstances. Although others have found that longer periods of exposure to NOS inhibition were associated with decreased renal GSH (11, 20), the observation of the potentially rate-limiting disturbance in this pathway at such an early stage is novel.

Surprisingly, this did not result in a decrease of renal GSH content. Possibly, NO depletion increases the mRNA half-life of the GCL subunits, as was found previously in vitro after exposure to chemical stress by diethyl maleate (35). Apparently antioxidative defense is still maintained for up to 4d even though NO deficiency is downregulating antioxidant systems. This notion is supported by the unchanged TBARS in plasma and urine. After 21d, several other genes related to GSH biosynthesis were decreased, suggesting that progressive GSH depletion may contribute to the increase in oxidative stress. Interestingly, vitamin E supplementation did not ameliorate the complex disturbance in gene expression contributing to GSH depletion. On the contrary, with vitamin E {gamma}-glutamyl transpeptidase expression, important for glutathione regeneration, also became depressed. An illustration has been drawn to clarify overall reduction in genes coding for enzymes of glutathione biosynthesis (Fig. 7A). The present observation suggests that the presence of a normal functioning NO system in the renal cortex is necessary for the normal regulation of genes involved in GSH generation.


Figure 7
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  		Fig. 7. A: glutathione (GSH) biosynthesis pathway (left) and the relevant microarray data (right). Ratios in bold type are suppressed genes. GSH biosynthesis genes were suppressed in the whole pathway. {gamma}-Glutamate-cysteine ligase was primarily disturbed by NO depletion at 4d. B: hemin degradation pathway (left), and the relevant microarray data (right). Ratios of regulated genes are indicated in bold type. There was induction of heme oxygenase-1 (HO-1), but biliverdin reductase A (BVRA) was suppressed, changes that could result in reduced availability of the potent antioxidant bilirubin.

 
Quantitative PCR showed that already after 4d of L-NNA BVRA was decreased, while after 21d HO-1 expression was induced and BVRA was further decreased. This is compatible with enhanced conversion of heme by HO-1 to CO, iron, and biliverdin, and inhibited subsequent conversion of biliverdin to bilirubin. The role of HO-1 and BVRA in heme degradation pathway is depicted in Fig. 7B. Indeed, severe BVRA depletion by RNA interference led to higher ROS levels than nearly complete glutathione depletion (4). NOS inhibition during a renal insult, such as hypoxia, strongly stimulates HO-1 (24), resulting in increased production of CO (34), which could act as backup for NO (29). Because chronic NOS inhibition induces renal vascular injury and glomerular collapse (2, 42, 45), this effect is reinforced. However, HIF-3{alpha}, a negative regulator of the hypoxia-inducible gene response (14), was downregulated by NO depletion after 4d, suggesting that functionally hypoxia is already present at an early stage. This may be a prelude to hypoxia-induced injury at a later stage (12), because HO-1 was strongly induced after 21d, but not at all after only 4d. It can be speculated that the increase in HO-1 is a response to increased oxidative stress, induced by prolonged decreased bilirubin formation and diminished scavenging of NO, because vitamin E ameliorated HO-1 induction. To assess whether renal injury induces HO-1 expression, both proteinuria and macrophage influx were correlated with HO-1 expression. All 21d NO-depleted rats were hypertensive, but of these rats the nonproteinuric ones did not show induced HO-1 gene expression. As expected the degree of inflammation as indicated by staining of macrophages by ED-1 in NO depleted rats is related to proteinuria. We also found a strong relationship of HO-1 gene expression to both tubulointerstitial inflammation and proteinuria. Considering the relative contribution of the tubulointerstitial compartment to renal tissue, it is likely that HO-1 is dependent on tubulointerstitial inflammation rather than to glomerular damage. This notion is supported by the study of Pedraza-Chaverri et al. (30) that showed induction of HO-1 gene in tubular but not glomerular proteinuria.

Thus, in hypertensive renal injury caused by NO depletion, renal HO-1 expression may result from renal injury.

Whether induction of renal HO-1 in proteinuric rats (including NO-depleted rats) could provide renal protection was investigated by inhibiting HO-1 with SnPP during NO depletion. We dissected hypertension from renal damage by inhibiting HO-1 synchronously with NO depletion or starting 2 wk after initiating NO depletion when hypertension was present without proteinuria. Inhibition of HO-1 during NO depletion had no impact on development of hypertension or proteinuria. In contrast, inhibiting HO-1 from a later point, when hypertension had already occurred, ameliorated the further development of proteinuria in the following 2 wk. These findings suggest that HO-1 is a protein that appears as a response to (imminent) injury rather than a back-up system for NO deficiency. This is in contrast to the concept that HO-1 has a primary protective function against diverse renal damage, for example caused by cyclosporine or ischemia/reperfusion (31, 37). Other studies have shown antioxidative and protective effects by administration of biliverdin. The administered biliverdin can be converted to bilirubin, a powerful water-soluble antioxidant. To observe the difference in antioxidative property between biliverdin and bilirubin, the expression of BVRA was reduced, which led to enhanced oxidative stress (4). In the present study, BVRA, which was already decreased after 4d of NO depletion, may fail to produce an adequate amount of bilirubin. This may explain why in NO depleted rats the enhanced HO-1 expression cannot protect the kidneys against damage.

Gene expression of the pro-oxidant, kidney-specific NOX4 (isoform of gp91phox; component of the membranous NADPH oxidase fraction) was suppressed after 21d and was normalized by vitamin E cotreatment. Moreover the NADPH oxidase component p47phox (component of the cytosolic NADPH oxidase fraction), which has been implicated in the development of hypertension in the Dahl rat (13) and the spontaneously hypertensive rat (SHR) (8), was not induced in the present study. The FMO family, another pro-oxidant enzyme system, was markedly suppressed. There is limited information about the role of the FMOs in the kidney with respect to oxidative stress, and the exact consequences of the observed regulation are unclear. Thus the primary transcriptional response to severe NO depletion was certainly not pro-oxidant, and enhancement of O2–· production by NADPH oxidase, as suggested in our previous study (2) may be mainly functional.

Regarding genes involved in renal damage, the microarray data indicated strong differential expression of KIM-1 and osteopontin, which were both confirmed by RT-PCR and by immunohistochemistry for osteopontin. KIM-1, specifically located in proximal tubular cells (16), has been reported to be expressed in renal damage in which proximal tubular cells underwent differentiation and proliferation, but also regeneration (19). Osteopontin gene and protein expression often precede renal tubulointerstitial injury (38) but appear to follow proteinuria (18). Histological examination revealed location of osteopontin in renal tubules and osteopontin protein expression appeared to be related to proteinuria. Meprin proteins are metalloproteinases that degrade various proteins and extracellular matrix components, such as osteopontin and fibronectin (43). Indeed in the present study meprin proteins were reduced in NO-depleted animals. The reduction of expression of meprin 1A and 1B may result in a longer half-life for these proteins. Thus, the microarray showed that renal tubulointerstitial injury in the course of chronic NO depletion is accompanied by strong induction of KIM-1 and osteopontin, which may indicate a response to injury, because KIM-1 and osteopontin are also associated with regeneration.

Vitamin E ameliorated hypertension in the present study and, as previously reported, reduced hypertension in uremic rats (41) and improved endothelial dysfunction in SHR (39), by NO generation and lowering O2–· activity (39, 41). Chronic antioxidant treatment in pigs with renovascular disease improved renal hemodynamics, NO bioavailability, and decreased structural injury but did not affect hypertension (9). In the present study vitamin E also ameliorated proteinuria and normalized plasma creatinine and the expression of many genes that were deranged at 21d. The corrected genes were mainly related to inflammation and damage, and the primary NO-dependent disturbances of GSH and bilirubin synthesis pathways were not substantially affected. Furthermore, vitamin E only slightly improved endothelial dysfunction and did not correct the expression of any genes that had already been affected at 4d. Hence, vitamin E cotreatment during chronic NO deficiency failed to normalize the severely disturbed antioxidant response.

In summary, long-term NOS inhibition resulted in a primary disturbance of genes related to antioxidant defense, notably bilirubin and glutathione synthesis, which was not improved by vitamin E cotreatment. Upregulation of HO-1 appears to follow proteinuria and also seems to be dependent on tubulointerstitial inflammation. Normalization of expression by vitamin E indicated that most inflammatory and some damage-related genes were driven by oxidative stress due to a primary loss of antioxidant defense. This was reflected by normalized plasma creatinine and ameliorated hypertension and proteinuria. The role of HO-1 in kidney appears to be more complex than has previously been suggested. Interaction between NO synthesis and heme degradation pathways with respect to blood pressure regulation is not supported by the present experiments.

Perspectives.
Transcriptome analysis of the renal cortex of NO-deficient rats, with hypertension and renal damage, points at failure of antioxidant defense rather than induction of pro-oxidative forces. Interestingly, NO seems to mediate the transcriptional regulation of the bilirubin and GSH synthesis-related systems. Dependency of defense systems upon the presence of NO may explain the severity of renal damage caused by chronic NO depletion.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
The Dutch Kidney Foundation financially supported this study (NS6013 and PC144).


   ACKNOWLEDGMENTS
 
Marian Bulthuis, Dionne v.d. Giezen, Paula Martens, and Ria de Winter-v.d. Broek provided expert technical assistance; we gratefully acknowledge their contributions to this study. Dr. Branko Braam was supported by a research fellowship of the Royal Dutch Academy of Arts and Sciences.


   FOOTNOTES
 
Address for reprint requests and other correspondence: B. Braam, Nephrology and Immunology, Univ. of Alberta, 11-107 Clinical Sciences Bldg., 8440-112 St., Edmonton, Alberta T6G 2G3, Canada (e-mail: braam{at}ualberta.ca).

Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
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