Antisense oligonucleotides for ICAM-1 attenuate reperfusion injury and renal failure in the rat

Kidney International, Vol. 50 (1996), pp. 473—480

Antisense oligonucleotides for ICAM-1 attenuate reperfusion injury and renal failure in the rat HERMANN HALLER, Dusi DRAGUN, ANNEGRET MIETHKE, JooN KEUN PARK, ANGELIKA WETS, ANDREA LIPPOLDT, VOLKMAR GR0I3, and FRIEDRICH C. Lun Franz Voihard Clinic at the Mwc DeibrOck Center for Molecular Medicine, Virchow Klinikum, Humboldt University of Berlin, Berlin, Germany

Antisense oligonucleotides for ICAM-1 attenuate reperfusion injury and renal failure in the rat. The leukocyte adhesion molecule ICAM-1 is implicated in ischemic renal reperfusion injury. We tested the utility of an TCAM-1 antisense oligodeoxyribonucleotide (ODN) with lipofectin, six

injury and protected renal function [17, i8]. However, antibody treatment has inherent problems that could be circumvented with antisense oligonucleotides. We tested the hypothesis that ICAM-1 hours prior to 30 minutes of bilateral renal ischemia in the rat. We antisense oligonucleotides can inhibit ICAM-1 expression in the measured ICAM-1 expression by immunohistochemistry and Western ischemic kidney, prevent neutrophil adhesion and infiltration, and blot. Our antisense ODN showed a specific ICAM-1 surface expression thereby ameliorate ischemia-induced renal failure. inhibition in vitro. We then assessed ICAM-1 expression, leukocyte

infiltration, serum creatinine, serum urea concentration, and renal histology in rats subjected to renal ischemia and controls. Serum creatinine and

Methods

urea concentrations 12 and 24 hours post-ischemia were increased in saline treated and reverse ODN treated rats, compared to antisense ODN treated or sham operated rats (P < 0.05). Western blotting showed decreased ICAM-1 protein in antisense ODN-treated kidneys, compared to reverse ODN treated and saline treated ischemic controls (P < 0.05). Antisense ODN also ameliorated the ischemia-induced infiltration of granulocytes and macrophages (P < 0.05), and resulted in less cortical

Oligonucleotides

Phosphorothioate oligodeoxyribonucleotides (ODN) were purchased (Tib Molbiol, Berlin, Germany). We selected an antisense

ODN (ISIS 1939) against the human 3' untranslated region derived from the rat ICAM sequence RSICAM and the human ICAM-1 sequence HSICAMO1 (European Molecular Biology Laboratories data base, Heidelberg, Germany) [19, 20]. For the rat experiments, we compared rat and human sequence data and used the rat homologue to ISIS 1939 (5' ACC GGA TAT CAC

renal damage as assessed by a quantitative pathological grading scale (P < 0.05), compared to reverse ODN or saline treatment. Thus, antisense

ODN for ICAM-1 protected the kidney against ischemic renal failure. The clinical applicability of these findings extends beyond ischemic acute renal failure.

ACC TIC CT 3'). The reverse ODN sequence was used as control. We used a cationic lipid solution (Lipofectin; GIBCO BAL, Life Technologies, Hamburg, Germany) to enhance the

Reperfusion injury involves activated leukocytes with an enhanced adhesiveness to the endothelium [1, 2]. In the kidney, the adherent leukocytes plug capillaries, generate free radicals and proteolytic enzymes, and release cytokines [3]. The vasa recta become congested, capillary perfusion is impaired [4], and endothelial permeability is increased [5]. Adhesion molecule mediated, neutrophil endothelial binding is inherent to this process [6, 7]. The leukocyte 2 integrin complex (CD11/CD18) interacts with

ODN uptake. In the in vitro experiments, lipofectin was added five

minutes prior to the ODN application, while in the animal experiments ODN and lipofectin were added together. Animal protocol Male Sprague-Dawley rats (Ivanovas, Kisslegg, Germany) aged

12 weeks, were maintained in cages at 24 2°C and were given

the endothelial ligand intercellular adhesion molecule-i tap water and a standard rat diet (C-1000; Altromin, Lage, (ICAM-1) [8]. The initial rolling of neutrophils is mediated by the selectins, while CD11/CD18-ICAM-1 interactions are responsible for leukocyte adhesion and diapedesis [9]. Studies in liver, brain, and myocardium [10—14] showed that ICAM-1 is up-regulated

Germany) containing 0.2% sodium by weight. They were fasted 12

hours before surgery. After thiohexital (Brevimytal; Bayer, Leverkuesen, Germany) anesthesia, a cathether was placed in the right femoral artery and vein. Lipofectin solution (1 ml) containduring ischemia-reperfusion. Antibodies against either CD1 if ing antisense ODN, reverse ODN control, lipofectin control, or CD18 or ICAM-1 prevented tissue damage and protect organ 0.9% saline control were administered to four groups of 12 rats function in other studies [10, 15, 16]. Antibodies against both each, via the venous catheter six hours prior to ischemia. We also CD11/CDII8 and ICAM-1 ameliorated ischemia-induced tissue included a sham-operated saline-injected group and a shamoperated lipofectin-injected group to control for any operative, or lipofectin-induced effects. Finally, we included a group of rats that Received for publication November 28, 1995 and in revised form February 1, 1996 Accepted for publication February 5, 1996

© 1996 by the International Society of Nephrology

were sacrificed to determine basal ICAM-1 expression in nonanesthetized, nonmanipulated rats. Ischemic injury was induced in

anesthetized rats by clamping the left and right renal hila with microaneurysm clamps for 30 minutes after a bilateral flank 473

474

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Hailer et al: Antisense oligonucleotides for repeifusion injuiy

incision. Blood samples (0.5 ml) for urea and creatinine determi- of ICAM-1 on cultured endothelial cells we used a modified nation were obtained from the femoral arterial cathether at —6, 0, ELISA protocol [21]. 12, and 24 hours after reperfusion. Rats were killed under chloral Western blotting hydrate anesthesia and the kidneys removed at 12 and 24 hours. The excised kidneys were divided with forceps and scissors into In vitro procedures and ELISA for ICAM-1 medulla and cortex. The tissue slices were treated with ice cold For the in vitro testing of ODN and lipofectin concentrations we buffer (10 mrvi Tris-HCI pH 7.4, 100 mvi NaCI, 300 mivi sucrose, I used cultured human umbilical vein endothelial cells, which were mM EDTA, 0.2 mrvi PMSF, I jig/mI leupeptin, 0.5% Triton X-100, isolated from umbilical cords by chymotrypsin treatment as 0.2% NaN3) and immediately homogenized. The homogenate was described previously [211. For measurement of surface expression then spun at 13 000 g for five minutes and the supernatant was

475

Holler et al: Antisense oligonucleotides for reperfusion injuty

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Fig. 1. Effect of sense and antisense oligonucleotides on JCAM-l expression

in rat kidney. A. (opposite page, top) Immunohistochemical staining for ICAM-l in ischemic animals with saline treatment (left, arrows on tubular cell and vascular endothelial cell surface), antisense oligonucleotides (ODN) treatment (middle) and reverse ODN treatment (right, arrows on same structures) (representative of 30 photomicrographs). B. Densitometnc data from these sections (N = 30, *P < 0.05). Symbols are: (LI) no treatment; () anti-sense ODN; () sense ODN. Ischemia induced a marked increase in ICAM-l expression along the endothelial cell lining of the blood vessels and in the peritubular area. Antisensc ODN prevented the ischemia-induced increase in IcAM-1 expression, both in the vasculature and in the penitubular area. Reverse ODN had no effect.

lschemia I

0

—6

I

I

12

24

B

200 180 160 140 120 100

ODN lschemia

a 80

B

lschemia

I —6

I

I

0

12

24

Time, hours Fig. 3. Effect of antisense and reverse ODN on serum creatinine concentra-

lschemia + as ODN

I

tions (A) and urea concentrations (B) before and after ischemia. Saline treated animals showed a significant increase in creatinine and urea

concentrations, peaking at 24 hours. Antisense ODN prevented the ischemia-induced increase in creatinine and urea concentrations. Reverse ODN had no effect. Symbols are: (0) ischemia; (+) ischemia + AS-ODN; (A) ischemia + rev-ODN; (LI) sham-operated. *p < 0.05 compared to ischemia without treatment.

lschemia + rev ODN

I 0 10 20 30 40 50 60 70 8090100 % Of vessels counted

Fig. 4. A. (opposite page, bottom) Immunohistochemical staining of isch-

(New England Nuclear, Boston, MA, USA). The membrane was

then treated according to a commercially available protocol (Serva, Heidelberg, Germany). The membrane was incubated

emic kidneys for granulocytes (arrows on representative cells) with saline

with a monoclonal antibody directed against rat ICAM-1 (clone

treatment (right) (representative of 30 photomicrographs). B. The number of

1A29; Medac, Hamburg, Germany), followed by a goat antimouse

granulocyte infiltrates qualitatively expressed as no infiltrates (s), 1 to 4 infiltrates (Lu, or 5 infiltrates (•) from these immunohistochemical sections (vessels counted N > 50; *p < 0.05). Antisense ODN decreased granulocyte infiltration.

antibody, linked to alkaline phosphatase (Dianova, Hamburg, Germany). After equilibration of the blot in assay buffer (pH

treatment (left), antisense ODN treatment (middle), and reverse ODN

10.0), the detection was carried out by incubation with the AP-substrate CDP Star (Serva, Heidelberg, Germany) for five

minutes. collected. After determination of protein content, the immunoDot blot and densitometry were used for quantitation of blotting was carried out as previously published [22]. Briefly, 25 ICAM-i expression. A total of 0.1 j.tg of each sample was dotted jxg of each sample were run on a 10% polyacrylamide gel and onto Poly-Screen PVDF Transfer-Membrane. The further treatelectro-blotted onto a Poly-Screen PVDF Transfer Membrane ment of the membrane was performed according to the above

476

HaIler et al: Antisense oligonucleotides for reperfusion injuly

lschemia

Cortex

Medulla

H

lschemia

lschemia

+asODN

+revODN

_

__

H

O.D.

Arbitrary units

Fig. 2. A. Western blot for ICAM-1 in the cortex and in the medulla of kidneys from saline treated ischemic animals (left), antisense ODN treated animals (middle), and reverse ODN treated animals (right) (representative experiment of 12 kidneys in each group). B. Densitometric data from these Western blots (N = 12 in each group). Ischemia induced a marked increase in ICAM-1 expression both in the renal cortex (LI) and in the renal medulla (s). Treatment with antisense ODN prevented the ischemia-induced increase in ICAM-1 expression both in the vasculature and in the peritubular area. Reverse

ODN had no effect. *P <

described protocol (Serva, Heidelberg, Germany). The signal was quantified densitometrically with a Biorad (Freiburg, Germany) video densitometer 620. The signals were then integrated and the results were expressed in arbitrary units.

0.05.

microdensitometric method [23]. The autoradiograms were digitized directly via a television camera (CCD 72/MTi-camera) on a screen, allowing the measurement of an entire section at once. Transmissions as well as optical densities were obtained utilizing post-processing programs. The degree of leukocyte infiltration was determined by visually counting the stained granulocytes and monocytes, respectively, in the perivascular areas across kidney sections, as described previ-

Immunohistochemistiy and pathology Immunohistochemistry was carried Out as previously described [23]. For immunohistochemical staining, the sections were incubated with the monoclonal antibody anti-rat ICAM-1, clone 1A29, ously [23]. To quantify the results we used a grading scale in which (Medac, Hamburg, Germany), monoclonal antibody anti-rat 0 indicated that no infiltrating cells were present, I indicated that monocytes, clone ED 1, (Camon, Wiesbaden, Germany) mono- one to four infiltrating leukocytes were identified, and 2 indicated clonal antibody anti-rat granulocytes, clone His 48, (Dianova, infiltrates of 5 or more cells. The infiltrates were expressed as % Hamburg, Germany) and diluted in RPMI (Seromed, Heidelberg, of the vessels analyzed. Kidneys from six animals in each group Germany) for 30 minutes at room temperature in a humid were used for this purpose. Multiple sections were examined, so chamber. After washing with TBS, the sections were incubated that at least 50 separate vessels were evaluated from each of the with a rabbit anti-mouse bridging antibody (Dako, Hamburg, three groups. Six half kidneys from each group at both 12 and 24 hours were Germany). The immunoreactivity was visualized with an alkaline sectioned, fixed in formalin, and stained with hematoxylin and phosphatase antialkaline phosphatase (APAAP) complex (Dako). For detection and development we used the neufuchsin-naphthol- eosin as described for our laboratory previously [24]. Without As-Bi-phosphate substrate (Merck, Darmstadt, Germany) as de- knowledge of the regimens, sections from each kidney were evaluated by a pathologist. Tubular necrosis was assessed with a scribed.

Semiquantitative data on the ICAM-1 expression were obtained by measuring the gray values of the film autoradiograms using the microdensitometrical program of the IBAS 2.5 (Kontron, Konstanz, Germany) according to a previously published

quantitative grading scale (0 to 3) corresponding to none, mild, moderate, or severe necrosis as outlined elsewhere [241. Cell swelling, vacuolization, nuclear pyknosis, necrosis and cellular infiltrate were evaluated.

Hailer et ai: Antisense oiigonucieotides for reperfusion injuly

Statistical analysis

477

staining, which was more prominent than in the antisense ODN

group. Reverse ODN treatment, on the other hand, did not

Statistical analysis was carried out on a Macintosh II computer ameliorate the granulocyte infiltration. Granulocyte infiltration (Apple Inc., Cupertino, CA, USA) with a commercially available was found in only 23 vessels in antisense treated kidneys, com-

program (Statview; Cricket Software Inc., Philadelphia, PA, pared to 56 in untreated ischemic kidneys and 57 in kidneys USA). The results (mean SEM) represent duplicate measure- treated with reverse ODN (P < 0.05). We also quantified the ments made on seven to ten separate rats from each group. numbers of infiltrates (Fig. 4B). Granulocyte infiltrates were Nonparametric (Wilcoxon test) and parametric (2 way ANOVA) significantly reduced by antisense treatment (P < 0.05), while were used as appropriate. P c 0.05 was considered significant.

Results

To select the most effective lipofectin concentration for the gene transfer, we examined lipofectin concentrations ranging from 0.1 to 5 mg/mg DNA on TNF-a induced expression of ICAM-1. The endothelial cells were incubated with increasing concentrations of lipofectin for four hours. Antisense oligonucleotides inhibited the TNF-n induced ICAM-1 expression (P < 0.01) at all concentrations used, while reverse oligonucleotides had only a minor, nonsignificant effect. At lipofectin concentrations 0.1 to 0.5 mg/mg DNA, we observed only a slight, nonsigificant inhibition of TNF-a induced ICAM-1 expression, while concentrations of 0.8 mgImgDNA inhibited ICAM-1 effectively.

Lipofectin at 2 mg/mg DNA damaged the cells. From these

reverse ODN did not influence the ischemia-induced granulocyte infiltration.

We conducted the same immunohistochemical staining and quantitative evaluation for monocytes. Figure 5A shows the monocyte perivascular staining in the renal cortex from ischemic animals with saline, compared to antisense ODN treated animals

and reverse ODN treated animals. Monocytes around blood vessels were more frequent in the saline and reverse ODN treated sections, compared to the antisense ODN treated sections. Mono-

cyte infiltration was found in 39 vessels of antisense treated kidneys, compared to 54 in untreated ischemic kidneys and 56 in kidneys treated with reverse ODN (P < 0.05). We also quantified the numbers of infiltrates (Fig. SB). Monocyte infiltrates were

significantly reduced by antisense treatment (P < 0.05), while reverse ODN did not influence the ischemia-induced granulocyte

experiments, a lipofectin concentration of 0.8 mg/mg DNA and a infiltration. Representative hematoxylin and eosin renal cortical sections ODN concentration of 2 mg/kg body wt was chosen for the in vitro studies.

from saline treated, antisense ODN treated, and reverse ODN

Figure 1A shows the effect of reverse and antisense oligonucle- treated rats subjected to renal ischemia are shown in Figure 6. otides on expression of ICAM-1 in renal cortical vessels 24 hours Rats with saline treated (Fig. 6, left) and reverse ODN treated after 30 minutes of ischemia. The saline injected control showed (Fig. 6, right) renal ischemia showed moderate to severe necrosis. ICAM-1 staining along the vascular intima. This staining was The sections show tubular cell swelling, cellular vacuolization, decreased in antisense ODN treated animals. The reverse ODN pyknotic nuclei, and round cell infiltration. Rats treated with treated animals, on the other hand, showed prominent ICAM-1 antisense ODN (Fig. 6, middle) showed prominent cell swelling staining. Figure lB shows the corresponding densitometric anal- but only mild necrosis. Only a few pyknotic nuclei were observed, ysis. Saline and reverse ODN treatment showed similar densities, and the round cell infiltration was less than in the other two while the antisense ODN treated animals showed significantly less groups. Analysis of the grading scales revealed signficantly less severe renal cortical damage in kidneys from antisense ODN ICAM-1 staining density. Figure 2A shows the Western blotting of these same kidneys for treated animals compared to the other two ischemic groups. ICAM-l protein. Both the renal cortex and medulla from saline Discussion treated and reverse ODN treated rats showed prominent amounts We were successful in inhibiting ICAM-1 expression in endoof ICAM-1, while the antisense ODN treated animals showed significantly less amounts of ICAM-1. Optical density (Fig. 2B) thelial cells in vitro with antisense ODN [25]. We used these illustrates the significant differences between these groups. results to estimate the lipofectin and antisense ODN concentraICAM-1 expression in nonischemic sham operated, saline, or tions necessary to inhibit ICAM-1 expression in the rat with renal lipofectin treated animals was not absent compared to unmanipu- ischemia. We found that antisense ODN indeed inhibited lated control rats. We observed a detectable increase in ICAM-1 ICAM-1 expression in vivo, and that it also ameliorated acute expression in kidneys from these rats, indicating that the manip- renal failure according to functional and histological criteria. The ulation itself up-regulates ICAM-1 (optical densities in arbitrary inhibited ICAM-1 expression was associated with decreased vasunits 11.9 1.3 vs. 8.5 1.7 in the medulla; 12.4 1.5 vs. 8.6 cular infiltration of both granulocytes and monocytes, which 2.3 in the cortex). We found no difference between lipofectin and supports the conclusion that adhesion molecules are important in saline treated, sham operated animals. the genesis of ischemic acute renal failure. We believe that our Figure 3 shows the effects on renal function. Creatinine (Fig. conclusions are supported by the fact that we showed the effect of 3A) and urea (Fig. 3B) concentrations in serum increased by 12 antisense treatment on ICAM-1 expression, production, and hours and 24 hours in rats with ischemia treated with saline or effect in terms of leukocyte infiltration, as well as demonstrating with reverse ODN, compared to rats given antisense ODN. Sham an effect on renal histology and function. operated rats had no increase in creatinine or urea concentraOur finding that ICAM-1 is important in reperfusion injury is tions, compared either to baseline values or to values from not new. Antibodies directed against ICAM-1 effectively block its role in cell-to-cell adhesion and can ameliorate ischemic reperfuunmanipulated rats (not shown). Figure 4A shows the effect on perivascular granulocyte infiltra- sion injury in several animal models [10, 15, 17, 18, 26—28]. A tion at 12 hours as shown by immunohistochemistry. Saline- modest but detectable up-regulation of ICAM-1 was present in treated ischemic rat kidneys showed considerable granulocyte our sham operated rats, indicating the sensitivity of the cell-to-cell

478

HaIler et a!: Antisense oligonucleotides for reperJhsion injuty

Isehemia

Ischemia

+ as ODN

Ischernia

Ischemia

Ischeniia + rev ODN

Ischemia

+ as ODN + rev ODN adhesion mechanism. However, our purpose was not to retest this ICAM-1 expression on the endothelial cell surface in vitro and hypothesis. Instead, we sought to develop a novel, nonantibody ischemia-induced up-regulation of ICAM-1 protein in vivo. The means of blocking ICAM-1 expression in vivo to specifically inhibitory effect seems to involve RNase H hydrolysis of the target ameliorate ischemic reperfusion injury. We used a phosphorothio- mRNA [191. Experience with antisense technology is controversial ate-stabilized ODN against the 3' untranslated region of ICAM-1. with regard to specificity of the inhibitory effect of phosphothi-

Bennett and coworkers have previously shown that antisense ODN against this region are highly specific and very effective [25]. Furthermore, the antisense ODN selectively inhibited ICAM-1

expression while other endothelial adhesion molecules were not affected [251. Our antisense ODN also inhibited TNF-a induced

orate ODN [29]. Our results support the notion that phosphorothioate ODN against endothelial adhesion molecules are a specific therapeutic tool. To enhance ODN uptake we used the cationic lipid preparation, lipofectin. We established the optimal dose in the in vitro

479

Hailer et al: Antisense oligonucleotides for reperfusion injury

transplant medicine. In experimental animals, both renal isografts

B

and kidneys exposed to transient ischemia show a long-term course of progressive proteinuria and gradual renal failure [32]. Thus, initial damage appears to foster a subsequent decline, even if conventional rejection mechanisms are not involved. Cadaveric donor kidneys are routinely stored in Collins or similar solutions for 12 to 72 hours before transplantation.Delayed graft function from ischemia and reperfusion injury is a major post-transplant

-1————-——--l

Ischemia + as ODN

111111111I I

lschemia + rev ODN

o 1020 304050 607080 90100

% Of vessels counted Fig. 5. A. (opposite page, top) Immunohistochemical staining of ischemic kidneys for monocytes (arrows on representative cells) with saline treatment (left), antisense ODN treatment (middle), and reverse ODN treated animal (right) (representative of 30 photo,nicrographs). B. The number of granulocyte infiltrates qualitatively expressed as no infiltrates (s), ito 4 infiltrates (Li), or 5 infiltrates (•) from these immunohistochemical sections (vessels counted N> 50; P < 0.05). Antisense ODN decreased monocyte infiltration.

problem and has a direct negative impact on long-term graft survival [33]. Transplanted hearts and livers also are subject to reperfusion injury and ICAM-1 seems to play a role in acute and chronic rejection [34—38]. The antisense ODN treatment could avoid any potential immunological problems that might accompany the use of antibodies directed against adhesion molecules, particularly if the therapy is required more than once [20]. We would envision a multiple antisense ODN treatment of transplant grafts directed against a variety of adhesion molecules associated with reperfusion injury and are currently pursuing experimental

studies of this nature. Finally, we believe the ODN approach opens new avenues to investigate pathogenetic mechanisms involved in acute renal failure and other renal diseases. Acknowledgment

This project was supported by the Deutsche Forschungsgemeinschaft (DFG Ha - 1388/2-3). Reprint requests to Hermann HaIler, M.D., Franz Volhard Clinic, Wiltberg Strasse 50, 13122 Berlin, Germany.

Fig. 6. (opposite page, below) Hematoxylin and casio-stained thin sections of fixed, paraffin-embedded, ischemic rat kidneys. Saline treatment (left),

References

antisense ODN treatment (middle) and reverse ODN treated animal

(right) (representative of 30 sections). Inflammatory infiltrate and tubular

necrosis manifest by swollen, vacuolated proximal tubular cells with pyknotic nuclei, necrotic cells and loss of the basal lam ma, was diminished by antisense ODN treatment. A quantitative grading scale verified these findings.

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