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NDT Advance Access originally published online on March 29, 2007
Nephrology Dialysis Transplantation 2007 22(7):1873-1881; doi:10.1093/ndt/gfm113
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© The Author [2007]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

N-acetylcysteine attenuates NSAID-induced rat renal failure by restoring intrarenal prostaglandin synthesis

Shai Efrati1,2, Sylvia Berman1,2, Yariv Siman-Tov2, Raffie Lotan2, Zhan Averbukh1, Joshua Weissgarten1 and Ahuva Golik3

1Nephrology Division, 2Research & Development Unit and 3Department of Internal Medicine A, Assaf Harofeh Medical Center, Zerifin 70300, affiliated to Sackler Faculty of Medicine, Tel Aviv University, Israel

Correspondence and offprint requests to: S. Efrati, MD, Head of the Research & Development Unit, Assaf Harofeh Medical Center, Zerifin 70300, Israel. Email: efratishai{at}013.net



   Abstract
Top
 Abstract
Introduction
 Methods
 Experimental design
 Statistical analysis
 Results
 Discussion
 References
 
Background. Renal failure is a threatening side-effect of NSAID administration, consequent to NSAID-mediated abrogation of prostaglandin synthesis and resultant renal ischaemia. N-acetylcysteine (NAC) has renoprotective properties. We examined effects of NAC in a rat model of NSAID-induced renal failure.

Methods. Renal failure was generated in 80 rats by 6-day water deprivation and 3-day 15 mg/kg/day diclofenac injection. The rats were concomitantly treated, or not, by NAC, 40 mg/kg/day. Renal function was evaluated by cystatin C, creatinine and urea. Intrarenal blood flow was measured by laser Doppler. The kidneys were subjected to pathological examination or evaluation of intrarenal NO, H2O2 and PGE2.

Results. NAC significantly attenuated deterioration of renal function in diclofenac-treated rats: cystatin C dropped from 2.8 ± 0.35 to 2.2 ± 0.67 mg/l, P = 0.016; creatinine from 1.2 ± 0.97 to 0.96 ± 0.19 mg/dl, P = 0.02; urea from 208.4 ± 57.9 to 157.6 ± 33.7 mg/dl, P = 0.028. Diclofenac-inflicted hystopathological damage was significantly reduced following NAC treatment. Intrarenal medullar blood flow dropped by 51 ± 12.4% in diclofenac-treated rats, but only by 14 ± 3.39% in those receiving NAC after diclofenac injection (P < 0.001). H2O2 was elevated in renal tissues of diclofenac-receiving rats, while decreased in NAC-treated animals. PGE2 release by diclofenac-treated rats dropped significantly, but was restored after NAC administration both in renal cortices (144.7 ± 10.4 vs 19.7 ± 1.5 pmol/ml, P < 0.001) and medullae (148.5 ± 7.3 vs 66.6 ± 7.3 pmol/ml, P < 0.001).

Conclusions. In this model of renal failure induced by NSAID administration combined with water deprivation, NAC treatment successfully attenuated the deterioration of renal function by inducing renal vasodilatation, decreasing oxidative stress via inhibition of intrarenal ROS content and restoration of intrarenal PGE2 release back to the basal levels.

Keywords: glutathione; ischaemia; nitric oxide; NSAID; oxidative stress; prostaglandins



   Introduction
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 Abstract
 Introduction
Methods
 Experimental design
 Statistical analysis
 Results
 Discussion
 References
 
Non-steroidal anti-inflammatory drugs (NSAID) are the most common prescription medicine, also freely available over-the-counter [1]. Unfortunately, one of the main side effects of NSAID administration is renal function damage. NSAID are accountable for 7% of all cases of acute renal failure and for 37% incidents of drug-associated acute renal failure [2].

Two different forms of acute renal failure have been shown to develop as a result of NSAID administration: haemodynamically-mediated ischaemic nephropathy and acute interstitial nephritis [3,4]. The former, and probably the latter, might be directly related to NSAID-mediated abrogation of prostaglandin synthesis. In general, renal prostaglandins act primarily as vasodilators. Normally, the basal rate of renal prostaglandin synthesis is relatively low. Therefore, prostaglandins do not play any important role in regulation of renal haemodynamics in healthy subjects. In contrast, release of these hormones is profoundly increased in situations of low effective blood volume, such as heart failure, cirrhosis or true volume depletion due to gastrointestinal or renal sodium and water loss [5–7]. In any of these situations, inhibition of prostaglandin synthesis by NSAID administration might lead to renal ischaemia, decline in glomerular hydraulic pressure and, consequently, to acute renal insufficiency [5–7]. Indeed, renal biopsies from patients with NSAID-induced acute renal failure disclose signs of acute tubular necrosis [2].

N-acetylcysteine (NAC) has been shown to effectively prevent nephrotoxicity induced by contrast media, hypoperfusion or in toxin-induced renal failure in humans and experimental animals [8–13]. In addition, NAC is known to exert a vasodilatatory effect on renal microcirculation [14]. The operative mechanisms have not yet been fully elucidated. Thus far, the probability of NAC playing a role in prevention or attenuation of NSAID-induced acute renal failure has never been approached. The purpose of the present study was to examine a possible renoprotective effect of NAC administration in a rat model of NSAID-induced acute renal failure.



   Methods
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 Abstract
 Introduction
 Methods
Experimental design
 Statistical analysis
 Results
 Discussion
 References
 
Animals
The experiments were conducted according to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. All the animals included in the study were born and bred at significant pathogen-free (spf) conditions in animal facilities of the Research & Development Unit of Assaf–Harofeh Medical Center. The maintenance was according to the guidelines of the Local Ethics Committee for Animal Experimentation, and the experimental protocol was approved by the latter. Rats were fed regular chow routinely purchased by our animal facilities via Harlan Laboratories.



   Experimental design
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 Abstract
 Introduction
 Methods
 Experimental design
Statistical analysis
 Results
 Discussion
 References
 
The study protocol was divided into three phases. Phase 1 was designed to evaluate the effects of NAC administration on kidney functions in a model of acute renal failure induced by combination of diclofenac treatment and water deprivation. The end point of this phase of experiment included pathological evaluation of the kidneys, and measurement of serum cystatin C, creatinine and urea. Phase 2 was designed to evaluate the physiological effects of NAC on renal microcirculatory blood flow. Phase 3 was dedicated to ex-vivo experimentations, which included assessment of intrarenal reactive oxygen species (ROS), represented by H2O2, as well as evaluation of NO bioavailability and measurement of PGE2 release by renal cortices and medullae.

Induction of renal failure
In 40 Sprague–Dawley male rats (age 2 months ± 1 week, weight 250 ± 30 g), renal failure was induced according to the following protocol:

  • 3 days—water deprivation;
  • 3 additional days—i.p. injection of diclofenac sodium, 15 mg/kg (Voltaren, Novartis Pharma, BU) combined with water deprivation.

In preliminary experiments, such regimen of 6-day water deprivation, combined with 3-day diclofenac administration, induced moderate renal failure: a 2-fold increase in serum cystatin C, about 3 mg/l, and serum creatinine, about 1.3 mg/dl. In rats with free access to water, 3 days of i.p. administration of diclofenac, in doses of up to 60 mg/kg body weight, had no significant effect on serum cystatin C or serum creatinine.

Experimental protocol
The animals were randomly divided into five equal groups, as follows:

  • Water deprivation group (n = 10): water deprivation for 6 days combined with i.p. administration of 0.1 ml saline 0.9% twice a day, at days 4 through 6 of the experiment.
  • NAC group (n = 10): water deprivation for 6 days combined with i.p. administration of NAC (Flumil Antidoto 20%, Zambon S.A., Spain), 40 mg/kg twice a day, at days 4 through 6 of the experiment.
  • Diclofenac group (n = 10): water deprivation for 6 days, combined with i.p. administration of diclofenac, 15 mg/kg in 0.1 ml saline 0.9%, twice a day, at days 4 through 6 of the study.
  • Diclofenac + NAC group (n = 10): water deprivation for 6 days, combined with i.p. administration of diclofenac, 15 mg/kg in 0.1 ml saline, and NAC, 40 mg/kg, twice a day, at days 4 through 6 of the study.

Phase 1: Renal function evaluation
Biochemical studies
At the end of the sixth day of the study protocol, 5 ml of blood was procured from each animal by cardiac puncture under light halothane anaesthesia, for biochemical evaluations of cystatin C, creatinine, urea and potassium. All the measurements were performed on Roche/Hitachi 717 autoanalyser (Roche Diagnostics, Mannheim, Germany). In brief, urea was determined using the Roche modification of Talke and Schubert enzymatic procedure optimized for UV kinetic measurements, with lower detection limits <0.8 mmol/l and CV <3.5%. Creatinine was assessed using a highly sensitive Roche modification of Jaffe method (lower detection limit 18 µmol/l; CV, coefficient of variability, <2.3%). Cystatin C was measured by a particle-enhanced immunoturbidimetry method, with a commercially available Dako Cystatin C PET Reagent Set (DAKO, Hamburg, Germany). A polyclonal rabbit antibody against human cystatin C was used for the immunoturbidimetric reaction, providing the lower detection limit <0.1 mg/l and CV <2.5%.

Pathological evaluation
Following blood withdrawal for biochemical studies, five rats from each group were randomly selected to be sacrificed by halothane overdose. Their kidneys were immediately removed, preserved in formalin and subsequently embedded in paraffin blocks. Large sections (1 µm) were cut perpendicularly to the renal capsule, in order to ensure that both cortex and medulla would be presented in each section. Samples were stained with haematoxylin–eosin dye, and 20 microscopic fields (magnification, 40x) were randomly selected for light microscope evaluation. Stratification of the kidney damage was established by differential count of percentage of necrotizing cells in damaged tubules per total count of the tubules, and tubular lumen obstruction was established by counting the percentage of granular casts or cellular fragments out of the total number of tubules counted.

Phase 2: Physiological evaluation
The 2nd phase of the study included 40 rats maintained on water deprivation regimen for 3 days. All the rats were anaesthetized with 1.5–2.5% halothane inhaled via insufflation mask. Portex catheters (REF 800/100/100) were inserted into the left external jugular vein, for drug administration.

The rats were divided into four equal groups (n = 10) treated as follows:

  1. Group 1—i.v. administration of 0.1 ml saline 0.9%.
  2. Group 2—i.v. administration of diclofenac, 15 mg/kg, in 0.1 ml saline.
  3. Group 3—i.v. administration of NAC, 40 mg/kg, in 0.1 ml bolus.
  4. Group 4—i.v. administration of diclofenac, 15 mg/kg, and following 15-min injection of NAC, 40 mg/kg.

Fifteen minutes after the respective injections, the rats were either subjected to renal microcirculation flow evaluation (20 animals), or sacrificed by halothane overdose under laminar flow for ex-vivo experimentations (the remaining 20 rats).

Microcirculation blood flow
Within the kidney, the physiological oxygen requirements in the renal outer medulla are the highest, while concentrations of oxygen in the blood stream prior to reaching the outer medulla are the lowest. Thus, the magnitude of the ischaemia-induced renal damage is far more devastating in medullar than in the cortical tissue [15]. For this reason, we chose to evaluate the physiological consequences of diclofenac-induced acute renal ischaemia directly in the medullar part of the kidney, using gluteal muscle, as control peripheral tissue. This part of the study included 20 rats, five from each of the four above delineated groups. Following anaesthetic procedure and insertion of jugular vein catheter, the left kidney was exposed through a midline incision, then decapsulated and mechanically fixed. The rat renal core temperature was monitored and maintained at ~37°C with a heating lamp and intermittent dripping of warm saline and paraffin oil. The urinary bladder was incised, to prevent urine retention. A laser Doppler probe (DRT-4, Moor Instruments Ltd, England) was inserted 4.5–5 mm in depth, into the renal medulla, and reduction in blood flow brought about by severe vasoconstriction, the main outcome of ischaemia, was recorded. The second laser Doppler probe was inserted through a small incision into the right gluteus muscle. On-line recordings of the haemodynamic studies were stored, displayed and analysed using a computerized system (DRT-4, Moor Instruments Ltd, England).

Phase 3: Ex-vivo experimentations
This part of the study included 20 rats, five from each one of the four experimental groups described previously (phase 2). The rats were anaesthetized and a jugular vein catheter inserted. Saline, diclofenac and/or NAC were administrated intravenously. Following 15 min, the rats were sacrificed. Both kidneys were immediately removed under laminar flow.

(1) Extraction of cytosolic/nuclear proteins from renal tissue samples

The kidneys assigned for this procedure were immediately placed on ice. All extractions were performed using the Nuclear Extraction Kit (Chemicon, USA and Canada). The obtained extracts were immediately frozen at –80°C, to be subsequently used for H2O2 assessment.

(2) Short-term culture preparation

The kidneys assigned for this part of the study were washed in phosphate-buffered saline (PBS) buffer, pre-warmed to the room temperature. Renal cortex was separated from medulla, and each part was cut into small (~1 mm2) pieces and washed twice in PBS. Viable cortical and medullar clusters were divided into equal triplicates and seeded separately in 6-well tissue culture plates in RPMI1640 supplemented with 20% serum, at 37°C, in a humid incubator with 5% CO2. One millilitre samples of culture medium were allocated from each well 15 min after seeding and stored at –80°C for PGE2 and NO measurements.

(2) Prostaglandin E2 (PGE2), NO and H2O2 measurement

PGE2 was determined using a high-sensitivity peptide enzyme immunoassay, EIA (R&D Systems, USA), based on a competitive binding of the sample PGE2 and the fixed amount of horseradish peroxidase-labelled PGE2 to the sites of a specific monoclonal antibody, according to the manufacturer's protocol.

Quantitative H2O2 assay acquired from Biotech (Oxis International, USA) was based on the oxidation of ferrous ions (Fe2+) to ferric ions (Fe3+) by H2O2. The ferric ions formed stable coloured complexes with xylenol orange. Total amounts of the formed Fe3+-xylenol orange complexes were subsequently measured at 560 nm wavelength in ELISA reader.

Total NO concentrations were determined by a highly sensitive two-step EIA based on enzymatic conversion of nitrates present in the milieu to nitrites using nitrate reductase, and subsequent colorimetric detection of total NO by Griess reaction.



   Statistical analysis
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 Abstract
 Introduction
 Methods
 Experimental design
 Statistical analysis
Results
 Discussion
 References
 
Statistical analysis was performed using SPSS-version 13 software. Parametric data were expressed as means ± SDs. Statistical differences between the groups were evaluated by unpaired Student's t-test, and the differences within each group by paired t-test. Non-parametric data were evaluated by ANOVA. Differences yielding P-values <0.05 were considered statistically significant.



   Results
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 Abstract
 Introduction
 Methods
 Experimental design
 Statistical analysis
 Results
Discussion
 References
 
Phase 1: Renal function evaluation
Biochemical studies
The results of the biochemical evaluation of renal function parameters are summarized in Table 1 and in Figure 1A through 1C. After 3 days of water deprivation, the daily i.p. injection of diclofenac, 15 mg/kg at days 4 through 6 (group 3), brought about a significant increase in serum cystatin C, creatinine and urea, as compared with 6 days of water deprivation without diclofenac treatment (group 1: cystatin C—1.62 ± 0.28 mg/l, creatinine—0.57 ± 0.08 mg/dl and urea—56 ± 7.6 mg/dl vs group 2: 2.8 ± 0.35 mg/l, 1.2 ± 0.27 mg/dl and 208.4 ± 57.9 mg/dl, respectively, P < 0.005 for each comparison). Administration of NAC 15 min after diclofenac injection (group 4) significantly attenuated the increase of all three parameters. In this group, mean serum cystatin C was 2.2 ± 0.67 mg/l compared with 2.8 ± 0.35 mg/l in group 2, (P = 0.016), while serum creatinine and urea were 0.96 ± 0.19 and 157.6 ± 33.7 mg/dl vs 1.2 ± 0.27 and 208.4 ± 57.9 mg/dl (P = 0.020 and P = 0.028, respectively). NAC administration alone (group 2) produced no statistically significant effect on serum cystatin C, creatinine or urea levels, as compared with rats from group 1, i.e. controls subjected only to water deprivation (Figure 1A through 1C).


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  		Table 1. Biochemical and histological parameters of renal function evaluation at the end of the 6-day study protocol (n = 10 in each group)

 

Figure 1
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  		Fig. 1. (A–C) Biochemical evaluation of renal function on the sixth day of the experiment (A, cystatin C; B, creatinine; C, urea).

 
Pathological examination
Light microscopy evaluation of kidney tissue samples from control group 1 (water deprivation only) and experimental group 2 (water deprivation + NAC) revealed no acute tubular necrosis or any other signs of tubular obstruction (Table 1; Figure 2A). Microscopic examination of renal tissues from group 3 (water deprivation + diclofenac) and from group 4 (water deprivation + diclofenac + NAC) revealed glomeruli with normal appearance of the basement membrane and without signs of cellular proliferation (Figure 2B and C). On the other hand, in the same diclofenac-treated groups 3 and 4, a visible tubular damage, represented by extensive protein deposition and sloughed epithelial cells, was demonstrable. This damage was, however, found significantly attenuated in group 4 (water deprivation + diclofenac + NAC) compared with group 3 (water deprivation + diclofenac only). Tubular lumen obstruction by broad granular casts and cell fragments was present in 39 ± 11.9% of counted tubules in group 3, compared with 14.6 ± 3.8% in group 4, P = 0.002 (Figure 2A and B and Table 1). Similarly, the percentage of necrotizing tubular cells was significantly lower in group 4: only 19.8 ± 5.5% of tubular cells was found damaged, compared to 58.6 ± 14.8% in group 3, P = 0.002 (Table 1).


Figure 2
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  		Fig. 2. (A–C) Close-up photographs of microscopic fields (magnitude x40) of renal tissue samples from kidneys of rats subjected to water deprivation only (A); water deprivation + diclofenac (B) or to water deprivation + diclofenac + N-acetylcysteine (C). Light microscopy reveals glomeruli with normal appearance of the basement membrane and no signs of cellular proliferation in all groups. Tubuli containing protein and sloughed epithelial cells, while not demonstrable in group A, are evident in groups B and C. As can be seen, the magnitude of the damage is much higher in the diclofenac group (B) compared with the diclofenac + N-acetylcysteine group (C).

 
Phase 2: Physiological evaluation
Microcirculation studies
The results of laser Doppler measurements are summarized in Figure 3. Application of laser Doppler probes 15 min after i.v. diclofenac administration (group 3) revealed a significant 56.7 ± 10.27% decrease of mean renal medulla blood flow, as opposed to a non-significant 8.1 ± 9.5% decrease of the gluteus muscle blood flow serving as control. NAC treatment (group 2) resulted in a significant 31 ± 8.3% increase of the medulla blood flow and a non-significant 1.6 ± 5.2% increase of the gluteus muscle blood flow. In group 4 (water deprivation + diclofenac + NAC), 15 min after diclofenac administration, mean medulla blood flow decrease was 51 ± 12.4% of the baseline (the control group 1). Following NAC administration, mean medulla blood flow decrease was only 14 ± 3.39% of the baseline, constituting a net 37 ± 8.9% improvement (P < 0.001). No significant effect on the control, gluteus muscle blood flow, was noticeable in any of the experimental groups.


Figure 3
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  		Fig. 3. (A and B) Changes in intrarenal blood flow recorded by laser Doppler in renal medullae (A) vs control gluteal muscle (B), expressed as percent from the baseline.

 
Phase 3: Ex-vivo experimentations
The results of the ex-vivo experimentations are summarized in Table 2 and in Figures 4 and 5. PGE2 release by cortices as well as by medullae from kidneys of diclofenac pretreated group 2 was significantly inhibited (in cortices 19.7 ± 1.5 pmol/ml compared with control 148.5 ± 8.8 pmol/ml, P < 0.001; in medullae 66.6 ± 7.0 pmol/ml compared with control 146.6 ± 12.0 pmol/ml, P < 0.0001). NAC injection following diclofenac treatment prevented the drop of PGE2 synthesis (144.7 ± 10.4 pmol/ml in cortices and 148.5 ± 7.3 pmol/ml in medullae, P < 0.001 compared with the respective diclofenac group values). In fact, in cultures from animals injected by NAC following diclofenac pretreatment, PGE2 release was statistically not different from controls (group 1). Furthermore, NAC administration to animals not receiving diclofenac resulted in significant augmentation of PGE2 release above the normal control values (581.6 ± 142.1 pmol/ml in cortices, 503.5 ± 109.4 pmol/ml in medullae, P < 0.0001 for both comparisons with the respective controls. See Table 2 and Figure 4).


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  		Table 2. The results of the ex-vivo experiments

 

Figure 4
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  		Fig. 4. The results of ex-vivo experiments: changes in PGE2 synthesis. PGE2, prostaglandin E2. #Significantly different compared to the values within the group. *Significantly different compared to the respective values in the rest of the groups.

 

Figure 5
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  		Fig. 5. The results of ex-vivo experiments: changes in H2O2 synthesis. H2O2, hydrogen peroxide. #Significantly different compared to the values within the group. *Significantly different compared to the respective values in the rest of the groups.

 
NO synthesis in the NAC-treated group was found moderately increased in the cortex (294.8 ± 74.5 µM/ml compared with control 255.7 ± 5.25 µM/ml), the difference, however, not reaching statistical significance. In contrast, NO synthesis in medullae from NAC-treated rats significantly exceeded the control values (301.7 ± 81.0 µM/ml compared with control 244.5 ± 28.3 µM/ml, P = 0.01). The NO release in both diclofenac-receiving groups was not significantly different from controls (group 1).

H2O2 concentrations were significantly increased in cytosolic extracts from diclofenac pretreated rats compared with their respective controls or, for that matter, other experimental groups (0.033 ± 0.007 vs control 0.023 ± 0.001 µM/ml and vs NAC + diclofenac 0.028 ± 0.003 µM/ml in cortices and 0.032 ± 0.004 vs control 0.025 ± 0.001 µM/ml and vs NAC + diclofenac 0.025 ± 0.001 µM/ml in medullae, P < 0.001 in all comparisons. See Table 2 and Figure 5).



   Discussion
Top
 Abstract
 Introduction
 Methods
 Experimental design
 Statistical analysis
 Results
 Discussion
References
 
The present study was designed to investigate a possible renoprotective effect of NAC in a novel rat model of NSAID-induced acute renal failure. The experimental protocol consisted of diclofenac administration combined with prolonged water deprivation. In this model, laser Doppler measurements demonstrated severe diclofenac-induced intrarenal vasoconstriction. Pathological examination revealed profound acute tubular necrosis (ATN). Biochemical analyses demonstrated elevated cystatin C, urea and creatinine along with a significant decrease in PGE2 release. NAC administration to animals similarly treated with diclofenac, resulted in a substantial decrease of serum cystatin C, creatinine, urea levels and significant attenuation of renal tubular damage, as evidenced by pathological examination. Furthermore, deleterious physiological consequences of diclofenac administration, represented by a steep drop in renal medullar blood flow rate, as compared to unaltered blood flow in control gluteal muscle, were significantly attenuated in animals treated by NAC. Most surprisingly, the results of the present study demonstrated that operating mechanisms regulating these NAC-induced renoprotective effects involved, in addition to well-expected antioxidant effects, augmentation of PGE2 release by renal tissue.

Thus far, beneficial effects of NAC treatment have been reported in several models of toxic and ischaemic injuries. The effects proved to be cell/organ specific, thus, it is not surprising that the underlying mechanisms are diverse. Some studies report NAC-induced decrease in oxidative stress via degradation of NO [9,16,17], while others demonstrate NAC-stimulated increase in NO production [9,16,18], improvement of NO tolerance [19] and, most importantly, amplification of NO-induced vasodilatation [20–23]. In a number of studies NO synthesis was, indeed, found enhanced in NAC-induced vasorelaxation [24]. The latter observation seems quite explicable, since elevated NO triggers augmentation of c-GMP production, thus decreasing availability of cytosolic-free calcium and altering the calcium-dependent signalling processes, including vasoconstriction [24–26]. In the present investigation, a moderate rise in NO synthesis by cortices, and more so by medullae, was observed in primary cultures of kidneys from rats treated solely by NAC. However, no increased NO production was evident in cultures from animals receiving NAC after diclofenac treatment. One must thus conclude that in this experimental setting the NAC-induced attenuation of deleterious physiological, pathological and biochemical consequences of diclofenac-induced ischaemia, was not mediated by NO.

Examination of cytosolic extracts from fresh cortical and medullar renal tissue portions revealed increased amounts of ROS, as represented by elevated concentrations of H2O2, in kidneys from diclofenac-treated animals. Excessive ROS production is strongly associated with various oxidative stress conditions, including the ischaemia-induced oxidative stress [27,28]. In particular, H2O2 has been demonstrated to serve as a second messenger triggering augmented synthesis of cyclooxygenase 2 (COX-2) and arachidonic acid, the operating mechanism being activation of NF-kB, with subsequent translocation of the latter to the nucleus [29]. Evidently, in the present study increased H2O2 production observed in fresh renal tissue samples from diclofenac-pretreated rats was terminated following NAC administration. Apparently, though not surprisingly, in this experimental setup NAC acted as an antioxidant exerting, at least in part, its renoprotective effects by decreasing the amounts of intrarenal H2O2 back to normal control values.

Prostaglandins are known to exert diverse physiologic effects, from protective to cytotoxic, and their modes of action are cell and organ specific. With respect to the kidney, strong renoprotective role of prostaglandins has been unequivocally proven in both in vivo and in vitro models of renal injury [30–32]. This is specifically true for PGE2, the major metabolite of arachidonic acid within the kidney [30,31]. Therefore, the most important outcome of the present study appears to be the finding that NAC administration restored renal PGE2 release in a model of NSAID-induced nephropathy. PGE2 synthesis, as expected, dropped significantly in kidneys of rats treated by diclofenac (about 3-fold in medullae and about 8-fold in cortices). However, in renal tissues from animals that received NAC following diclofenac treatment, PGE2 production increased back to the levels observed in control rats, which had been subjected only to water deprivation. Moreover, PGE2 release dramatically exceeded the control values both in cortices and in medullae from kidneys of rats treated solely by NAC.

Metabolic breakup of arachidonic acid molecule, culminating in PGE2 formation, is a process involving transformation of reduced glutathione (GSH) to its oxidized form (GSSG). By this, GSH serves as electron donor in the process of conversion of PGG2 to PGH2. Furthermore, in conversion of PGH2 to PGE2, oxidation of GSH to GSSG serves as a cofactor of PGE2 synthase [33,34]. NAC is a precursor of the GSH molecule [35]. As already mentioned, ischaemia-injured renal tissue contains excessive amounts of arachidonic acid and COX. Combined with surplus of glutathione provided by NAC administration, this would inevitably result in an outburst of PGE2 release as, indeed, happened in kidneys of animals subjected to prolonged water deprivation prior to NAC administration. In turn, in kidneys from diclofenac pretreated rats, conversion of arachidonic acid to PGE2 was significantly inhibited at the stage of its breakup to PGG2. In this situation, wherein less PGG2 was available for conversion to PGH2 and subsequently to PGE2, NAC administration restored PGE2 synthesis close to the levels found in normal control kidneys. To the best of our knowledge, this is the first study demonstrating that renoprotective effect of NAC treatment is mediated, at least in part, via augmentation of PGE2 production within the kidney.

Our study provided the following conclusions:

  1. NSAID treatment combined with water deprivation provided an easily available, reliable and convenient model of rat renal insufficiency manifested by severe vasoconstriction, drop of intrarenal blood flow, extensive acute tubular necrosis and steep deterioration of vital renal functions.
  2. In this experimental model, NAC successfully attenuated the deterioration of renal function by inducing renal vasodilatation, decreasing oxidative stress via inhibition of intrarenal ROS content and, most importantly, restoration of intrarenal PGE2 release back to the normal levels.

Conflict of interest statement. None declared.



   References
Top
 Abstract
 Introduction
 Methods
 Experimental design
 Statistical analysis
 Results
 Discussion
 References
 

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Received for publication: 27.11.06
Accepted in revised form: 9. 2.07


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