Gadph

Fig. 3. Effect of renal ischemia on chemokine and ICAM-1 expression. (A) Twenty micrograms of total RNA was applied in each lane for the Northern blot analysis of KC expression. Data from two independent animals at 2- and 6-hr time points are shown. A significantly lower level of KC mRNA expression was detected in kidneys of GPx 1 and GPxP mice in comparison with nontransgenic animals. For comparison, results of Northern analysis with ICAM-1-labeled probe, showing no difference between the same groups of animals, are also presented. As an internal control hybridization with /i-actin was performed. (B) Five micrograms of total RNA was used for an RNase protection assay with the mCK-5 multiprobe template set (PharMingen). A probe set not treated with RNase is shown on the right. Corresponding RNase-protected fragments are highlighted. S, Sham-operated animals, which were killed 6 hr after operation. [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.l dehydrogenase (GAPDH)] are generated in vitro with a T7 polymerase transcription kit and [a-32P]UTP as a label. Total RNA (5 fig) is incubated overnight at 56° with a mixture of labeled antisense probes (5 x 105 Cherenkov counts) in 80% (v/v) formamide, 1 mM EDTA, 400 mM NaCl, and 40 mM piperazine-/V,/V'-bis 2-ethanesulfonic acid (PIPES) (pH 6.7) for 12-16 hr. Samples are then treated with an RNase A-Tl mixture, following proteinase K digestion for 15 min at 31°. Samples are phenol extracted and precipitated by ethanol. The protected RNA is then dissolved in loading buffer [80% (v/v) formamide, 1 mM EDTA, 50 mM Tris-borate (pH 8.3), 0.05% (w/v) xylene cyanol, and 0.05% (w/v) bromphenol blue], denatured for 3 min at 90°, and electrophoresed on a 5% (w/v) polyacryl-amide gel with urea. A standard curve is made by using undigested probes as markers and the identity of "RNase-protected" bands is established. To compare RNA amounts in the protected bands, the films are scanned with an imaging densitometer, and final values are factored relative to GAPDH levels. A similar approach may be used for any other chemokines not yet included in a kit, if analogous template plasmid with corresponding DNA fragment is created.

The results of a typical analysis by RNase protection assay of chemokine mRNA expression in kidneys from normal and GP transgenic mice subjected to ischemia-reperfusion are shown in Fig. 3B. Comparison of the level of activation of different chemokines indicates that they might be divided into several groups. Eotaxin was induced at 2 and 6 hr reperfusion in similar quantities in all three groups. MIP-l/J, IP-10, MCP-1, and TCA-3 were equally activated at 2 hr in normal, GPx 1, and GPxP mice, whereas at 6 hr a high level of expression persisted only in the first group. The most significant difference, however, was observed in the level of expression of MIP-2. This chemokine was highly inducible in normal mice. At 6 hr after ischemia, its level was 7.5 and 4.3 times higher in these animals in comparison with GPxl and GPxP mice, respectively (Fig. 3B). Importantly, only a few polymorphonuclear cells (PMNs) might be detected in kidneys of normal mice by this time point, indicating that induction of this chemokine precedes massive migration of leukocytes. KC and MIP-2 belong to the family of CXC chemokines, which have been shown to cause PMN activation and mediate chemotaxis and induction of the respiratory burst.30

Immunostaining

Frozen tissue sections (10 /¿m) are used for immunostaining. Sections stored in a freezer at —70° are removed and immediately covered with ice-cold fixation solution [2% (v/v) formaldehyde in phosphate-buffered saline, pH 7.4]. Sections are incubated for 10 min at 4° and then washed three times for 3 min each with wash buffer-saponin solution, containing 0.1% (w/v) saponin (Sigma, St. Louis, MO)

and Earl's buffered saline, pH 7.4 (EBSS; GIBCO-BRL, Gaithersburg, MD). Endogenous peroxidase activity is blocked by incubating sections in a blocking buffer [3M NaN3, 1% (v/v) H202, 0.1% (w/v) saponin in EBSS] for 60 min in the dark. Sections are then washed three times as described previously with wash buffer-saponin solution. Endogenous biotin activity is eliminated by washing with two blocking buffers (avidin-biotin blocking kit; Vector Laboratories, Burlingame, CA) sequentially, 1 hr each. Biotin solution is supplemented with 10% (v/v) donkey serum. After washing with wash buffer-saponin solution, sections are incubated overnight at room temperature with primary antibodies [goat anti-mouse antibodies (R&D Systems, Minneapolis, MN), 1:1000 to 1:2500 dilutions). Sections are then washed three times and incubated for 1 hr with biotin-donkey anti-goat IgG as a secondary antibody diluted 1:4000 (Jackson ImmunoResearch, West Grove, PA). Slides are washed three times in EBSS and incubated with Vectastain Elite ABC-peroxidase reagent (Vector Laboratories) supplemented with 0.1 % (w/v) saponin, for 30 min in the dark. Samples are developed with 3,3'-diaminobenzidine tetrahy-drochloride (DAB liquid substrate; Sigma) prepared according to the manufacturer instructions. After developing, sections are counterstained with hematoxylin, dehydrated, and mounted. Slides should be coded and interpreted by a pathologist who is blind to treatment. Each experiment is conducted several times and interpreted separately.

This method allowed us to demonstrate that MIP-2 and KC expression in a model of kidney ischemia-reperfusion injury is seen throughout the cortex, predominantly in tubules.19 This conclusion is supported by results of experiments with cultured tubular and mesangial cell lines exposed to chemical anoxia-ATP repletion as a model of ischemia-reperfusion.19 Mesangial cells play a key role in the inflammatory response in many pathological conditions, especially immunemediated glomerular diseases. In contrast, in the ischemia-reperfusion model these cells produced small amounts of CXC chemokines and therefore tubular cells play a central role in their synthesis.

Preparation of Whole Cell Extract for Protein Analysis and Immunoprecipitations

Kidney tissues (100 mg) are homogenized in 10 volumes of radioimmunopre-cipitation assay (RIPA) buffer [50 mMTris-HCl (pH 7.6), 150 mMNaCl, 1% (v/v) Nonidet P-40,0.5% (v/v) sodium deoxycholate, 0.1 % (w/v) SDS, I mMdithiothre-itol (DTT), 0.5 mM phenylmethylsulfonyl fluoride (PMSF), leupeptin, aprotinin, and pepstatin (10 ¿¿g/ml each), and the phosphatase inhibitors NaF (10 mM), NaV04 (1 mM), Na2Mo04 (1.5 mM), benzamidine (1 mM), glycerophosphate (20 mM), and p-nitrophenyl phosphate (20 mM)], using a Tissumizer (Tekmas, Cincinnati, OH). SDS-polyacrylamide gel electrophoresis sample buffer (5x) is added to afinal concentration of 50 mMTris-HCl (pH 6.8), 2% (w/v) SDS, 100 mM

DTT, 0.006% (w/v) bromphenol blue, and 10% (v/v) glycerol. Samples are boiled for 10 min and cooled on ice, and DNA is sheared by sonication. Lysates are finally centrifuged at 13,000g for 20 min at 4°, and supernatants are used for polyacrylamide gel electrophoresis and Western blotting. For immunoprecipita-tion analysis, RIPA homogenates are centrifuged at 13,000g for 20 min at 4°. The supernatants are transferred to new tubes. Anti-chemokine antibodies are added to 250 fig of protein extract and the mixture is incubated at 4° overnight. Thirty microliters of a 50:50 (w/v) slurry of protein A-Sepharose (Pharmacia, Piscataway, NJ) in RIPA buffer is then added to the samples. Immunopellets are collected and washed and protein is eluted by boiling for 1-2 min in SDS-polyacrylamide gel electrophoresis sample buffer. Samples are used for electrophoresis and Western blotting.

Involvement of Transcriptional and Posttranscriptional Mechanisms in Reactive Oxygen Species-Mediated Chemokine Activation

As noted in the Introduction, steady state levels of chemokine mRNAs may be modulated by transcriptional or posttranscriptional mechanisms. Transcriptional activation is mediated by increasing the activity of transcription factors. If transcription factors are induced in part by oxidative stress, in transgenic mice over-expressing antioxidant enzymes their level of activation should be significantly reduced. In this section we describe methods for analysis of activation of one of those factors, NF-/cB, which plays one of the key roles in chemokine response in general, as well as during ischemia-reperfusion in particular.

Preparation of Nuclear Extracts and Electrophoretic Mobility Shift Assay

To prepare nuclear extracts, kidneys are homogenized in 4 volumes of buffer A [10 mM HEPES (pH 7.9), 0.25 M sucrose, 15 mM KC1, 5 mM EDTA, 1 mM EGTA, 0.15 mM spermine, 0.16 mM spermidine, 1 mM DTT, 0.2 mM PMSF, and leupeptin, aprotinin, and pepstatin (10 /ig/ml each)], using eight strokes of a Dounce homogenizer (B-type pestle). After 10 min of incubation on ice, Nonidet P-40 is added to a final concentration 0.5% (v/v). Homogenates are centrifuged at lOOOg for 10 min at 4° and the pellets are washed twice by suspension in buffer B (the same as buffer A, but without sucrose). Nuclear extracts are then extracted with 4 volumes of buffer C [100 mM HEPES (pH 7.9), 1.5 mM MgCl2, 0.45 M KC1, 1 mM EDTA, 1 mM DTT, 10% (v/v) glycerol, 0.2 mM PMSF, and leupeptin, aprotinin, and pepstatin (10 /ig/ml each)]. Mixtures are allowed to stay for 30 min at 4° and nuclear extracts are obtained by centrifugation at 14,000g for 20 min at 4°. Aliquots of the extract should be stored at —80°. Protein content is assayed with the Bio-Rad (Hercules, CA) protein reagent. Oligonucleotides used in the electrophoretic mobility shift assay (EMSA) are annealed, end labeled with polynucleotide kinase and [y-32P]ATP (New England Nuclear), and purified by polyacrylamide gel electrophoresis. For detection of NF-/cB-binding activity the following oligonucleotides are used: 5'-AGCTCAGGGAATTTCCCTGGTCC-3' (containing the mouse MIP-2 NF-/cB-binding site) and 5'-GGCCAGGGAATTT CCCGGAGTA-3' (containing the kBI site of the mouse KC promoter region).21 For EMSA, 5-10 fig of extract is incubated in a reaction mixture containing 20 mM HEPES (pH 7.9), 60 mM KC1,5 mM MgCl2, 1 mM DTT, 0.2 mM EDTA, 0.5% (v/v) Nonidet P-40, 1 fig of poly(dI/dC), and 8% (v/v) glycerol in a final volume of 20 fil for 20 min at 4°. After preincubation, 105 cpm of radiolabeled DNA probe is added and incubation is continued for 15 min at room temperature. The DNA-protein complexes are separated on native 5% (w/v) polyacrylamide gels in 0.25 x Tris-borate-EDTA buffer. Supershift assays to prove the presence of specific proteins in shifted complexes are carried out after incubation of the nuclear extracts with 0.5 fig of rabbit polyclonal antibodies against p65 and p50 (sc-372 and sc-1192, respectively; Santa Cruz Biotechnology, Santa Cruz, CA) for 20 min at 4° followed by EMSA. For the competition assay, unlabeled double-stranded oligonucleotides should be added to the reaction mixtures at 10 or 25 molar excess over radiolabeled probes.

Results of a typical EMSA experiment with labeled MIP-2 NF-k B-binding site are shown in Fig. 4. At 6 hr of reperfusion NF-kB activity is significantly higher in kidneys from normal mice than from either GPxl or GPxP transgenic animals. In both cases, binding was specific and the composition of binding complexes from p65/p50 proteins was proved by supershift assay. The presence of c-Rel was not observed in complexes. These data indicate that GP overexpression in kidneys of transgenic mice modulated DNA binding in the regulatory regions of at least two chemokines during ischemia-reperfusion, thereby affecting activation and migration of PMNs.

In the majority of cells, NF-kB exists in an inactive form in the cytoplasm by being bound to an inhibitory protein, such as I-kBq; and I-kB/?.31 Treatment of cells with various inducers, including ischemia-reperfusion, results in the degradation of I-kB proteins, thus releasing the bound NF-kB, which translocates to the nucleus and upregulate chemokine expression. To measure the presence of inhibitory proteins in kidney extracts and the dynamics of their degradation, Western blotting analysis is used.

Protein Electrophoresis and Western Blotting

After measuring the protein concentration, protein samples are denatured in a boiling bath of sample buffer containing 250 mMTris-HCl (pH 6.8), 2% (w/v) SDS,

31 S. Ghosh, M. May, and E. Kopp, Annu. Rev. Immunol. 16, 225 (1998).

GPxl

GPxP

Competitor/ oligo:

GPxl

GPxP

^tmSmmmmmrnJi ^ttSmmmmmmim

^tmSmmmmmrnJi ^ttSmmmmmmim

GPxl

GPxP

Fig. 4. Binding of proteins to the kBI motif of the KC promoter region induced by ischemia-reperfusion. Kidney nuclear extracts from sham-operated animals (S) and mice after 32 min of ischemia and 2 or 6 hr of reperfusion were incubated with 32P-labeled oligonucleotides, and then the EMSA was performed. Extracts from two independent animals are shown for the 6-hr time point. The specificity of the binding was verified in 6-hr extracts in the presence of a 25-fold excess of non-labeled noncompetitive oligonucleodites (NS) or a 10- and 25-fold excess of nonlabeled probe. The binding proteins were identified by incubating the reaction mixtures in the presence of p65, p50, or c-Rel antibodies as well as with normal serum (MAb). [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.]

Competitor/ antibody:

p65/p50 p50/p50

Fig. 4. Binding of proteins to the kBI motif of the KC promoter region induced by ischemia-reperfusion. Kidney nuclear extracts from sham-operated animals (S) and mice after 32 min of ischemia and 2 or 6 hr of reperfusion were incubated with 32P-labeled oligonucleotides, and then the EMSA was performed. Extracts from two independent animals are shown for the 6-hr time point. The specificity of the binding was verified in 6-hr extracts in the presence of a 25-fold excess of non-labeled noncompetitive oligonucleodites (NS) or a 10- and 25-fold excess of nonlabeled probe. The binding proteins were identified by incubating the reaction mixtures in the presence of p65, p50, or c-Rel antibodies as well as with normal serum (MAb). [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.]

10% (v/v) glycerol, and 2% (v/v) 2-mercaptoethanol, and separated in minigels (7 cm; Bio-Rad) by SDS-15% (w/v) polyacrylamide gel electrophoresis. After electrophoresis, gels are electroblotted onto polyvinylidene difluoride (PVDF) membrane (Immobilon; Millipore, Bedford, MA). Membranes are blocked in a BLOTTO solution containing lx TBS [10 mM Tris-HCl (pH 8.0), 150 mM NaCl], 5% (w/v) milk, and 0.1% (v/v) Tween 20 for 1 hr at room temperature. Membranes are then incubated with rabbit polyclonal antibodies against I-zcBa and I-/cB/S (sc-371 and sc-969, respectively; Santa Cruz Biotechnology) diluted in BLOTTO for 12 hr at 4°. After washings, membranes are incubated in BLOTTO with secondary antibodies (1:1000-1:20,000 dilution). After extensive washings in 1 x TBS with 0.1 % (v/v) Tween 20, proteins are detected with a Phototope-HRP Western blot detection kit and LumiGlo reagent (both from New England BioLabs, Beverly, MA). Autoradiographs are quantitated by densitometry.

As shown in Fig. 5, at 30 and 45 min after ischemia-reperfusion, fast degradation of 1-kBg! and 1-kB/j was detected, although the level of depletion was significantly higher in normal mice. The degradation of I-kB« and I-kB/3 correlates well with the level of nuclear p65. Therefore, overexpression of GPs seems to inhibit the influx of NF-/cB into the nucleus by affecting degradation of its inhibitors.

To substantiate the transcriptional mechanism of CXC chemokine activation in vivo, the run-on transcriptional rate might be evaluated by using nuclei from kidneys isolated from sham-operated normal and transgenic mice as well as from nTg GPx1 GPxP

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