S 30 45 360 S 30 45 360 S 30 45 360

Fig. 5. Western blot analysis of degradation of I-zcBa andI-fcB/8 induced by ischemia-reperfusion. Overexpression of GPxl and GPxP in transgenic mice inhibits I-x Bor and I-kB/î degradation in kidney extracts at 30 and 45 min of reperfusion in comparison with nontransgenic mice. S, Sham-operated animals. These data correlate with p65 protein accumulation in the nuclear extracts from the same animals. [Reprinted with permission from N. Ishibashi, M. Weisbrot-Lefkowitz, K. Reuhl, M. Inouye, and O. Mirochnitchenko, J. Immunol. 163, 5666 (1999). Copyright © 1999 by The American Association of Immunologists.]

Fig. 5. Western blot analysis of degradation of I-zcBa andI-fcB/8 induced by ischemia-reperfusion. Overexpression of GPxl and GPxP in transgenic mice inhibits I-x Bor and I-kB/î degradation in kidney extracts at 30 and 45 min of reperfusion in comparison with nontransgenic mice. S, Sham-operated animals. These data correlate with p65 protein accumulation in the nuclear extracts from the same animals. [Reprinted with permission from N. Ishibashi, M. Weisbrot-Lefkowitz, K. Reuhl, M. Inouye, and O. Mirochnitchenko, J. Immunol. 163, 5666 (1999). Copyright © 1999 by The American Association of Immunologists.]

animals after ischemia-reperfusion. Another mechanism of posttranscriptional activation of chemokines involves an elevation in translational efficiency. To address this possibility, polysomal fractionation experiments are carried out in mouse kidneys after renal ischemia-reperfusion. The abundance of KC and MIP-2 transcripts is measured in the polysomal fractions, using RNA slot-blot analysis and chemokine-specific probes. These amounts should be adjusted by the amount of total mRNA in kidney homogenates. If increased amounts of polysomal bound mRNA are detected, it suggests an increased translatability for chemokines as one of the mechanisms for their activation during ischemia-reperfusion.

Nuclear Run-on Transcriptional Assay

To prepare nuclei from kidney cortex of mice after ischemia-reperfusion, ultra-centrifugation in sucrose gradients is used as already described in detail.32 Nuclei are resuspended in 50 mM HEPES (pH 8.0), 5 mM MgCl2, 5 mM DTT, BSA (1 mg/ml), 25% (v/v) glycerol and stored in liquid nitrogen. On thawing, nuclei are diluted in reaction buffer [20 mM Tris-HCl (pH 7.9), 20% (v/v) glycerol, 140 mM KC1, 5 mM MgCl2, 1 mM MnCl2, 14 mM 2-mercaptoethanol, ATP, GTP, and CTP (1 mM each), 10 mM creatine phosphate, 20 units/ml creatine phosphokinase] with 1 mCi of [32P]UTP (3000 Ci/mM) and incubated at 30° for 30 min, followed by DNase I treatment (150 units/ml). In pilot experiments, optimization of the incubation time may be needed. After proteinase K treatment, RNA is extracted and equal amounts of newly transcribed RNA are hybridized with 5 ¡ig of denatured, linearized chemokine and /J-actin cDNAs cross-linked to membranes. This method allows measurement of the rate of chemokine RNA transcription in kidney extracts from normal and transgenic mice after ischemia-reperfusion.

Quantification of Chemokine mRNA Bound to Polysomes

The polysome profile analysis is performed as follows: kidneys after ischemia-reperfusion are homogenized in 10 mM Tris-HCl (pH 7.4), 100 mM KC1, 10 mM MgCl2, and 1 mM dithiothreitol. The homogenate is centrifuged at 13,000g for 30 min at 4° to remove the mitochondria. Ten absorbance units (OD26o) of the postmitochondrial supernatant is layered onto 12.5 ml of 10 to 45% (w/v) sucrose density gradient prepared in the above-described buffer. The gradients are centrifuged in a Beckman (Fullerton, CA) SW40 Ti rotor at 38,000 rpm for 100 min at 4°. Fractions (0.5 ml) are collected by upward displacement, and the OD254 is measured and plotted. RNA isolated from the fractions is slot-blotted onto nylon

32 D. L. Spector, R. D. Goldman, and L. A. Leinwand, "Cells: A Laboratory Manual." Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998.

membrane and hybridized with random primer P-labeled chemokine cDNA probes and washed under stringent conditions.

Conclusion

Interaction between oxidative stress and inflammatory response is not a new concept. One of the best recognized sources of the ROS themselves is activated phagocytes, which under many pathological conditions can cause direct damage to the surrounding tissues. Several cytokines (such as TNF-a), released by the activated cells after interaction with target cells, are known to produce an oxidative stress response that might lead to dramatic changes in cellular fate (e.g., induce apoptosis). It has also become apparent that low levels of ROS play an important role in activation and recruitment of inflammatory cells through induction of adhe-sionreceptors and regulation ofthe activity of lipoxygenases andcyclooxygenases, transcription factors, protein kinases, and other mediators. All this indicates that there are many ways in which ROS can influence the function of the immune system as well as its interaction with other tissues. In this chapter we have presented methods by which to use genetically engineered animal models, which are able to modulate the level of oxidative stress, to study the mechanisms of chemokine activation in pathological processes.

Overexpression of human GPs in transgenic mice significantly protects animals against kidney ischemia-reperfusion injury.19 Both enzymes have similar substrate specificity, and are overproduced in the kidneys of our transgenic mice. The protective effect was accompanied by a significant decrease in chemokine expression (KC and MIP-2 in particular) and neutrophil migration. Although it might not be the only mechanism of protection in transgenic mice, the existence of compelling evidence for the role of neutrophils in renal ischemia-reperfusion indicates the ability of GPs to influence the outcome of ischemia-reperfusion injury by modulation of early ROS production.

Although we19 and others33 have shown that several chemokines are induced during ischemia-reperfusion, the biological significance of the activation of different chemokines on the development of renal injury during ischemia-reperfusion has not been directly tested yet. Selective activation of certain chemokines and their cell type specificity warrants detailed analysis of the mechanisms of activation, including identification and study of the interaction of different transcription factors and posttranscriptional mechanisms. A series of transcription factors that participate in chemokine activation have been reported. Even though the most indispensable among them was NF-kB, synergistic participation of other transcription

33 R. Safirstein, J. Megyesi, S. J. Saggi, P. M. Price, M. Poon, B. J Rollins, and M. B. Taubman, Am. J. Physiol. 261, f1095 (1991).

factors also was shown. Importantly, various combinations of these factors mediate cell type-specific activation of a particular chemokine.

In this chapter we have focused mainly on analysis of the activation of different chemokines and characterization of factors directly affecting this activation. The next objective will be the study of upstream mediators, such as Rac proteins, stress-and mitogen-activated kinases, and ceramide and sphingomyelinase pathways. All of them are activated during ischemia-reperfusion and are sensitive to ox/redox regulation, and have been implicated in the activation of transcription factors interacting with chemokine promoters. Methods to study those mechanisms have been described.34

The possibility that modulation of chemoattractant activation via antioxidants can significantly affect tissue injury warrants further investigation and will lead to the development of new techniques.

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