Renal ischemia 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 years1,2. The prognosis of ARF is complicated by the fact that reperfusion, although essential for the survival of ischemic renal tissue, causes additional damage (reperfusion-injury)3. Renal ischemia/reperfusion (I/R) also contributes to the considerable morbidity associated with surgery and anesthesia4,5 where renal dysfunction occurs in up to 50% of patients undergoing aortovascular surgery (for example, for supra- or juxtarenal abdominal aortic aneurysms)5. Renal ischemia also occurs during renal transplantation (especially during organ retrieval and storage) and reperfusion-injury in the early transplant period is associated with late allograft failure6.
Exposure of the kidney to sub-lethal ischemic, hypoxic, thermal or chemical insults stimulate intrinsic mechanisms that can protect the kidneys against a subsequent insult, a phenomenon termed "preconditioning." Generally, preconditioning of the kidney affords greater protection against renal injury than most pharmacological interventions7. Preconditioning of the kidney can be stimulated by several factors including brief periods of sublethal ischemia (ischemic preconditioning)8,9,10,11, hypoxia12 or heat (heat-shock preconditioning)13. Recently, "remote preconditioning" also has been described where ischemic preconditioning of the kidney can protect against sustained renal I/R up to 15 days later14 or against I/R injury in other remote organs such as the heart15. Chemical preconditioning of the kidneys also can be induced by triggers including exogenous adenosine, low-dose cyclosporine, FK506 or sodium arsenite16,17,18. Protection against subsequent renal I/R injury can be produced by administration of sublethal doses of lipopolysaccharide (LPS or endotoxin), a wall-fragment of Gram-negative bacteria19. However, administration of LPS is problematic in that it can produce undesirable side effects due to the excessive generation of pro-inflammatory cytokines [interleukin (IL)-1
, tumor necrosis factor (TNF)-
], induction of pro-inflammatory genes such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and the release of inflammatory mediators of cellular injury such as nitric oxide (NO) and prostaglandins (PGs)20. Within the kidney, LPS can mediate renal toxicity that contributes to acute renal failure (ARF)21. Concerns regarding this toxicity have prompted the development of less toxic derivatives of LPS such as monophosphoryl lipid A (MLA), which can provide protection against subsequent I/R injury22. However, although better tolerated in humans23, intravenous (IV) administration of MLA can still cause fever and the increased release of cytokines24. The recently developed synthetic structural analog of MLA, RC-552, also provides protection against I/R injury but without eliciting fever or cytokine release25. The effects of MLA or RC-552 against renal I/R injury have not been investigated, although it is known that an intraperitoneal (IP) dose of MLA (5 mg/kg) administered to rats localizes to the renal tubules and medulla after 24 hours but is absent from the glomerulus26.
Although Gram-positive organisms do not contain LPS27, Gram-positive bacteria can still cause septic shock and multiple organ failure without causing endotoxemia28. The cell wall of Gram-positive bacteria contains lipoteichoic acid (LTA), which is a macroamphiphile equivalent to LPS in Gram-negative bacteria27. Studies from this laboratory and others have previously demonstrated that LTA from Staphylococcus aureus can cause a moderate induction of iNOS (and subsequent NO production) via the activation of tyrosine kinases and nuclear factor (NF)-
B29,30 with a concomitant induction of GTP cyclohydrolase I and subsequent tetrahydrobiopterin synthesis31. Interestingly, at higher concentrations, LTA also induces COX-2 expression and PGE2 release via activation of NF-B in human pulmonary epithelial cells32. LTA also causes a moderate hypotension in the rat, but unlike LPS and S. aureus itself, it does not cause multiple organ failure or death in this species33. LTA has further advantages over LPS in that (1) it localizes to the (mouse) kidney after a 24 hour pretreatment and can be detected in urine throughout the 48 hours after administration34; (2) it does not induce iNOS in the rat kidney35; and (3) it does not cause renal inflammation when administered at doses as high as 15 mg/kg36. Although we have recently shown that pretreatment of rats with LTA can protect against subsequent myocardial I/R injury37,38, the ability of LTA to protect the kidney against I/R injury has not been investigated. Therefore, the aim of this study was to use an established in vivo rat model of renal I/R injury to investigate whether pretreatment with a low, sub-lethal dose of LTA (1 mg/kg) from S. aureus could protect the kidney against subsequent I/R injury. Furthermore, the mechanism(s) by which LTA reduces renal I/R injury was investigated and compared with that of LPS pretreatment.
METHODS
Experimental protocol
In vivo studies were carried out using 78 male Wistar rats (Tuck, Rayleigh, Essex, UK) weighing 212 to 337 g. Rats received a standard diet and water ad libitum and were cared for in accordance with both the Home Office Guidance in the Operation of the Animals (Scientific Procedures) Act 1986, published by Her Majesty's Stationery Office, London, U.K. and the Guiding Principles in the Care and Use of Animals published by the American Physiological Society. Animals were randomly allocated into eleven groups.
Sham Group
Rats were subjected to surgical procedures except for renal I/R, and were maintained under anesthesia for the duration of the experiment (that is, 45 min + 6 h, N = 12).
Control Group
Rats were subjected to surgical procedures and underwent renal ischemia for 45 minutes followed by reperfusion for 6 hours (N = 12).
1-hour SAL Group
Rats were administered saline (vehicle for LPS/LTA, 2 mL/kg IP) one hour prior to renal I/R (N = 6).
1-hour LPS Group
Rats received LPS (1 mg/kg IP) one hour prior to renal I/R (N = 6).
1-hour LTA Group
Rats received LTA (1 mg/kg IP) one hour prior to renal I/R (N = 6).
24-hour SAL Group
Rats were administered saline (vehicle for LTA/LPS, 2 mL/kg IP) 24 hours prior to renal I/R (N = 6).
24-hour LPS Group
Rats received LPS (1 mg/kg IP) 24 hours prior to renal I/R (N = 6).
24-hour LTA Group
Rats received LTA (1 mg/kg IP) 24 hours prior to renal I/R (N = 6).
24-hour Tempol Group
Rats received a bolus of tempol (30 mg/kg IV) five minutes prior to reperfusion and 30 mg/kg/h IV throughout reperfusion (N = 6).
24-hour LPS + Tempol Group
Rats received LPS (1 mg/kg IP) 24 hours prior to renal I/R followed by a bolus of tempol (30 mg/kg IV) five minutes prior to reperfusion and 30 mg/kg/h IV throughout reperfusion (N = 6).
24-hour LTA + Tempol Group
Rats received LTA (1 mg/kg IP) 24 hours prior to renal I/R followed by a bolus of tempol (30 mg/kg IV) five minutes prior to reperfusion and 30 mg/kg/h IV throughout reperfusion (N = 6).
Rats allocated to the pretreatment groups were administered an IP injection of one of the following, either 1 or 24 hours before commencement of renal I/R, after which they were returned to cages and allowed free access to rat chow and water: (1) LPS (1 mg/kg IP in 2 mL/kg saline); (2) LTA (1 mg/kg IP in 2 mL/kg saline); or (3) saline (2 mL/kg IP; vehicle for LTA and LPS).
The time course and doses of LPS and LTA used were based on those previously shown by our group and others to provide protection against subsequent I/R injury in the rat37,38,39,40. The protocol for tempol administration was based on that previously shown by us to significantly reduce renal I/R injury in the rat41.
Animal surgery
All rats were anesthetized with sodium thiopentone (Intraval® Sodium, 120 mg/kg IP; Rhone Merieux Ltd., Essex, UK) and anesthesia was maintained by supplementary IV bolii of sodium thiopentone as required. Rats were prepared surgically for renal I/R as described previously41,42. Briefly, anesthetized rats were placed onto a thermostatically controlled heating mat (Harvard Apparatus Ltd., Kent, UK) and body temperature maintained at 38 
1°C by means of a rectal probe attached to a homoeothermic blanket. A tracheotomy was performed to maintain airway patency and to facilitate spontaneous respiration. The right carotid artery was cannulated and connected to a pressure transducer (Senso-Nor 840, Horten, Norway) for the measurement of mean arterial blood pressure (MAP) and derivation of the heart rate (HR) from the pulse waveform, which were displayed on a data acquisition system (MacLab 8e, AD Instruments, Hastings, UK) installed on an Apple Macintosh computer. MAP and HR were monitored for the duration of each experiment. The jugular vein was cannulated for the administration of saline or anesthesia as required. A midline laparotomy was performed and the bladder was cannulated and allowed to drain freely without leakage into the peritoneum. Both kidneys were located and the renal pedicles, containing the artery, vein and nerve supplying each kidney, were carefully isolated.
Renal ischemia/reperfusion
As described previously, rats were subjected to bilateral renal occlusion for 45 minutes using non-traumatic Dieffenbach artery clips to clamp the renal pedicles41,42. Reperfusion commenced once the artery clips were removed. Occlusion was verified visually by change in the color of the kidneys to a paler shade, and reperfusion by a blush. Rats subjected to sham-operation (Sham-operated) underwent identical surgical procedures to Control rats, but were not subjected to bilateral renal clamping and were maintained under anesthesia for the duration of the experiment. All groups described above received a continuous infusion of 0.9% (wt/vol) saline (2 mL/kg/h, IV) throughout the I/R period, and at the end of all experiments rats were killed by an overdose of sodium thiopentone.
Measurement of biochemical parameters
At the end of the reperfusion period, blood (1 mL) samples were collected via the carotid artery into tubes containing serum gel. The samples were centrifuged (6,000 r.p.m. for 3 min) to separate serum. All serum samples were analyzed for biochemical parameters within 24 hours after collection (Vetlab Services, Sussex, UK). Serum creatinine concentrations were used as an indicator of renal function41,42. Serum concentrations of aspartate aminotransferase (AST), an enzyme located in the proximal tubule, was used as an indicator of reperfusion-injury41. Urine samples were collected during the reperfusion period and the volume of urine produced recorded. Urine concentrations of Na+ were measured (Vetlab Services) at the end of the reperfusion period and used in conjunction with serum Na+ concentrations to estimate fractional excretion of Na+ (FENa) using standard formulae, and which was used as an indicator of tubular function41,42. Concentrations of urinary N-acetyl--D-glucosaminidase (NAG), a specific indicator of tubular damage41, was also measured (Clinica Medica é Diagnóstico Dr. Joaquim Chaves, Lisbon, Portugal) and used as a marker of tubular injury.
Histological evaluation and scoring
Upon completion of experiments, kidneys were removed and fixed in 10% (wt/vol) formaldehyde, buffered with phosphate buffered saline (PBS; 0.01 mol/L, pH 7.4). For histological evaluation and scoring, a 5 mm section of kidney was removed and processed through to wax, after which 5 
m sections were cut and stained with hematoxylin and eosin. Histological assessment of tubular necrosis was determined semi-quantitatively using a method modified from McWhinnie et al43. As previously described, random cortical fields were observed using a 
20 objective41. A graticule grid (25 squares) was used to determine the number of line intersects involving tubular profiles. One hundred intersections were examined for each kidney 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 = same for score 1, but greater than 1/3 and less than 2/3 of the tubular profile shows nuclear loss; 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 with a maximum score of 300.
Immunohistochemical analysis of P-selectin
Immunohistochemical localization of P-selectin in kidney sections was performed as previously described44. Briefly, sections were fixed in 10% (wt/vol) PBS-buffered formalin, permeabilized using 0.1% (vol/vol) Triton X-100 in PBS for 20 minutes and incubated in 2% (vol/vol) normal rat serum (DBA, Milan, Italy) for two hours to minimize non-specific adsorption. Sections were then incubated overnight at 4°C with rabbit anti-human polyclonal antibody directed against P-selectin (DBA). Controls included kidney sections incubated with buffer alone or non-specific purified IgG (DBA). After blocking endogenous avidin and biotin, specific labeling of antigen-antibody complex was visualized using an avidin-biotin peroxidase complex immunoperoxidase technique using chromogen diaminobenzidine.
Immunohistochemical analysis of inducible nitric oxide synthase expression
The expression of iNOS protein in renal sections was evaluated using an immunohistochemical protocol described previously45. At the end of the experiment, kidneys were bisected, snap-frozen in liquid nitrogen and stored at -70°C. When required, samples were thawed, dehydrated using graded ethanol and embedded in Paraplast, after which 8 m sections were cut. After deparaffinization, endogenous peroxidase was quenched using 0.3% (vol/vol) hydrogen peroxide in 60% (vol/vol) methanol for 30 minutes. Sections were permeablized using 0.1% (vol/vol) Triton X-100 in PBS for 20 minutes. Non-specific adsorption was minimized by incubating the section in 2% (vol/vol) normal goat serum (DBA) in PBS for 20 minutes. Sequential incubation for 15 minutes with avidin and biotin (DBA) was used to block endogenous binding sites. The sections were then incubated overnight with 1:1000 dilution of primary antibody against iNOS (DBA) or with control solutions. Controls included buffer alone or non-specific purified rabbit IgG (DBA). Specific labeling was detected with a biotin-conjugated goat anti-rabbit IgG (DBA) and avidin-biotin peroxidase complex (DBA). To verify the binding specificity for iNOS (negative controls), some sections also were incubated with only the secondary antibody (no primary antibody). Positive controls for iNOS binding were performed on sections of kidney obtained from rats six hours after administration of lipopolysaccharide (LPS, 6 mg/kg, IV).
Immunohistochemical localization of nitrotyrosine
Tyrosine nitration, which was used as an index of the nitrosylation of protein by peroxynitrite, was determined using immunohistochemistry as previously described46. Briefly, sections were incubated overnight with a 1:1000 dilution of primary anti-nitrotyrosine monoclonal antibody (DBA). Separate sections also were incubated with control solutions consisting of PBS alone or a 1:500 dilution of non-specific purified rabbit IgG (DBA). Specific labeling was detected using a biotin-conjugated goat anti-rabbit IgG (DBA) and avidin-biotin peroxidase (DBA). Samples were then viewed under a light microscope.
Measurement of myeloperoxidase activity
Myeloperoxidase (MPO) activity in kidneys was used as an indicator of PMN infiltration into renal tissues using a method previously described41,45. Briefly, at the end of the experiments, kidney tissue was weighed and homogenized in a solution containing 0.5% (wt/vol) hexadecyltrimethylammonium bromide dissolved in 10 mmol/L potassium phosphate buffer (pH 7.4) and centrifuged for 30 minutes at 20,000 g at 4°C. An aliquot of supernatant was then removed and added to a reaction mixture containing 1.6 mmol/L tetramethylbenzidine and 0.1 mmol/L hydrogen peroxide. The rate of change in absorbance was measured spectrophotometrically at 650 nm. MPO activity was defined as the quantity of enzyme required to degrade 1 mol of hydrogen peroxide at 37°C and was expressed in mIU/100 mg wet tissue.
Determination of malondialdehyde levels
Levels of malondialdehyde (MDA) in kidneys were determined as an indicator of lipid peroxidation following a protocol described previously41,45. Briefly, kidney tissue was weighed and homogenized in a 1.15% (wt/vol) KCl solution. A 100 L aliquot of homogenate was then removed and added to a reaction mixture containing 200 L 8.1% (wt/vol) lauryl sulfate, 1.5 mL 20% (vol/vol) acetic acid, 1.5 mL 0.8% (wt/vol) thiobarbituric acid and 700 L distilled water. Samples were then boiled for one hour at 95°C and centrifuged at 3000 g for 10 minutes. The absorbance of the supernatant was measured spectrophotometrically at 650 nm. MDA levels were expressed as mol/L MDA/100 mg wet tissue.
Measurement of plasma nitrite/nitrate concentrations
Nitrite and nitrate are the primary oxidation products of NO reacting with oxygen and, therefore, the nitrite/nitrate concentration in plasma was used as an indicator of NO synthesis and was measured as described previously46. Blood was collected into heparin-treated microcentrifuge tubes and centrifuged (6000 r.p.m. for 3 min) to separate cells and plasma. Nitrate in the plasma sample was then enzymatically converted to nitrite. Briefly, nitrate was stoichiometrically reduced to nitrite by incubation of the sample aliquot (50 L) for 15 minutes at 37°C in the presence of nitrate reductase (0.1 IU/mL, E.C. 1.6.6.2; Roche Diagnostics Ltd., Lewes, East Sussex, UK), -nicotinamide adenine dinucleotide phosphate (-NADPH; 50 mol/L) and flavin adenine dinucleotide (FAD; 50 mol/L) in a final volume of 80 L. When the nitrate reduction was complete, unused -NADPH, which interferes with the subsequent nitrite determination, was oxidized by lactate dehydrogenase (LDH, 100 IU/mL; Roche) and sodium pyruvate (100 mmol/L) in a final reduction volume of 100 L and incubated for five minutes at 37°C. Subsequently, total nitrite in the plasma was assayed by adding 100 L Griess reagent [0.05% (wt/vol) naphthalethylenediamine dihydrochloride and 0.5% (wt/vol) sulphanilamide in 2.5% (vol/vol) phosphoric acid] to each sample. Optical density at 550 nm (OD550) was measured using a Molecular Devices microplate reader (Richmond, CA, USA). Total nitrite/nitrate concentration for each sample was calculated by comparison of the OD550 of a standard solution of sodium nitrate (also stoichiometrically converted to nitrite) prepared in saline.
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% wt/vol NaCl; Baxter Healthcare Ltd., Thetford, Norfolk, UK). The LTA used in this study was from S. aureus (Sigma) whereas LPS was obtained from E. coli serotype 0.127:B8 (Sigma).
Statistical analysis
All values described in the text and figures are expressed as mean standard error of the mean (SEM) for N observations. Each data point represents biochemical measurements obtained from up to six separate animals. For histological scoring, each data point represents analysis of kidneys taken from six individual animals. For immunohistochemical analysis, the figures shown are representative of at least three experiments performed on different experimental days. 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 ANOVA followed by Dunnett's post hoc test and a P value of less than 0.05 was considered to be significant.
RESULTS
The mean SEM for the weights of the rats used in this study was 271 4 g, N = 78). On comparison with Sham animals, renal I/R produced significant increases in serum and urinary markers of renal and tubular dysfunction and tubular and reperfusion injury as described in detail later in this article. Renal I/R (with or without tempol), either alone or subsequent to pretreatment with LPS or LTA, did not have a significant effect on urine flow (0.017 0.001 mL/min, N = 78).
Effect of LPS or LTA pretreatment on renal dysfunction mediated by I/R
Animals that underwent renal I/R exhibited significant increases in the serum concentrations of creatinine compared to Sham-operated animals Figure 1a, suggesting a significant degree of renal dysfunction. Compared to Control animals (I/R-only), pretreatment of rats with either LPS or LTA one hour prior to renal I/R did not produce any changes in serum levels of creatinine Figure 1a. However, on comparison with control animals, 24 hour pretreatment with LPS or LTA produced modest, but significant, reductions in the serum levels of creatinine Figure 1a. The reductions in serum creatinine obtained after pretreatment of rats with LPS or LTA were not significantly different from one another. Administration of saline (vehicle for LPS and LTA) to rats 1 or 24 hours prior to renal I/R did not result in any significant alterations of serum levels of creatinine compared to Control animals Figure 1a.
Figure 1.
Lipopolysaccharide (LPS)/lipoteichoic acid (LTA) pretreatment and ischemia/reperfusion (I/R)-mediated renal and tubular dysfunction. Alterations in (A) serum creatinine concentrations and (B) fractional excretion of Na+ (FENa) subsequent to renal I/R after pretreatment for 1 or 24 hours with saline (SAL, 2 mL/kg IP), LPS (1 mg/kg IP) or LTA (1 mg/kg IP). *P < 0.05 vs. control group (I/R only), 
P < 0.05 vs. SAL 24-hour pretreatment group.
Effect of LPS or LTA pretreatment on tubular dysfunction mediated by I/R
Renal I/R produced a significant increase in FENaFigure 1b, suggesting a marked increase in tubular dysfunction. On comparison with FENa measured in Control animals, administration of LPS or LTA one hour prior to renal I/R did not have a significant effect on FENaFigure 1b. However, pretreatment of rats with LPS or LTA 24 hours prior to renal I/R produced significant reductions in FENa, suggesting improvement in tubular function Figure 1b. The reductions in FENa obtained were not significantly different between the LTA and LPS pretreated groups Figure 1b. Administration of saline to rats 1 or 24 hours prior to renal I/R did not significantly alter FENa compared with Control animals Figure 1b.
Effect of LPS or LTA pretreatment on I/R-mediated tubular and reperfusion-injury
On comparison with values obtained from Sham-operated animals, renal I/R produced significant increases in the urinary levels of NAG (suggesting significant tubular injury; Figure 2a) and serum concentrations of AST (suggesting significant reperfusion-injury; Figure 2b). Urinary NAG and serum AST concentrations were not significantly affected by pretreatment with LPS or LTA one hour prior to I/R Figure 2, but were significantly reduced subsequent to the 24 hour pretreatment with LPS or LTA prior to renal I/R Figure 2. In each case, similar reductions in either urinary NAG or serum AST were obtained after pretreatment with LPS or LTA Figure 2. Administration of saline to rats 1 or 24 hours prior to I/R did not result in any significant alterations in urinary NAG or serum levels of AST compared to Control animals Figure 2.
Figure 2.
LPS/LTA pretreatment and I/R-mediated tubular and reperfusion injury. Effects of renal I/R on (A) urinary N-acetyl--D-glucosaminidase (NAG) concentrations and (B) serum aspartate aminotransferase (AST) levels after 1 or 24 hours pretreatment with saline (SAL, 2 mL/kg), LPS (1 mg/kg IP) or LTA (1 mg/kg IP). *P < 0.05 vs. control group (I/R only), P < 0.05 vs. SAL 24-hour pretreatment group.
Effects of LPS or LTA pretreatment on renal histopathology and scoring
On comparison with the normal renal histology observed in kidneys taken from Sham-operated rats Figure 3a, animals that underwent renal I/R demonstrated the recognized features of tubular damage Figure 3b. These features included tubular dilation, brush border loss, nuclear condensation, cytoplasmic swelling and consistent loss of significant numbers of nuclei from tubular profiles Figure 3b. Kidneys obtained from rats pretreated with LPS or LTA 24 hours prior to renal I/R demonstrated reduced histological features of renal injury on comparison with kidneys obtained from control animals Figure 3 c, d. Markedly reduced tubular cell loss and tubular dilation was observed in kidneys obtained from LPS or LTA pretreated animals Figure 3 c, d.
Figure 3.
Histological evaluation of renal I/R injury subsequent to pretreatment with LPS or LTA. Rats were subjected to (A) Sham operation (B) I/R only (control group), or 24-hour pretreatment with (C) LPS (1 mg/kg IP) or (D) LTA (1 mg/kg IP) followed by renal I/R. Sections stained with trichromic Van Gieson (magnification 400). (E) Total severity score; (out of a total score of 300) obtained from renal sections from rats pretreated for 1 or 24 hours prior to renal I/R with either saline (SAL, 2 mL/kg), LPS or LTA. Please refer to Methods section for an explanation of the scoring system. *P < 0.05 vs. control group, P < 0.05 vs. SAL 24-hour pretreatment group.
Compared with the total severity score measured from kidneys obtained from Sham-operated animals, renal I/R produced a significant increase in total severity score Figure 3e. Administration of LPS or LTA one hour prior to renal I/R did not have a significant effect on total severity score when compared to that obtained from Control animals Figure 3e. However, the total severity score was significantly reduced by pretreatment with either LPS or LTA 24 hours prior to renal I/R Figure 3e. Administration of saline to rats 24 hours prior to renal I/R did not have a significant effect on total severity score when compared to that obtained from Control animals Figure 3e.
Effect of LPS and LTA pretreatment on P-selectin expression subsequent to renal I/R
Kidneys obtained from rats subjected to I/R demonstrated marked staining for P-selectin when compared with kidneys obtained from Sham-operated rats Figure 4 a, b, suggesting adhesion molecule expression during reperfusion. Kidneys obtained from rats pretreated with LPS or LTA demonstrated markedly reduced staining for P-selectin Figure 4 c, d when compared with kidneys obtained from Control animals, suggesting a reduction in the expression of P-selectin during reperfusion.
Figure 4.
Immunohistochemical localization of P-selectin in rat kidney sections. (A) Sham-operated group, (B) Control group (I/R only), and 24-hour pretreatment with (C) LPS (1 mg/kg IP) or (D) LTA (1 mg/kg IP) followed by renal I/R. Sections were incubated with rabbit anti-human polyclonal antibody directed at P-selectin and specific labeling of antigen-antibody complex visualized using an avidin-biotin peroxidase complex immunoperoxidase technique using chromogen diaminobenzidine. Magnification 125.
Effect of LPS and LTA pretreatment on kidney myeloperoxidase activity and malondialdehyde levels
Rats subjected to renal I/R exhibited a substantial increase in kidney MPO activity Figure 5a, suggesting increased PMN infiltration into renal tissues. However, pretreatment of rats with LPS or LTA 24 hours prior to I/R produced a significant reduction of MPO activity on comparison with MPO activity obtained from Control rat kidneys Figure 5a. MPO activities obtained from rats pretreated with LPS or LTA were not significantly different from one another Figure 5a and both MPO activities were not significantly different from MPO activity measured from kidneys obtained from Sham animals Figure 5a.
Figure 5.
Effects of LPS/LTA pretreatment followed by renal I/R on (A) kidney myeloperoxidase (MPO) activity and (B) kidney malondialdehyde (MDA) levels after 24 hours of pretreatment with LPS (1 mg/kg IP) or LTA (1 mg/kg IP). *P < 0.05 vs. control group (I/R only);
P < 0.05 vs. LTA pretreatment group.
Rats subjected to renal I/R exhibited a substantial increase in kidney MDA levels Figure 5b, suggesting increased formation of reactive oxygen species (ROS) and consequent lipid peroxidation. However, pretreatment of rats with LPS or LTA 24 hours prior to renal I/R produced a significant reduction in MDA levels on comparison with MDA levels obtained from Control rat kidneys Figure 5b. MDA levels obtained from rats pretreated with LTA were significantly lower than that obtained after pretreatment with LPS, suggesting a greater effect on lipid peroxidation and ROS production Figure 5b.
Comparison of the effects of tempol and LPS or LTA pretreatment on renal I/R
Administration of tempol to rats during renal I/R produced a significant reduction in both serum creatinine levels and FENa in comparison with values obtained from Control animals Figure 6, suggesting improvement of renal and tubular dysfunction. Tempol, when administered to rats pretreated with LPS or LTA, did not provide any additional improvement of serum creatinine levels or FENa in comparison with levels obtained from rats pretreated with LPS or LTA only Figure 6.
Figure 6.
Effects of tempol on LPS/LTA pretreatment and I/R-mediated renal and tubular dysfunction. Alterations in (A) serum creatinine concentrations and (B) fractional excretion of Na+ subsequent to renal I/R after pretreatment for 24 hours with LPS (1 mg/kg IP) only, LPS (1 mg/kg) followed by tempol (30 mg/kg IV bolus injection followed by infusion of 30 mg/kg/h throughout the reperfusion period), LTA (1 mg/kg IP) only, LTA (1 mg/kg) followed by tempol (30 mg/kg IV bolus injection followed by infusion of 30 mg/kg/h throughout the reperfusion period), or tempol only (30 mg/kg IV bolus injection followed by infusion of 30 mg/kg/h throughout the reperfusion period). *P < 0.05 vs. control group (I/R only).
Full figure and legend (27K)Effects of LPS or LTA pretreatment on expression of iNOS and NO production
When compared to kidney sections obtained from Sham-operated rats Figure 7a, immunohistochemical analysis of sections obtained from rats subjected to renal I/R revealed positive staining for iNOS Figure 7b. In contrast, reduced staining was observed in the kidney sections obtained from rats administered LPS Figure 7c and substantially reduced staining was observed in kidney sections obtained from rats pretreated with LTA 24 hours prior to renal I/R Figure 7d. The positive control for iNOS staining, performed on sections of kidneys obtained from rats subjected to endotoxemia, demonstrated marked positive staining for iNOS protein (data not shown). No positive staining for iNOS protein was observed in kidney sections from rats subjected to renal I/R, which were incubated with the secondary antibody only (no primary antibody; negative control; data not shown).
Figure 7.
LPS/LTA pretreatment and renal I/R-mediated iNOS expression. Kidney sections were incubated overnight with 1:1000 dilution of primary antibody directed against iNOS. (A) Sham operation, (B) I/R-only (control group), and 24-hour pretreatment with (C) LPS (1 mg/kg IP) or (D) LTA (1 mg/kg IP) prior to renal I/R. Magnification: 125. (E) Plasma nitrite/nitrate concentrations. Griess assay performed on plasma collected from rats 1 or 24 hours after pretreatment with saline (SAL, 2 mL/kg), LPS (1 mg/kg IP) or LTA (1 mg/kg IP). *P < 0.05 vs. control group (I/R only), P < 0.05 vs. SAL 24-hour pretreatment group, 
P < 0.05 vs. sham-operated group.
Renal I/R resulted in a significant increase in the plasma levels of nitrite/nitrate (an indicator of the formation of NO) in comparison with values obtained from the plasma of Sham-operated animals Figure 7e. Increased plasma nitrite/nitrate levels mediated by renal I/R were not significantly affected by pretreatment with LTA or LPS one hour prior to I/R, but were significantly reduced subsequent to 24 hours of pretreatment with LPS or LTA prior to renal I/R Figure 7e. In each case, similar reductions in either plasma nitrite/nitrate levels were obtained after pretreatment with LTA or LPS Figure 7e. However, plasma nitrite/nitrate levels obtained rats pretreated with LPS or LTA and subjected to renal I/R were still significantly higher than levels obtained from Sham-operated animals Figure 7e. Administration of saline only to rats 1 or 24 hours prior to I/R did not result in any significant alterations of plasma levels of nitrite/nitrate compared to Control animals Figure 7e.
Effect of LPS or LTA on nitrotyrosine formation during renal I/R
In comparison to renal sections obtained from Sham-operated rats that were administered saline only Figure 8a, immunohistochemical analysis of renal sections obtained from rats subjected to renal I/R revealed positive staining for nitrotyrosine Figure 8b. In contrast, substantially reduced nitrotyrosine staining was observed in the kidney sections obtained from rats, which were pretreated with LPS Figure 8c or LTA Figure 8d 24 hours prior to renal I/R.
Figure 8.
LPS/LTA pretreatment and renal I/R-mediated nitrotyrosine formation. Kidney sections were incubated overnight with 1:1000 dilution of primary antibody directed against nitrotyrosine. (A) Sham operation, (B) I/R only (control group), and 24 hours of pretreatment with (C) LPS (1 mg/kg IP) or (D) LTA (1 mg/kg IP) prior to renal I/R. Magnification 125.
DISCUSSION
Renal I/R is a common problem that occurs during aortic surgery or renal transplantation or is caused by cardiovascular anesthesia, leading to renal dysfunction and injury4,5,47,48,49,50. Thus, early therapeutic intervention that reduces the consequences of renal I/R has been a topic of intense research interest, motivated by the fact that previous interventions against ARF have proven to be largely negative and that dialysis still remains the only effective therapy1. To date, preconditioning of the kidney (whether ischemic, hypoxic, chemical or by heat-shock) has generally provided a greater degree of protection against renal I/R injury than most other pharmacological interventions7. The results presented here demonstrate, to our knowledge for the first time, that 24 hour pretreatment (or pharmacological preconditioning) with a non-lethal dose of LTA (1 mg/kg) significantly reduces the renal and tubular dysfunction, and tubular and reperfusion injury associated with a subsequent episode of I/R of the rat kidney. Furthermore, LTA pretreatment produced a marked reduction in the histological evidence of renal injury including tubular dilatation and congestion.
It is interesting to consider the mechanism(s) that may be involved in the protection afforded by 24 hours of pretreatment of rats with LTA. Our study found that the renoprotective effects of LTA were time-dependent, that is, one hour of pretreatment did not afford protection but 24 hours was required for protection to be conferred, suggesting a modulation of gene expression. After 24 hours of pretreatment with LTA: (1) expression of the adhesion molecule P-selectin caused by renal I/R was reduced; (2) renal MPO activity was reduced to levels observed from Sham-operated animals, suggesting a reduction in PMN infiltration into renal tissues; (3) reduced renal MDA levels were observed, suggesting decreased lipid peroxidation due to reduced production of ROS; (4) a reduction in the expression of iNOS and NO production (compared to I/R-only rats) was found; and (5) there was evidence of reduced nitrotyrosine formation, indicating reduced peroxynitrite production. Further studies found that the protective effects of LTA against renal and tubular dysfunction were comparable to that obtained using tempol, a ROS-scavenger that removes both superoxide and hydroxyl radicals, which we have previously shown to provide beneficial actions against renal I/R injury in the rat41. Additive effects were not observed when tempol was administered to rats pretreated with LTA, indicating that LTA pretreatment was effective in reducing the formation of ROS (as reflected in the ability of LTA pretreatment to reduce lipid peroxidation to levels not significantly different from Sham-operated animals). Finally, although iNOS expression was substantially reduced and NO production significantly attenuated by pretreatment with LTA, levels of nitrite/nitrate in the plasma remained significantly higher than those measured in the plasma of Sham-operated animals. Thus, we propose that the beneficial actions of LTA pretreatment are mediated by a significant reduction in the production of ROS, NO and peroxynitrite. However, we propose that the significant levels of NO production measured after renal I/R contribute to the beneficial actions of LTA, a hypothesis discussed in detail in the next section.
LTA pretreatment reduces adhesion molecule expression, PMN infiltration and production of ROS
In this study, LTA pretreatment markedly reduced the expression of P-selectin, the expression of which, among other adhesion molecules, has been associated with renal I/R-injury51,52. Several studies have demonstrated that P-selectin is expressed during renal I/R and ARF, and ligands that block P-selectin or antibody directed against P-selectin can reduce renal I/R injury53. Expression of adhesion molecules such as P-selectin and intercellular adhesion molecule (ICAM)-1 is a fundamental requirement for the recruitment of PMNs into renal tissues during renal reperfusion51,52,53 and studies using specific markers of PMNs indicate their accumulation in postischemic kidneys54,55. Our current study showed that LTA pretreatment reduced renal MPO activity to levels that were not significantly different to levels measured in the kidneys of Sham-operated rats, suggesting an almost total reduction in PMN infiltration into renal tissues. This finding is in keeping with previous studies reporting that depletion of PMN activity or numbers reduces renal I/R injury52,54,55. Activated PMNs are generally considered to be the principal effectors of renal I/R-injury as they can release (1) superoxide, which can be converted to hydroxyl radicals and (2) NO, which can combine with superoxide to form peroxynitrite (see below)56. Hydroxyl radicals and peroxynitrite are highly reactive and cause tissue injury, for example, via lipid peroxidation, DNA damage and activation of poly (ADP-ribose) polymerase42,52,54,55,56. PMNs also release myeloperoxidase, which catalyses the formation of another potent oxidant, hypochlorous acid56. PMNs also generate cytokines and interact with the renal endothelium leading to further pathophysiology21. In this study, the beneficial role of attenuation of PMN infiltration into renal tissues was reflected by a significant reduction in MDA levels in the kidneys of rats pretreated with LTA (almost to levels measured in renal tissues obtained from Sham-operated rats), suggesting significant reduction in lipid peroxidation (subsequent to reduced production of ROS).
Taken together, the evidence presented in our study suggests that renal I/R produces moderate glomerular dysfunction, but a substantial increase in tubular dysfunction and injury. This is in keeping with the notion that renal I/R causes both glomerular and tubular dysfunction57. Several reports have described the vulnerability of the outer medulla and thick ascending limb cells to structural damage during renal I/R, which has been attributed to the imbalance between the metabolic demand of this region of the kidney and oxygen delivery to these cells58. Although not directly investigated in our study, distribution of PMNs in the cortex, outer and inner medulla during I/R previously has been investigated, and after two hours of reperfusion a gradient of PMN accumulation has been described (cortex < outer medulla < inner medulla)59. After a two-hour reperfusion, the outer medulla contains very few PMNs and in the cortex itself, the peritubular regions contain double the numbers of PMNs compared to intraglomerular regions. However, although it is important to consider the role of the medulla during episodes of renal I/R where it has been reported that PMNs may contribute indirectly to tubular injury by exacerbating medullary hypoperfusion during reperfusion58,60, it is also accepted that PMNs play an important role in direct injury to the tubular injury associated with renal reperfusion51,60. Furthermore, early observations have reported that the damage to kidney during I/R is primarily to the S3 segment of the PT61. This was further reinforced by the demonstration in our study that renal I/R caused relatively greater tubular dysfunction and injury than glomerular dysfunction and that the beneficial effects of LTA pretreatment were more pronounced on tubular dysfunction and injury (FENa and urinary NAG). However, we are confident that the effects of LTA on adhesion molecule expression (observed in the cortex) and on PMN recruitment (measured as MPO activity) will also prevail in medullary regions.
LTA pretreatment reduces iNOS expression, NO production and peroxynitrite formation
In this study, LTA pretreatment markedly reduced the I/R-mediated expression of iNOS in the rat kidney and subsequently, plasma nitrite/nitrate levels, indicating reduced NO production. There is now good evidence that formation of high levels of NO subsequent to the expression of iNOS plays an important role in the pathophysiology of renal injury mediated by hypoxia and I/R of the kidney46,60,62. NO mediates cytotoxicity via several mechanisms including DNA damage, inhibition of mitochondrial respiration and degradation of the actin cytoskeleton as well as by modulation of the expression of cytokines and adhesion molecules60,62. Several studies by our and other laboratories have shown that inhibition of the expression or activity of iNOS, or the absence of iNOS itself can ameliorate or prevent renal I/R injury46,63,64. We demonstrate here that LTA pretreatment is associated with a marked reduction in iNOS expression and subsequent NO production, and propose that this contributes to the observed renoprotective actions of LTA. Furthermore, NO reacts with superoxide anion to form the highly reactive and injurious species peroxynitrite65, which causes injury via direct oxidant injury and protein tyrosine nitration, as well as via the production of hydroxyl radicals66,67. Specifically in the kidney, peroxynitrite generation is implicated in the pathophysiology of both renal I/R and hypoxia-reoxygenation injury, respectively41,46,60,62,68, and recent studies by our group and others have demonstrated the beneficial actions of inhibiting the formation or presence of peroxynitrite (such as using ebselen)41,46,69. In our current study, LTA pretreatment markedly reduced nitrotyrosine formation, which is indicative of peroxynitrite production, presumably via inhibition of superoxide anion and reduction of NO production.
Heeman and colleagues demonstrated the beneficial actions of LPS pretreatment against renal I/R injury in mice19, while IL-1, interferon (IFN)-
and iNOS mRNA expression did not change during the pretreatment and reperfusion periods. However, increased TNF- (2 hours after reperfusion) and decreased IL-6 (before ischemia and shortly after commencement of reperfusion) mRNA expression were observed and reported as potential mechanisms for the observed beneficial effects19. Although we did not study this in the rats here, the ability of LTA to modulate cytokine production certainly warrants further investigation.
Our results showed that LTA provided beneficial actions that were not significantly different to those obtained using LPS. However, a major advantage of using LTA at the concentration used is that it did not produce any of the obvious 'endotoxic' side-effects associated with a similar administration of LPS, mediated by excessive generation of pro-inflammatory cytokines, induction of pro-inflammatory genes and release of mediators of cellular injury20,21. This advantage of LTA administration over LPS is reflected in our earlier studies in which LPS pretreatment (1 mg/kg) activated the coagulation cascade and caused liver injury in the rat37. In rats, higher doses of LTA (10 mg/kg) causes only a moderate induction of iNOS and hypotension in the rat, without producing the multiple organ failure or death31,33. Additionally, it has also been shown that—unlike LPS—LTA administered at 10 mg/kg to rats does not induce iNOS in the kidney35 and does not cause renal inflammation34, even though in mice it localizes to the kidney 24 hours after administration, producing high local concentrations36.
Role of NO in the protection against renal I/R injury afforded by LTA pretreatment
Although LTA pretreatment substantially reduced iNOS expression and significantly attenuated NO production subsequent to renal I/R, plasma levels of NO remained significantly higher than levels measured in the plasma of Sham-operated rats Figure 7e. The beneficial role of NO in preconditioning of the kidney, brain and heart been well been characterized7,70,71,72. In the heart and brain, a biphasic response has been described in which NO from eNOS is utilized as an immediate but short-term response, and NO from iNOS as a delayed but long-term defense71,72. Ogawa and colleagues recently demonstrated that NO, produced from eNOS, mediated ischemic preconditioning of the kidney, which could be blocked using N
-nitro-L-arginine methyl ester (L-NAME) or enhanced by administration of L-arginine73. Additionally, NO (produced via the NO donor sodium nitroprusside) has been shown to attenuate PMN retention in renal tissues and reduce the deleterious effects of PMNs on glomerular and tubular function74. Thus, we propose that the reduced levels of NO produced during I/R subsequent to LTA pretreatment partially mediates the beneficial effects observed in our current study.
In conclusion, our results show, to our knowledge for the first time, that 24 hours of pretreatment of rats with a non-lethal dose of LTA significantly reduces the renal dysfunction and injury in rats subjected to I/R of the kidneys. The mechanism(s) underlying the observed protective effects of LTA is mediated in part by NO, but also involves a reduction in the expression of P-selectin and iNOS, attenuation of PMN recruitment, reduced ROS and nitrotyrosine formation. We suggest that LTA is useful in enhancing the tolerance of the kidney against renal dysfunction and injury in situations where renal tissues are subject to I/R, for example, during aortovascular surgery or renal transplantation.
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Acknowledgments
This work was supported in part by the Clínica Médica é Diagnóstico Dr. Joaquim Chaves, Lisbon, Portugal. PKC is funded by the National Kidney Research Fund (R41/2/2000).
