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Cardiovascular Research 2004 61(3):630-643; doi:10.1016/j.cardiores.2003.10.024
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Copyright © 2003, European Society of Cardiology

The cyclopentenone prostaglandin 15-deoxy-{Delta}12,14-prostaglandin J2 ameliorates ischemic acute renal failure

Prabal Kumar Chatterjeea,1, Nimesh S.A Patela, Salvatore Cuzzocreab, Paul A.J Brownc, Keith N Stewartd, Helder Mota-Filipee, Domenico Brittif, Wolfgang Eberhardtg, Josef Pfeilschifterg and Christoph Thiemermann*,a

aDepartment of Experimental Medicine, Nephrology and Critical Care, William Harvey Research Institute, Queen Mary-University of London, Charterhouse Square, London EC1M 6BQ, UK
bDepartment of Clinical and Experimental Medicine and Pharmacology, University of Messina, Messina, Italy
cDepartment of Pathology, University of Aberdeen, Aberdeen, UK
dDepartment of Medicine and Therapeutics, University of Aberdeen, Aberdeen, UK
eLaboratory of Pharmacology, University of Lisbon, Lisbon, Portugal
fDepartment of Veterinary and Agricultural Science, University of Teramo, Teramo, Italy
gPharmazentrum Frankfurt, Klinikum der Johann Wolfgang Goethe-Universitat, Frankfurt am Main, Germany

* Corresponding author. Tel.: +44-20-7882-6118; fax: +44-20-7251-1685. c.thiemermann{at}qmul.ac.uk

1 Present address: Department of Pharmacology, School of Pharmacy and Biomolecular Sciences, University of Brighton, Moulsecoomb, Brighton BN2 4GJ, UK.

Received 20 May 2003; revised 5 September 2003; accepted 15 October 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Here we investigate the effects of the endogenous prostaglandin D2 metabolite 15-deoxy-{Delta}12,14-prostaglandin J2, on the renal dysfunction and injury caused by ischemia/reperfusion of the kidney. Methods: Male Wistar rats, subjected to bilateral renal ischemia for 45 min followed by reperfusion for up to 48 h, were administered 15-deoxy-{Delta}12,14-prostaglandin J2 (1 mg/kg, intravenously) 5 min prior to and again after 3 or 12 h reperfusion. Results: 15-deoxy-{Delta}12,14-prostaglandin J2 significantly reduced (i) renal and tubular dysfunction (serum urea and creatinine levels, creatinine clearance, fractional excretion of Na+ (FENa)), (ii) tubular and reperfusion-injury (urinary N-acetyl-β-D-glucosaminidase, aspartate aminotransferase (ASP) and {gamma}-glutamyltransferase ({gamma}-GT)) and (iii) histological evidence of renal injury. 15-deoxy-{Delta}12,14-prostaglandin J2 also improved renal function (plasma creatinine levels) and reduced the histological signs of renal injury (after 48 h reperfusion). Administration of 15-deoxy-{Delta}12,14-prostaglandin J2 markedly reduced the expression of inducible nitric oxide synthase (iNOS) and intercellular adhesion molecule-1 during reperfusion (determined using immunohistochemistry). Immunohistochemical analysis of p65 translocation and Western blot analysis of I{kappa}B-{alpha} degradation revealed that 15-deoxy-{Delta}12,14-prostaglandin J2 inhibited the activation of nuclear factor (NF)-{kappa}B in renal cells. Subsequently, 15d-PGJ2 was able to significantly reduce nitric oxide production during renal ischemia/reperfusion and by primary cultures of rat proximal tubular (PT) cells incubated with interferon-{gamma} and bacterial lipopolysaccharide (LPS) in combination. Conclusions: We demonstrate here, for the first time, that 15-deoxy-{Delta}12,14-prostaglandin J2 significantly reduces renal ischemia/reperfusion-injury via reduction of pro-inflammatory gene expression during reperfusion subsequent to the inhibition of the activation of NF-{kappa}B.

KEYWORDS Renal function; Ischemia; Reperfusion; Prostaglandins; Rat


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Renal ischemia (I) is a major cause of acute renal failure (ARF), which despite significant advances in critical care medicine, remains a major clinical problem, producing grave morbidity and mortality that has not decreased significantly over the last 50 years [1]. The prognosis of ARF is complicated by the fact that reperfusion (R), although essential for the survival of ischemic renal tissue, causes additional damage (reperfusion-injury) [2]. To date, no specific therapy has been shown to alter the course or outcome of ischemic ARF [3] and many reasons have been proposed for the inability of these interventions to improve the prognosis of ARF, including an incomplete understanding of the pathophysiology underlying the development of ARF [1,3].

Several members of the cyclopentenone family of prostaglandins (PGs) possess anti-neoplastic, anti-viral and anti-inflammatory properties [4]. PGJ2 is formed by dehydration within the cyclopentane ring of the endogenous prostaglandin, PGD2, and is further metabolized to yield {Delta}12-PGJ2 and 15-deoxy-{Delta}12,14-PGJ2 (15d-PGJ2) [4] (Fig. 1). 15d-PGJ2 is a high affinity natural ligand for peroxisome proliferator-activated receptor (PPAR)-{gamma}; a nuclear hormone receptor belonging to the superfamily of ligand-dependent transcription factors related to retinoid, steroid and thyroid hormone receptors, which regulate gene expression by heterodimerising with the retinoid X receptor (RXR) [5]. Activation of PPAR-{gamma} results in trans-repression of the expression of pro-inflammatory mediators at the transcriptional level by inhibition of nuclear factor (NF)-{kappa}B, STAT-1 and activator protein-1 (AP-1) [6]. Specifically, 15d-PGJ2 attenuates the activation of NF-{kappa}B by preventing the phosphorylation of its inhibitor protein by IK kinase (IKK) [7]. It is now widely accepted that 15d-PGJ2 attenuates the NF-{kappa}B-mediated transcriptional activation of many pro-inflammatory genes and mediators by mechanisms which are both dependent and independent of PPAR interactions [4–7], including inducible nitric oxide (NO) synthase (iNOS) [6,8], cyclooxygenase-2 (COX-2) [9], intercellular cell adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1 [10], tumor necrosis factor (TNF)-{alpha} and interleukin (IL)-12 [8]. Recently is has been reported that 15d-PGJ2 can specifically induce hemeoxygenase (HO)-1 in neuronal cells and cardiac myocytes [11,12]. In vivo, there is now good evidence that 15d-PGJ2 can provide beneficial actions against different forms of acute and chronic inflammation via substantial reduction of the activation of NF-{kappa}B, expression of pro-inflammatory genes and cytokine formation [13,14].


Figure 1
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  		Fig. 1 Chemical structure of the cyclopentenone prostaglandin, 15d-PGJ2.

 
Several in vitro renal studies using human, rat and murine mesangial cells have shown that both PGJ2 and 15d-PGJ2 possess anti-inflammatory properties, including the ability to inhibit NF-{kappa}B activation by IL-1β, subsequently reducing the expression of iNOS, COX-2 and monocyte chemoattractant protein-1 (MCP-1) [15–17]. Recently, 15d-PGJ2 has also been reported to reduce pro-inflammatory IL-1β-induced intracellular lipid accumulation in mesangial cells [18]. 15d-PGJ2 can also activate of MAP kinase independent of interaction at the PPAR-{gamma} [19] and regulate mesangial cell proliferation and death [20]. Interestingly, PGJ2 production is reduced in mice suffering immune complex glomerulonephritis [21]. Overall, manipulation of these particular PGs may be relevant to the treatment of progressive renal disease [20].

PPARs are differentially expressed within the kidney [22,23]. PPAR-{gamma} expression is abundant in the renal inner medulla where it is localized to the collecting duct and interstitial cells [23] and endogenous PPAR-{gamma} activity is also associated with the glomerular and medullary microvasculature [22] as well as in the renal mesangium [24–26]. However, PPAR-{gamma} expression is up-regulated by glomerular injury suggesting a possible renoprotective role for PPAR-{gamma} ligands [26]. The role of the PPAR-{gamma} both in renal and cardiovascular disease has been reviewed [27,28]. Recently it has been discovered that synthetic ligands of the PPAR-{gamma} (rosiglitazone and ciglitazone) can reduce the renal injury and dysfunction caused by I/R of the rat kidney [29] and that troglitazone can protect against non-diabetic glomerulosclerosis in the rat [30]. However, the use of these thiazolidinediones as pharmacological agents has some drawbacks, including incidences of toxicity (e.g. severe liver damage in patients administered troglitazone) and possible hepatotoxicity of rosiglitazone and pioglitazone [31], risk of carcinogenicity [32], absence of PPAR-{gamma}-independent beneficial anti-inflammatory effects [11,33] and significant loss of potency at the nuclear receptor in whole cell preparations [34]. Recently, acute treatment with troglitazone has been shown to increase susceptibility to ventricular fibrillation during myocardial I/R in pigs [35].

There are currently only two studies investigating the effects of 15d-PGJ2 on organ injury caused by I/R in vivo in which we recently reported that 15d-PGJ2 could provide protection against myocardial infarction and gut I/R injury [11,36]. Surprisingly, even with evidence from many in vitro studies that 15d-PGJ2 provides renoprotective effects in models of progressive renal disease, to our knowledge there are no studies which have investigated the beneficial actions of the natural PPAR-{gamma} ligand 15d-PGJ2 on renal I/R injury (and associated ischemic ARF) in vivo. To achieve this goal, we have investigated the effects of 15d-PGJ2 on the renal injury and dysfunction caused by bilateral clamping of both renal arteries (for 45 min) followed by reperfusion (for up to 48 h) in the rat. In order to gain a better insight into the mechanism(s) of action of 15d-PGJ2, we have also measured its effects on (i) the expression of iNOS and NO production in vivo and in vitro by primary cultures of rat proximal tubular (PT) cells, (ii) the expression of adhesion molecule ICAM-1 and (iii) the activation of the transcription factor NF-{kappa}B using a combination of immunohistochemical and Western blot analysis.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 In vivo procedures
2.1.1 Renal ischemia/reperfusion (short-term model)
The in vivo investigations described in this study conformed to both the UK Home Office Guidance in the Operation of the Animals (Scientific Procedures) Act 1986, published by Her Majesty's Stationery Office, London, UK and the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Fifty-two male Wistar rats (Tuck, Rayleigh, Essex, UK) weighing 215–305 g and receiving a standard diet and water ad libitum, were anesthetized with sodium thiopentone (Intraval® Sodium, 120 mg/kg intraperitoneally; Rhone Merieux Ltd., Essex, UK) and anesthesia was maintained by supplementary infusions of sodium thiopentone. Sodium thiopentone was chosen as the anesthesia in the short-term model due to its long duration of action, i.e. administration of one dose at 120 mg/kg provided that anesthesia lasting was for 4–6 h.

Rats were subjected to Sham-operation or bilateral renal ischemia for 45 min followed by reperfusion for 6 h as described previously [37,38] after being allocated into the following five groups: (i) I/R+Saline group, rats were subjected to 45 min renal ischemia followed by reperfusion for 6 h (n=12); (ii) I/R+15d-PGJ2 group, same as the I/R+Saline group but administered 15d-PGJ2 (1 mg/kg intravenous bolus) 5 min prior to commencement of reperfusion and again after 3 h reperfusion (n=12); (iii) I/R+Vehicle group, same as the I/R+Saline group but which received 10% (v/v) dimethylsulfoxide (DMSO, vehicle for 15d-PGJ2, 2 ml/kg intravenous bolus) 5 min prior to commencing reperfusion and again after 3 h reperfusion (n=12); (iv) Sham+Saline group, rats were subjected to identical surgical procedures as the above groups except for renal I/R, and were maintained under anesthesia for the duration of the experiment (45 min+6 h, n=12); (v) Sham+15d-PGJ2 group, identical to Sham-operated rats except for the administration of 15d-PGJ2 (1 mg/kg intravenous bolus) 5 min prior to commencing reperfusion and again after 3 h reperfusion (n=4). All rats received an intravenous saline infusion (2 ml/kg per h) throughout the I/R period.

2.1.2 Renal ischemia/reperfusion-injury (long-term model)
To assess the effects of 15d-PGJ2 administration on renal function and injury in the later stages of ischemic ARF, further studies were performed using 30 male Wistar rats (Charles River, Milan, Italy) weighing 170–260 g. Rats were allowed access to food and water ad libitum and were cared for in compliance with Italian regulations on protection of animals used for experimental and other scientific purposes (DM 116192), as well as with the European Economic Community regulations (OJ of EC L358/1 12/18/1986) and the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

Rats were anesthetized using chloral hydrate (400 mg/kg, intraperitoneal injection) and core body temperature maintained at 37 °C using a homoeothermic blanket. Chloral hydrate was chosen as the anesthesia for the long-term model as, at a dose of 400 mg/kg, it provided a short anesthesia from which the rats recovered after approximately 1–2 h. Rats were then divided into the following four groups before being subjected to either Sham-operation or bilateral renal ischemia for 45 min followed by reperfusion for up to 48 h as previously described [38]: (i) I/R+Saline group, rats were subjected to renal ischemia (45 min) followed by reperfusion for 48 h and were administered 2 ml/kg saline as an intraperitoneal bolus 30 min before Sham-operation and after 12 h (n=10); (ii) I/R+Vehicle group, rats were subjected to renal ischemia (45 min) followed by reperfusion for 48 h and were administered 2 ml/kg 10% (v/v) DMSO/saline (vehicle for 15d-PGJ2), intraperitoneally 30 min before Sham-operation and after 12 h (n=5); (iii) I/R+15d-PGJ2 group, rats subjected to renal ischemia (45 min) followed by reperfusion for 48 h rats were administered 15d-PGJ2 (1 mg/kg intraperitoneal bolus) 30 min prior to commencement of I/R and after 12 h reperfusion (n=10); (iv) Sham+Saline group, rats were subjected to identical surgical procedures except for renal I/R (n=5).

After performing flank incisions, rats belonging to the I/R groups were subjected to bilateral renal ischemia for 45 min during which the renal arteries and veins were occluded using microaneurysm clips. After the renal clips were removed, the kidneys were observed for a further 5 min to ensure reflow after which 1 ml saline at 37 °C was injected into the abdomen and the incision was sutured in two layers. Rats were then returned to their cages, allowed to recover from anesthesia and observed for 48 h. Sham-operated rats underwent identical surgical procedures to I/R rats except for the application of microaneurysm clips.

The time course and doses of 15d-PGJ2 used in both these models of renal I/R were based on those previously shown by us, and others, to provide beneficial actions against I/R injury or inflammation in the rat and mouse [11,13,14,39].

2.2 Measurement of biochemical parameters
After 6 h (short-term model) or 24 and 48 h reperfusion (long-term model), blood samples were collected via the carotid artery or tail vein into tubes containing serum gel. The samples were centrifuged (6000 rpm for 3 min) to separate serum from which biochemical parameters were measured (Vetlab Services, Sussex, UK or by DB, University of Teramo, Italy). Serum urea and creatinine levels were used as an indicator of renal function [37,38]. Aspartate aminotransferase (AST) and {gamma}-glutamyltransferase ({gamma}-GT), enzymes both located in the PT, were used as indicators of reperfusion-injury [37,38]. Urine samples were collected throughout the reperfusion period for 20 min intervals and the volume of urine produced recorded. Urine concentrations of creatinine and Na+ were measured (Vetlab Services) and used in conjunction with serum creatinine and Na+ levels to estimate creatinine clearance (used as an indicator of glomerular filtration rate) and fractional excretion of Na+ (FENa), using standard formulae, which were used as indicators of renal function [37,38]. Urinary N-acetyl-β-D-glucosaminidase (NAG) activity, a specific indicator of tubular injury [37,38], was also measured (Clinica Medica é Diagnóstico Dr. Joaquim Chaves, Lisbon, Portugal). Urinary NAG activity was standardized using urinary levels of creatinine to take account of urinary flow rate and were expressed as iu/mmol creatinine [38].

2.3 Histological evaluation
Renal sections were prepared as described previously [38] and used for histological assessment of I/R injury. Briefly, 100 intersections were examined and a score from 0 to 3 was given for each tubular profile involving an intersection: 0: normal histology; 1: tubular cell swelling, brush border loss, nuclear condensation, with up to 1/3 of tubular profile showing nuclear loss; 2: as for score 1, but greater than 1/3 and less than 2/3 of tubular profile showing nuclear loss and 3: greater than 2/3 of tubular profile shows nuclear loss. The total score for each kidney was calculated by addition of all 100 scores (maximum score 300).

2.4 Immunohistochemical localization of iNOS, ICAM-1 and p65
Evidence of iNOS and ICAM-1 expression was determined using immunohistochemistry as previously described [11,13,14]. Localization of p65 (Rel A) was used as an indicator of NF-{kappa}B activation in vivo as previously described [40] where its presence in the cytoplasm indicated that the NF-{kappa}B heterotrimeric complex was still in its "dormant" or inactive form in association with p50 and I{kappa}B in the form of an ‘I{kappa}B–NF-{kappa}B complex’. In contrast, localization of p65 to the nucleus indicated that the NF-{kappa}B (p50 and p65) had translocated from the cytoplasm into the nucleus from where it could activate transcription of NF-{kappa}B-dependent genes.

Renal sections were scored for the presence of p65 in the nucleus of renal cells. Briefly, 25 intersections were examined for each kidney and evaluated for the presence of p65-positive staining in the nucleus of cells. The final score obtained was expressed as percentage (%) NF-{kappa}B-positive nuclei.

2.5 Western blot analysis of I{kappa}B{alpha} degradation
Rat kidneys were removed and immediately frozen in liquid N2 and stored at –70 °C until preparation of cytosolic fractions as described previously [41]. The protein content in the cytosolic fractions of the homogenates was determined using the Bradford assay as described previously [41]. Western blot analysis of I{kappa}B{alpha} proteins was performed as described previously [42]. Immunocomplexes were visualized using enhanced chemiluminescence and the intensity of the bands evaluated using a GS-700 imaging densitometer (Bio-Rad, Munich, Germany) and expressed graphically as relative densitometric units (% Sham+Saline control).

2.6 Isolation and culture of rat proximal tubular cells
Pure populations of rat PT cells were isolated from kidneys obtained from 12 male Wistar rats (250–350 g) using collagenase digestion, differential sieving and Percoll density centrifugation as described previously [43]. PT cells were grown in Minimum Essential Medium (MEM) containing 10% (v/v) FCS in a humidified 5% CO2/95% air atmosphere at 37 °C and the medium was changed every 48 h until the cells reached confluence.

2.7 In vitro experimental design
Confluent primary cultures of rat PT cells were incubated with 100 iu/ml interferon (IFN)-{gamma} and 10 µg/ml bacterial lipopolysaccharide (LPS) in combination for 24 h in the absence or presence of increasing concentrations of 15d-PGJ2 (1–100 nM). The range of 15d-PGJ2 concentrations used was determined from previous studies that have shown 15d-PGJ2 to effectively inhibit NF-{kappa}B activation, iNOS expression and NO production by renal cells [16,17,20]. 15d-PGJ2 was added to incubation medium 10 min before the addition of IFN-{gamma} and LPS and again after 12 h incubation. Upon completion of incubations (24 h after the addition of IFN-{gamma}/LPS), NO levels in incubation medium was measured as described below.

2.8 Measurement of nitrite/nitrate concentrations
Concentrations of NO2 and nitrate NO3, the primary oxidation products of NO subsequent to reaction with oxygen, were measured in samples of rat plasma and incubation medium from PT cells and used as an indicator of NO synthesis after enzymatic conversion of NO3 to NO2 using nitrate reductase as previously described [38,39].

2.9 Materials
Unless otherwise stated, all compounds used in this study were purchased from Sigma-Aldrich Company Ltd. (Poole, Dorset, UK). All stock solutions were prepared using non-pyrogenic saline [0.9% (w/v) NaCl; Baxter Healthcare Ltd., Thetford, Norfolk, UK]. 15d-PGJ2 was purchased from Calbiochem (Beeston, Nottingham, UK). LPS was obtained from E. coli serotype 0.127:B8 (Sigma).

2.10 Statistical analysis
All values described in the text and figures are expressed as mean±standard error of the mean (S.E.M.). Statistical analysis was carried out using GraphPad Prism 3.02/Instat 1.1 (GraphPad Software, San Diego, CA, USA). Data were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett's post hoc test and a P value of less than 0.05 was considered to be significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
The mean±S.E.M. for the weights of the rats used in the short-term study was 267±6 g (n=52) and for the long-term study was 217±4 g (n=30). On comparison with Sham animals, renal I/R produced significant increases in serum, urinary and histological markers of renal dysfunction and injury as described in detail below (Figs. 1–5GoGoGoGo). When compared to rats used as Shams, renal I/R (with or without administration of 15d-PGJ2 or its vehicle), did not have a significant effect on urine flow (0.017±0.001 ml/min, n=52).


Figure 2
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  		Fig. 2 Effect of 15d-PGJ2 on renal dysfunction caused by I/R; alterations in (A) serum urea, (B) serum creatinine concentrations, (C) creatinine clearance and (D) FENa+ subsequent to renal I/R after administration of 15d-PGJ2. * P<0.05 vs. I/R+Vehicle group; {blacksquare} P<0.05 vs. Sham+Saline group.

 

Figure 3
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  		Fig. 3 Effect of 15d-PGJ2 on renal and reperfusion-injury caused by renal I/R; alterations in (A) urinary N-acetyl-β-D-glucosaminidase levels; (B) total severity score, (out of a total score of 300); (C) serum levels of AST and (D) serum levels of {gamma}-GT subsequent to renal I/R after administration of 15d-PGJ2. * P<0.05 vs. I/R+Vehicle group or I/R-only group; {blacksquare} P<0.05 vs. Sham+Saline group.

 

Figure 4
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  		Fig. 4 Effect of 15d-PGJ2 on the histological signs of renal I/R injury. Renal sections obtained from a Sham-operated rat (A, D) are compared to that taken from a rat subjected to renal I/R (B, E). Renal sections taken from rats subjected to renal I/R after administration of 15d-PGJ2 are also represented (C, F). Rats that underwent renal I/R demonstrated the recognized features of renal injury (E). Specifically, this included glomerular degeneration (GD), tubular dilatation (TD), tubular congestion (TC) and the presence of eosinophilia (E). Hemotoxylin and eosin, original magnification x40 (A, B, C), x125 (D, E, F); figures are representative of at least three experiments performed on different days; ctx: cortex, mda: medulla.

 

Figure 5
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  		Fig. 5 Effect of 15d-PGJ2 on the course of renal ischemia/reperfusion-injury. (A) Serum creatinine levels were measured 24 and 48 h after renal ischemia in rats subjected to I/R and which were administered either saline (bullet, I/R+Saline group) or 15d-PGJ2 ({circ}, I/R+15d-PGJ2) 30 min prior to ischemia and after 12 h reperfusion. Data represent mean±S.E.M. for N observations, * P<0.05 vs. I/R+Saline group. Histological examination of kidney sections prepared from rats subjected to ischemia followed by reperfusion for 48 h demonstrated recognized features of renal injury (B), including glomerular degeneration (GD), tubular dilatation (TD), tubular congestion (TC) and the presence of eosinophilia (E). Rat subjected to I/R but which were administered 15d-PGJ2 displayed reduction in renal injury (C). Hemotoxylin and eosin, original magnification x125, figures are representative of at least three experiments performed on different days.

 
3.1 Effect of 15d-PGJ2 on renal dysfunction mediated by I/R
Animals, which underwent renal I/R, exhibited a significant increase in serum levels of urea and creatinine compared to Sham-operated animals (Fig. 2A,B) suggesting a significant degree of renal dysfunction. This was reflected by a significant reduction in creatinine clearance in rats subjected to I/R (Fig. 2C). Compared to control animals (I/R+Vehicle group), administration of 15d-PGJ2 produced a significant reduction in serum levels of urea and creatinine (Fig. 2A,B) and a significant improvement of creatinine clearance (Fig. 2C).

Renal I/R produced significant increases in FENa (Fig. 2D) suggesting a marked increase in tubular dysfunction. On comparison with FENa measured in control (I/R+Vehicle group) rats, administration of 15d-PGJ2 produced a significant reduction in FENa (Fig. 2D) suggesting improvement in tubular function.

3.2 Effect of 15d-PGJ2 on renal injury caused by renal I/R
Rats subjected to renal I/R produced significant increases in urinary NAG (Fig. 3A) suggesting a marked increase in tubular injury. Administration of 15d-PGJ2 significantly reduced urinary NAG levels associated with renal I/R (Fig. 3A) suggesting reduction of tubular injury. This renoprotective effect of 15d-PGJ2 was reflected in the histological scoring of renal injury. On comparison with the total severity score measured from kidneys obtained from Sham-operated animals, renal I/R produced a significant increase in total severity score (Fig. 3B). Administration of 15d-PGJ2 significantly reduced total severity score when compared to that obtained from rats subjected to renal I/R only (Fig. 3B).

When compared with values obtained from Sham-operated animals, renal I/R produced significant increases in serum concentrations of AST and {gamma}-GT (suggesting significant reperfusion-injury, Fig. 3C,D). Administration of 15d-PGJ2 caused a significant reduction in both serum AST and {gamma}-GT levels indicating a reduction in reperfusion-injury (Fig. 3C,D).

3.3 Effects of 15d-PGJ2 on the histological features of renal I/R
When compared with the renal histology observed in kidneys taken from Sham-operated rats (Fig. 4A,D), rats that underwent renal I/R demonstrated recognized features of renal injury (Fig. 4B,E). Specifically, this included degeneration of glomerular structure (Fig. 4E: GD), tubular dilatation (Fig. 4E: TD), luminal congestion (Fig. 4E: TC) and the presence of eosinophilia (Fig. 4E: E). In contrast, renal sections obtained from rats subjected to renal I/R which were administered 15d-PGJ2, demonstrated marked reduction of the histological features of renal injury on comparison with kidneys obtained from rats subjected to I/R only (Fig. 4C,F).

3.4 Effect of 15d-PGJ2 on the course of renal ischemia/reperfusion-injury
In the long-term model of ischemic ARF, renal function (assessed by serum creatinine levels) was significantly improved after administration of 15d-PGJ2 to rats subjected to renal ischemia followed by reperfusion for 24 and 48 h (Fig. 5A). Histological examination of kidney sections prepared from rats subjected to renal I/R and treated with vehicle for 15d-PGJ2 (Fig. 5B) demonstrated greater renal injury than kidneys obtained from Sham-operated rats (Fig. 5C). Rats subjected to I/R but which were administered 15d-PGJ2 demonstrated marked reductions in renal injury (Fig. 5D).

3.5 Effects of 15d-PGJ2 on iNOS expression and NO production in vivo and in vitro
When compared to kidney sections obtained from Sham-operated rats (Fig. 6A), immunohistochemical analysis of sections obtained from rats subjected to renal I/R revealed positive staining for iNOS (Fig. 6B). In contrast, reduced staining was observed in the kidney sections obtained from rats subjected to renal which were administered 15d-PGJ2 (Fig. 6C).


Figure 6
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  		Fig. 6 Effect of 15d-PGJ2 on iNOS expression and NO production; kidney sections were incubated overnight at 4 °C with primary monoclonal antibody directed at iNOS (1:500 [v/v] in PBS). Specific labeling of antigen–antibody complex was visualized using an avidin–biotin peroxidase complex immunoperoxidase technique using chromogen diaminobenzidine. (A) Sham-operation, (B) I/R-only, (C) I/R with administration of 15d-PGJ2. Magnification: x125. Staining for iNOS was localized to the PT in renal sections prepared from rats subjected to I/R (arrows, Fig. 7B). (D) Nitrite/nitrate concentrations in plasma obtained from rats subjected to Sham-operation or subjected to I/R and which were administered either saline or 15d-PGJ2. (E) Nitrite/nitrate levels measured in incubation medium collected from rat PT cells incubated for 24 h with IFN-{gamma} and LPS in the presence or absence of increasing concentrations of 15d-PGJ2. * P<0.05 vs. I/R+Vehicle or cytokine-only group, {blacksquare} P<0.05 vs. Sham+Saline or untreated group.

 
Renal I/R resulted in a significant increase in the plasma levels of NO2/NO3 (an indicator of the formation of NO) on comparison with values obtained from the plasma of Sham-operated animals (Fig. 6D). Increased plasma NO2/NO3 levels caused by renal I/R were significantly reduced by administration of 15d-PGJ2 to rats (Fig. 6D). Incubation of primary cultures of rat PT cells with IFN-{gamma} and LPS in combination for 24 h produced a significant increase in NO production (Fig. 6E). Incubation of PT cells with IFN-{gamma} and LPS in the presence of increasing concentrations of 15d-PGJ2 (1–100 nM) resulted in a significant, concentration-dependent inhibition of NO production (Fig. 6E).

3.6 Effect of 15d-PGJ2 on ICAM-1 expression subsequent to renal I/R
Kidneys obtained from rats subjected to I/R demonstrated marked staining for ICAM-1 when compared with kidneys obtained from Sham-operated rats (Fig. 7A,B), suggesting adhesion molecule expression during reperfusion. Kidneys obtained from rats administered 15d-PGJ2 demonstrated markedly reduced staining for ICAM-1 (Fig. 7C) when compared with kidneys obtained from rats subjected to renal I/R only, suggesting a reduction in the expression of this adhesion molecule during reperfusion.


Figure 7
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  		Fig. 7 Immunohistochemical localization of ICAM-1 in rat kidney sections; (A) Sham-operated group (B) I/R-only group, (C) I/R with administration of 15d-PGJ2. Sections were incubated overnight at 4 °C with primary mouse anti-rat monoclonal antibody directed at ICAM-1 (CD54) (1:500 [v/v] in PBS). Specific labeling of antigen–antibody complex was visualized using an avidin–biotin peroxidase complex immunoperoxidase technique using chromogen diaminobenzidine. Magnification: x125.

 
3.7 Effect of 15d-PGJ2 on NF-{kappa}B activation (p65 translocation and I{kappa}B-{alpha} Western blot)
Immunohistochemical (brown) staining for p65 was located in the cytoplasm in kidney sections obtained from Sham-operated rats (Fig. 8A) indicating that NF-{kappa}B was present in its inactive or dormant form as a heterotrimeric complex with p50 and I{kappa}B. Immunohistochemical analysis of kidney sections obtained after renal I/R demonstrated positive staining for p65 which was localized in the nucleus of PT cells (Fig. 8B), suggesting translocation of p65 from the cytoplasm to the nucleus. In the kidney sections obtained from rats administered 15d-PGJ2, positive staining for p65 was mostly located in the cytoplasm of PT cells indicating an inhibitory effect of 15d-PGJ2 on p65 translocation (and thus inhibition of NK-{kappa}B activation) (Fig. 8C). Scoring of renal sections for assessment of p65 present in the nucleus of renal cell nuclei revealed that compared to kidneys obtained from Sham-operated rats, almost half of all nuclei were positive for p65 in renal sections obtained from rats subjected to I/R (Fig. 8D). This number was markedly reduced in renal sections obtained from rats subjected to renal I/R but which were administered 15d-PGJ2 (Fig. 8D).


Figure 8
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  		Fig. 8 Effect of 15d-PGJ2 on p65 translocation and I{kappa}B-{alpha} degradation. Representative immunohistochemistry sections of kidney obtained from (A) rats subjected to Sham treatment (saline), (B) rats subjected to renal I/R and (C) rats subjected to I/R and administered 15d-PGJ2. In the renal section from Sham-treated rat (A), positive (brown) staining for p65 was found in the cytoplasm (indicated by arrows). After renal I/R (B), positive staining for p65 was located in the nucleus of renal cells (indicated by arrows). In the renal section obtained from rats administered 15d-PGJ2 (C), positive staining was localized to the cytoplasm suggesting that 15d-PGJ2 attenuates the translocation of p65 from the cytoplasm into the nucleus of renal cells during I/R. Original magnification: x1200. Figure is representative of at least three experiments performed on different experimental days. (D) Renal sections were scored for the presence of p65-positive staining in the nucleus of cells and expressed as percent NF-{kappa}B positive nuclei (of total numbers of nuclei counted). (E) Western blot analysis shows the effect of 15d-PGJ2 on I{kappa}B-{alpha} degradation in rat kidney tissues. Sham: basal level of I{kappa}B-{alpha} in cytosolic fractions obtained from the kidneys of Sham-operated rats. I/R-only: in the cytosolic fraction prepared from the kidneys of rats subjected to renal I/R, the intensity of the band corresponding to I{kappa}B-{alpha} protein is markedly reduced compared to the Sham-operated group. I/R+15d-PGJ2: the degradation of I{kappa}B-{alpha} caused by renal I/R is markedly attenuated by pre-treatment of rats with 15d-PGJ2. Each immunoblotting is from a single experiment and is representative of three separate experiments. (F) Densitometric analysis of Western blot analysis, expressed as relative densitometric units (%Sham+Saline control).

 
To elucidate whether renal I/R leads to the degradation of I{kappa}B{alpha} (which is a prerequisite for the subsequent activation of NF-{kappa}B), we investigated how renal I/R and administration of 15d-PGJ2 modulated cytosolic levels of I{kappa}B{alpha} using immunoblotting (Western blot) analysis. A basal level of I{kappa}B{alpha} was detectable in the cytosolic fraction prepared from the kidneys of Sham-operated rats (Fig. 8E). However, in the cytosols prepared from the kidneys of rats subjected to renal I/R, levels of I{kappa}B{alpha} were significantly increased (Fig. 8E). Administration of 15d-PGJ2 prior to, and during reperfusion, attenuated the degradation of I{kappa}B{alpha} during renal reperfusion (Fig. 8E). These findings were reflected in the densitometric analysis of Western blots which revealed (i) significantly greater levels of I{kappa}B{alpha} in the cytosol of renal cells obtained from the kidneys rats subjected to I/R when compared to Sham-operated rats (Fig. 8F) and (ii) a significant reduction in I{kappa}B{alpha} levels in the cytosol of renal cells prepared from the kidneys of rats subjected to I/R but which were administered 15d-PGJ2 when compared with the I{kappa}B{alpha} measured in the cytosolic fraction prepared from the kidneys of I/R-only rats (Fig. 8F). Taken together, these results suggest that 15d-PGJ2 can attenuate the activation of NF-{kappa}B during reperfusion, subsequently modulating the expression of pro-inflammatory genes such as iNOS and ICAM-1.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
In the present study, we have shown that renal I/R of the rat kidney results in a reduction in renal function as demonstrated by increased serum levels of urea, creatinine and a significant reduction in creatinine clearance. This renal dysfunction correlated with increased FENa indicating tubular dysfunction. Renal I/R injury was documented using histological analysis and correlated both with tubular injury that was measured as increased NAG enzymuria and with increased levels of serum AST and {gamma}-GT, indicating reperfusion-injury. All these data, together with expression of iNOS, the adhesion molecule ICAM-1 and increased NO production, confirmed a well known pattern of renal dysfunction and injury associated with I/R of the kidney and ischemic ARF [1,2]. We demonstrate here, for the first time, that administration of the cyclopentenone PG, 15d-PGJ2 produces a significant reduction of renal dysfunction and injury caused by I/R of the kidney of the rat in vivo. Furthermore, these effects are still evident after 48 h reperfusion suggesting that 15d-PGJ2 provides a beneficial effect on the development and course of ischemic ARF.

It is interesting to consider the mechanism(s) that may be involved in the beneficial actions provided by 15d-PGJ2. 15d-PGJ2 is a potent agonist of PPAR-{gamma} in vitro [4] and recent evidence suggests that in addition to the therapeutic potential PPAR-{gamma} agonists have demonstrated in the treatment of diabetes, inflammation and cancer [4], PPAR-{gamma} agonists such as rosiglitazone and pioglitazone can reduce I/R injury of the heart and gastrointestinal system [11,45–48]. We have recently demonstrated that the PPAR-{gamma} ligands rosiglitazone and ciglitazone can ameliorate I/R injury of the rat kidney via reduction of the expression of ICAM-1 and subsequent decrease in the infiltration of polymorphonuclear leukocytes (PMNs) into reperfused renal tissues and reduction of oxidative stress [29]. In the same study we demonstrated that PPAR-{gamma} is expressed in the rat kidney and that this expression is maintained throughout renal I/R [29]. As 15d-PGJ2 has been recognized as the most potent known natural ligand of PPAR-{gamma}, and in view of the expression of PPAR-{gamma} throughout renal I/R, it is highly likely that a major proportion of the beneficial actions afforded by 15d-PGJ2 are mediated by interactions at the PPAR-{gamma}. Interestingly, 15d-PGJ2 has also been reported to have some agonist activity at the PPAR-{alpha}, although with much weaker potency than that observed for the PPAR-{gamma} [5,27]. We have recently reported that the PPAR-{alpha} agonists such as clofibrate and WY14643 can also significantly reduce renal I/R in rats [49]. However, in view of the fact that expression for PPAR-{alpha} is diminished during renal I/R and that these PPAR-{alpha} ligands did not modulate the expression of ICAM-1 [49], it is unlikely that any significant proportion of the beneficial action of 15d-PGJ2 reported here was mediated via interactions at the PPAR-{alpha}.

However, it is now known that 15d-PGJ2 also exerts effects which are independent of PPAR activation [4,15,17,19]. For instance, 15d-PGJ2 directly inhibits the activation of the transcription factor NF-{kappa}B by preventing the phosphorylation of IKK, thereby preventing the degradation of I{kappa}B [7]. We demonstrate here that (i) 15d-PGJ2 reduces the translocation of p65 from the cytoplasm into the nucleus of renal cells during I/R and (ii) 15d-PGJ2 attenuates the degradation of I{kappa}B-{alpha} within the cytoplasm of renal cells during I/R—both of which indicate an inhibitory role for 15d-PGJ2 on the activation of NF-{kappa}B during renal I/R. Whether this effect is mediated via the PPAR-{gamma} or is independent of PPAR-{gamma} activation certainly warrants further investigation. However, it should be noted here that 15d-PGJ2 was able to reduce IFN-{gamma}/LPS-stimulated NO production by pure populations of rat PT cells; a cell type in which there is minimal expression of PPAR-{gamma} [23,27], suggesting a mechanism of action of 15d-PGJ2 in this study which is largely independent of PPAR-{gamma} activation. Activation of NF-{kappa}B leads to the expression of numerous genes and mediators implicated in the development of renal I/R injury, the pathophysiology of ARF and different acute and chronic renal disease states, including iNOS, ICAM-1, VCAM-1 and MCP-1 [43,50]. We report here that renal I/R results in the expression of iNOS in the rat kidney (as determined using immunohistochemistry), subsequently followed by production of NO. NO, derived from NOS, plays an important role in renal function, both under normal and pathophysiological conditions [51,52]. Induction of iNOS in the kidney by cytokines/LPS and during I/R results in renal toxicity [51,52] with several in vivo and in vitro investigations demonstrating that inhibition of iNOS activity, or absence of iNOS itself, can ameliorate or prevent renal I/R in vivo and hypoxia-induced injury to PT cells in vitro [38,53,54]. In this study, it is clear that the substantial reduction of both iNOS expression and NO production by 15d-PGJ2 played a significant role in the reduction of renal I/R injury. This was reflected in the ability of 15d-PGJ2 to significantly reduce IFN-{gamma}/LPS-stimulated NO production by primary cultures of rats PT cells.

Recently it has been suggested that an imbalance between the expression and activity of iNOS and eNOS is an important contributor to the pathophysiology of ARF [55]. A recent study has reported that in the early stages of renal I/R injury (up to 24 h after ischemia), activity of eNOS is significantly attenuated whereas iNOS activity increases [56]. Specifically, in the cortex of rats subjected to I/R, eNOS activity decreased by 60% after 12 h reperfusion and by 87% after 24 h, whereas iNOS activity increased by 43% after 12 h and by 80% after 24 h reperfusion [56]. Such a decline in eNOS activity, although likely to contribute to reperfusion-injury by causing excessive vasoconstriction and promoting microvascular thrombosis, is unlikely to have contributed significantly to the increased plasma levels of NO measured in this study from rats subjected to renal I/R. However, increasing iNOS activity subsequent to ischemia certainly supports our observation of iNOS expression during reperfusion and suggests that the source of NO measured in our study was iNOS rather than eNOS.

The contribution of adhesion molecules such as ICAM-1, VCAM-1 and P-selectin to renal I/R injury has been recognized [57,58], the expression of which leads to the PMN recruitment and infiltration into reperfused tissues and consequent oxidative stress-mediated injury [57]. In this study, renal I/R increased expression of ICAM-1 expression that was markedly reduced by administration of 15d-PGJ2. This reduction of I/R-mediated ICAM-1 expression is similar to that obtained using the PPAR-{gamma} agonist BRL-49653 (rosiglitazone) during intestinal I/R in the mouse [44] and myocardial and renal I/R in the rat [11,29]. However, whether the reduction of ICAM-1 expression by 15d-PGJ2 reported here is mediated via the PPAR-{gamma} or is independent of this receptor remains to be determined.

The results presented here demonstrate that (i) renal I/R results in the activation of NF-{kappa}B and subsequent expression of pro-inflammatory genes, specifically, renal expression of iNOS and ICAM-1, which are dependent on the activation of NF-{kappa}B, and (ii) for the first time in vivo, that 15d-PGJ2 attenuates the activation of NF-{kappa}B and the subsequent expression of downstream target genes. This certainly supports reports that 15d-PGJ2 can reduce the expression of inflammatory genes (iNOS) in glomerular mesangial cells cultured from human, rat and murine kidneys and evidence that agents which inhibit the activation of NF-{kappa}B reduce renal injury subsequent to I/R of the kidney. The results obtained in this study were certainly comparable with those obtained using synthetic PPAR-{gamma} ligands such as rosiglitazone and ciglitazone [29]. However, at a dose of 1 mg/kg used in this study, 15d-PGJ2 was more potent than rosiglitazone which has been recognized as the most potent and selective synthetic PPAR-{gamma} agonist [59], suggesting that a significant part of the beneficial effects obtained using 15d-PGJ2 in this study were independent of the PPAR-{gamma}. However, the use of synthetic thiazolidinediones have been associated with some degree of toxicity, e.g. severe liver damage in patients administered troglitazone, possible hepatotoxic effects of rosiglitazone and pioglitazone [31], risk of carcinogenicity [32], and other pharmacological features such as an absence of PPAR-{gamma}-independent beneficial anti-inflammatory effects [11,33] and a significant loss of potency at the nuclear receptor in whole cell preparations [34]. Recently, acute treatment with troglitazone has been shown to increase susceptibility to ventricular fibrillation during myocardial I/R in pigs [35] and we have observed a loss of beneficial effect of ciglitazone at higher concentrations when administered to rats prior to renal I/R, possibly due to toxicity or non-specific effects [29]. To our knowledge, similar effects have not been reported for 15d-PGJ2 in vitro or in vivo. In support of a hypothesis suggesting, differing actions of synthetic thiozolidinediones and 15d-PGJ2, the most recent study has shown that both troglitazone and ciglitazone, but not 15d-PGJ2, enhance the expression of MCP-1 and subsequent monocyte and macrophage recruitment in the induction phase of mesangial proliferative glomerulonephritis [60]. Infact, a separate study has shown 15d-PGJ2 to reduce MCP-1 expression in mesangial cells [16].

In this study, 15d-PGJ2 ameliorated renal I/R injury and the development and course of ischemic ARF when administered prior to reperfusion. Levels of 15d-PGJ2 may be reduced in patients suffering ischemic ARF, which could contribute to the pro-inflammatory state, associated with this condition. Although this warrants further investigation, it would certainly be in keeping with the observation that PGJ2 production is reduced in mice suffering from immune complex glomerulonephritis [21]. We propose that 15d-PGJ2, or compounds utilizing similar mechanisms of action, may be useful prophylactically in enhancing the tolerance of the kidney against the renal dysfunction and injury experienced in situations where renal tissues are subject to ischemia followed by an extended period of reperfusion-injury, e.g. during aortovascular surgery or during and subsequent to renal transplantation. In conclusion, we demonstrate here, for the first time using an in vivo model of renal I/R injury in the rat, that 15d-PGJ2 significantly reduces renal I/R injury and ameliorates the development of ischemic ARF via reduction of pro-inflammatory gene expression during reperfusion subsequent to the inhibition of the activation of nuclear factor NF-{kappa}B.


   Acknowledgements
 
The authors would like to thank Dr. ssa Tiziana Genovese and Dr. ssa Rosanna Di Paola, Department of Clinical and Experimental Medicine and Pharmacology, University of Messina, Italy, for expert technical assistance and Dr. Rui Pinto, Laboratory of Pharmacology, University of Lisbon, Portugal, for measurement of urinary NAG activities. P.K.C. was funded by the National Kidney Research Fund (Grant R41/2/2000). This work was, in part, supported by the Clínica Médica é Diagnóstico Dr. Joaquim Chaves, Lisbon, Portugal.


   Notes
 
Time for primary review 27 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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