HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 16/8/908 01-0880fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by LLOBERAS, N. Articles by GRINYÓ, J. M. Search for Related Content PubMed PubMed Citation Articles by LLOBERAS, N. Articles by GRINYÓ, J. M.

Postischemic renal oxidative stress induces inflammatory response through PAF and oxidized phospholipids. Prevention by antioxidant treatment¹

Laboratory of Experimental Nephrology, Department of Medicine, University of Barcelona, and Nephrology Service, Hospital Bellvitge, L’Hospitalet, Barcelona, Spain

²Correspondence Laboratory of Experimental Nephrology (4122). Unit of Medicine. Pavelló del govern, 4^a plta. Hospital de Bellvitge, CSUB, Feixa Llarga s/n. E-08907. L’Hospitalet, Barcelona, Spain. E-mail: jmgrinyo{at}medicina.ub.es

SPECIFIC AIMS

Reperfusion injury is considered primarily an inflammatory response to oxidative stress. In this study, we examine 1) whether platelet-activating factor (PAF) and PAF-like lipids are released during reperfusion in an in vivo model of warm renal ischemia; 2) the relationship between these phospholipids and oxidative damage on the one hand and leukocyte polymorphonuclear (PMN) recruitment in renal tissue on the other; and finally 3) whether antioxidant treatment influences the behavior of these phospholipids, the renal inflammatory response, and the outcome of postischemic acute renal failure.

PRINCIPAL FINDINGS

1. Antioxidant treatment decreases PAF release after renal ischemia
After 60 min of warm ischemia in rabbits, blood from the renal vein was collected at predetermined time intervals to determine PAF and PAF-like lipid release timing. After ischemia, all the rabbits showed a single peak of total PAF release in the first 15 min of reperfusion.

Ischemic vitamin C-treated rabbits (150 mg/kg 24 h and 1 h before surgery) showed a marked reduction in total PAF activity, reaching levels markedly lower than those in ischemic untreated rabbits (3.86±0.85 vs. 24.28±2.96 nmol/L, respectively, P<0.0001).

2. Isolation and characterization of PAF-like lipids in postischemic renal plasma: effect of vitamin C treatment in rabbits and of PAF receptor antagonist blockade
Plasma samples from the identified peak of PAF after ischemia were further extracted and purified in order to characterize the different sort of phospholipids using HPLC. Fractions were collected every minute and bioassayed. Ischemic rabbits presented a mean value of native PAF and PAF-like lipids of 5.26 ± 1.11 and 4.94 ± 0.70 nmol/L, respectively. As shown in Fig. 1 , after antioxidant treatment with vitamin C both phospholipids presented a notable decrease to 1.43 ± 0.54 nmol/L of native PAF and 0.62 ± 0.20 nmol/L of PAF-like.

View larger version (10K): [in this window] [in a new window]   Figure 1. Isolation and characterization of native PAF and PAF-like lipids from plasma of renal warm ischemic animals with or without vitamin C treatment. HPLC chromatogram of bioactive lipids was gathered in different intervals. Lipids were extracted and the polar lipid fraction was isolated, characterized by ODS reverse-phase HPLC, and bioassayed by platelet 3H serotonin release. Separately injected PAF standards were eluted at 8–10 min. Samples were processed by the Heery method and subjected to reverse-phase HPLC in a 5 µm 4.6 mm x 25 cm ODS column (Pharmacia Smart System, Uppsala, Sweden). Samples were injected in 200 µL of the mobile phase and eluted isocratically with a solvent system of (MeOH: CH3CN: H2O containing 1 mM NH4 acetate; 84:15:1) at a flow rate of 1 mL/min to develop the column. Compounds eluting from the column were detected by UV absorbance at 206 nm. Fractions were collected every minute, evaporated to dryness under a nitrogen stream, and bioassayed. Recovery of [3H]-labeled PAF internal standards was 60–70%.

In vitro, PAF and PAF-like fractions were notably reduced after pretreatment with the PAF receptor antagonist (6.70±3.10 vs. 1.08±0.97 nmol/L, and 6.02±0.65 vs. 0.59±0.37 nmol/L), PAF and PAF-like with and without UR 12670, respectively).

3. Decrease in DNA oxidation and renal postischemic inflammatory response after antioxidant treatment
To assess the involvement of free radical formation in kidney after ischemia-reperfusion, we identified 8 oxo 2' deoxyguanosine, a sensitive marker of DNA damage, under a fluorescence microscope and semiquantitatively graded on a scale from 0 to 3+. Ischemic animals showed intense renal DNA oxidation whereas vitamin C-treated ischemic animals presented a clear decrease in oxidative parameters (2.2±0.3 vs. 0.9±0.2, respectively, P=0.008).

To examine renal PMN recruitment in the postischemic inflammatory response, MPO activity was measured. Vitamin C-treated ischemic animals showed a significantly lower renal MPO level than that of the ischemic untreated group (0.06±0.04 vs. 0.34±0.07 ODS·min^-1·g tissue^-1, respectively, P=0.009).

4. Antioxidant treatment reduces functional and histological damage after warm ischemia in rats
To define the functional and histological effect of antioxidant treatment on ischemia-reperfusion, two groups of rats underwent warm renal ischemia with or without vitamin C treatment and were followed for 3 days. The serum creatinine profile is shown in Fig. 2 . Rats from the ischemic untreated group presented severe renal failure, which was maintained during the 3 day follow-up. Treatment with vitamin C improved renal function on the first day and continued to improve on the second and third days of follow-up. Serum creatinine in vitamin C-treated rats was significantly lower than that in ISCH untreated animals throughout the follow-up period (plasma day 1: 277±17.58 vs. 219±19.94 µmol/L, P=0.04; plasma day 2: 272±39.8 µmol/L vs. 143±25, P=0.02; plasma day 3: 226±41 vs. 99±13.3 µmol/L, P=0.01).

View larger version (17K): [in this window] [in a new window]   Figure 2. Serum creatinine (µmol/L) profile in warm renal ischemic animals with or without antioxidant treatment. Rats from the ischemic group presented severe renal failure, which was maintained on the 3 day follow-up. Treatment with vitamin C led to improved function on the first, second, and third day. Serum creatinine in vitamin C-treated rats was significantly lower than in ischemic untreated animals throughout the follow-up (day 1, P=0.04; day 2, P=0.02; day 3, P=0.01).

At the end of the study, renal histological damage was assessed using a semiquantitative scale from 0 to 4+. The histological evaluation revealed severe tubulointerstitial damage in kidneys from the ischemic untreated group, whereas vitamin C-treated animals had a significantly lower degree of histological damage (ISCH+VITC group, 0.91±0.16 vs. ISCH group, 1.89±0.36, P=0.02).

CONCLUSIONS AND SIGNIFICANCE

Ischemia-reperfusion injury leads to the massive generation of oxygen free radicals, which oxidize the cellular structures with a local PAF production through PLA_2, inducing leukocyte recruitment. PAF-like lipids resulting from the unregulated oxidation of the cellular membrane and from circulating lipoprotein phospholipids have been implicated in inflammatory states. Our in vivo data show the release of PAF and, in particular, PAF-like lipids from venous effluent in a model of warm renal ischemia. The release of those phospholipids from the renal vein, coupled to the oxidation of renal DNA and the increase in leukocyte accumulation, substantiates the triggering of an inflammatory response involving oxygen free radicals. Antioxidant treatment with vitamin C attenuated the postischemic oxidative changes, reduced PAF and PAF-like lipids, and consequently minimized leukocyte recruitment in renal tissue.

Here we confirm the increase in PAF production in vivo after renal ischemia that occurred at the onset of reperfusion and was extremely transient, since we were unable to detect PAF activity beyond the first 15 min. This corresponds to the action of PAF as the initial adhesion molecule that signals and tethers circulating leukocytes. Therefore, our data suggest an active role of PAF in the initiation of the inflammatory response in renal ischemia-reperfusion.

In our study, a large amount of total PAF from these early postischemic venous effluents contained phospholipids with PAF-like lipid activity. Preincubation of this PAF-like fraction with the specific PAF antagonist abolished PAF activity, thus confirming on the one hand that it has a biological activity on platelets through the PAF receptor and, on the other, that this phospholipid fraction increases the PAF signaling after ischemia. The first demonstration of phospholipid oxidation products in vivo was in a cigarette-smoking hamster model. We suggest that in ischemia-reperfusion damage, initial inflammatory mechanisms are mediated not only by PAF, but also by the concurrence of PAF-like lipids. Parallel to the appearance of these PAF-like compounds, an increase of 8-oxo-2'-deoxyguanosine reflects the oxidation of renal structures after ischemia. We suggest that PAF-like lipids account for the local inflammation immediately after ischemia-reperfusion and, therefore, that oxidized phospholipid can be generated in vivo by oxidative stress. Modification of these early postischemic oxidative events with this initial antioxidant treatment may interfere with the subsequent amplification of the oxidative cascade, thus aborting the inflammatory mechanisms.

The increase in leukocytes in the ischemic kidney evaluated by MPO activity confirms this event after warm renal ischemia. As our results show, ischemic tissue was oxidized after the delivery of PAF-like lipids in venous effluent, resulting in the recruitment of leukocytes. In vitamin C-treated ischemic kidneys, leukocyte accumulation and the presence of PAF compounds decreased, suggesting again that antioxidant treatment blocked the inflammatory response after ischemia through reduction of the postischemic oxidative stress.

Several mechanisms by which vitamin C may intervene in the oxidative-induced interaction between inflammatory cells and endothelial cells have been described. As P-selectin can be affected by aqueous-phase oxygen radicals, vitamin C might attenuate the up-regulation of this critical adhesion molecule by neutralizing these radicals. The water-soluble antioxidant intercepts aqueous-phase oxygen radicals before they attack lipoprotein lipids, thus preventing the initiation of lipid peroxidation. In our ischemia-reperfusion study, vitamin C reduced the production of PAF and PAF-like lipids, confirming that lipoperoxidation was affected and that this is related to the suppression of aqueous-phase oxygen radical generation. Reducing the production of oxygen free radicals with an antioxidant may not only block enzymatic PAF synthesis, but might also prevent the unregulated generation of circulating phospholipids with PAF-activity, thus impeding subsequent oxidative events.

Vitamin C administration improved renal function during the 3 day follow-up and renal structure was preserved. As the improvement in tubulointerstitial damage in ischemic-treated kidneys 3 days after ischemia paralleled the reduction in leukocyte recruitment, we hypothesize that the antioxidant protects kidneys against ischemic insult by preventing inflammatory cell infiltration.

View larger version (104K): [in this window] [in a new window]   Figure 3. Schematic diagram. Oxidative stress is the main mechanism for triggering tissue ischemia-reperfusion damage. At the onset of reperfusion, organs are exposed to an injurious burst of oxygen free radicals (OFR). Within minutes of injury, ranges of inflammatory molecules are expressed on activated endothelial cells (EC). OFR induce PAF synthesis through an enzymatic pathway, PAF becomes anchored to the endothelial cell surface where, coupled to P-selectin (P-sel), it acts as the first juxtacrine signaling molecule, tethering and priming leukocytes. OFR generate PAF-like lipids by the unregulated oxidation (nonenzymatic pathway) of membrane and circulating lipoprotein (LDL) phospholipids. After their generation, PAF-like lipids circulate in the bloodstream and act on leukocytes in a paracrine manner through the PAF receptor, increasing their activation and adhesiveness. The antioxidant water-soluble vitamin C interferes ischemia-reperfusion damage at the early step of the inflammatory cascade. Vitamin C administration minimizes OFR generation and subsequently the production of PAF and PAF-like lipids, preventing EC and leukocyte PMN activation.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0880fje; to cite this article, use FASEB J. (April 23, 2002) 10.1096/fj.01-0880fje.

This article has been cited by other articles:

				A. Djamali Oxidative stress as a common pathway to chronic tubulointerstitial injury in kidney allografts Am J Physiol Renal Physiol, August 1, 2007; 293(2): F445 - F455. [Abstract] [Full Text] [PDF]

				K. Doi, K. Okamoto, K. Negishi, Y. Suzuki, A. Nakao, T. Fujita, A. Toda, T. Yokomizo, Y. Kita, Y. Kihara, et al. Attenuation of Folic Acid-Induced Renal Inflammatory Injury in Platelet-Activating Factor Receptor-Deficient Mice Am. J. Pathol., May 1, 2006; 168(5): 1413 - 1424. [Abstract] [Full Text] [PDF]

				H. Sugiura, T. Yoshida, K. Tsuchiya, M. Mitobe, S. Nishimura, S. Shirota, T. Akiba, and H. Nihei Klotho reduces apoptosis in experimental ischaemic acute renal failure Nephrol. Dial. Transplant., December 1, 2005; 20(12): 2636 - 2645. [Abstract] [Full Text] [PDF]

				P. Miketova, K. Kaemingk, M. Hockenberry, A. Pasvogel, J. Hutter, K. Krull, and I. M. Moore Oxidative Changes in Cerebral Spinal Fluid Phosphatidylcholine during Treatment for Acute Lymphoblastic Leukemia Biol Res Nurs, January 1, 2005; 6(3): 187 - 195. [Abstract] [PDF]

				K. Spargias, E. Alexopoulos, S. Kyrzopoulos, P. Iacovis, D. C. Greenwood, A. Manginas, V. Voudris, G. Pavlides, C. E. Buller, D. Kremastinos, et al. Ascorbic Acid Prevents Contrast-Mediated Nephropathy in Patients With Renal Dysfunction Undergoing Coronary Angiography or Intervention Circulation, November 2, 2004; 110(18): 2837 - 2842. [Abstract] [Full Text] [PDF]

				C. Berruyer, F. M. Martin, R. Castellano, A. Macone, F. Malergue, S. Garrido-Urbani, V. Millet, J. Imbert, S. Dupre, G. Pitari, et al. Vanin-1-/- Mice Exhibit a Glutathione-Mediated Tissue Resistance to Oxidative Stress Mol. Cell. Biol., August 15, 2004; 24(16): 7214 - 7224. [Abstract] [Full Text] [PDF]

				E. F. de Assis, A. R. Silva, L. F. C. Caiado, G. K. Marathe, G. A. Zimmerman, S. M. Prescott, T. M. McIntyre, P. T. Bozza, and H. C. de Castro-Faria-Neto Synergism Between Platelet-Activating Factor-Like Phospholipids and Peroxisome Proliferator-Activated Receptor {gamma} Agonists Generated During Low Density Lipoprotein Oxidation That Induces Lipid Body Formation in Leukocytes J. Immunol., August 15, 2003; 171(4): 2090 - 2098. [Abstract] [Full Text] [PDF]

				I. Herrero-Fresneda, J. Torras, J. M. Cruzado, E. Condom, A. Vidal, M. Riera, N. Lloberas, J. Alsina, and J. M. Grinyo Do Alloreactivity and Prolonged Cold Ischemia Cause Different Elementary Lesions in Chronic Allograft Nephropathy? Am. J. Pathol., January 1, 2003; 162(1): 127 - 137. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 16/8/908 01-0880fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by LLOBERAS, N. Articles by GRINYÓ, J. M. Search for Related Content PubMed PubMed Citation Articles by LLOBERAS, N. Articles by GRINYÓ, J. M.