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ORIGINAL ARTICLES

Characterization of Renal Ischemia-Reperfusion Injury in Rats

Patricia Williams, Henry Lopez, Deborah Britt, Christine Chan, Alan Ezrin, and Rod Hottendorf

Glycomed Incorporated, Alameda, California U.S.A.

The purpose of this study was to characterize the time course of renal &hernia-reperfusion injury in the rat. Male Sprague-Dawley rats were subjected to bilateral renal clamping for 45 min. At reestablishment of blood flow, the rats were divided into nine groups (representing 0, 0.5, 1, 2, 4, 6, 9, and 24 h, and 1 week post-ischemia). At each time point, blood samples were taken for analysis of blood urea nitrogen (BUN) and creatinine, and both kidneys were harvested for histopathology and myeloperoxidase activity (MPO) assays. An intracellular adhesion molecule (ICAM-1) monoclonal antibody (IMAb) was tested in a separate group of animals (1 mg/rat) to confirm that it may provide renal protection previously reported by Kelly et al. (1994). Following renal ischemia, significant increases in serum BUN and creatinine were observed compared to levels in normal animals. Serum BUN and creatinine increased 2,4, and 6 h post-ischemia leading to peak elevations 24 h post-ischemia. Values returned to normal at the 1 week time point. MPO activity was slightly increased 2 and 4 h following ischemia, with peak elevations occurring at the 6-h and 9-h time points. Histopathologic examination of kidneys revealed that the most severe damage occurred at the 24-h time point, which correlated with the peak elevations in serum BUN and creatinine. Evidence of renal injury was still evident histologically 1 week following ischemia, although renal function tests (BUN and creatinine) had returned to normal. In summary, renal injury following ischemia may be demonstrated as early as 4 h post-ischemia as judged by changes in renal function, MPO levels, and renal histopathology. However, based upon renal function tests and histology, peak injury is observed approximately 24 h following ischemia. The ICAM- monoclonal antibody, ICAM-Ab, provided some renal protection against ischemia-reperfusion injury in this study as measured by serum creatinine, BUN and renal histopathology. However, in contrast to the results reported by Kelly et al., the magnitude of the protective effects was not as dramatic in the present study, and furthermore, no reductions in renal MPO activity were observed. 0 1997 Elsevier Science Inc.

Key Words: Renal ischemia; ICAM antibody; Rat

Introduction Shock and reperfusion injury are associated with

hospitalization of nearly 4 million patients annually in the United States. The kidney, in particular, is at risk in situations of low-flow ischemia/reperfusion following aortic aneurysm repair (Paller, 1994). The mechanisms underlying ischemia/reperfusion damage to kidneys are likely multifactorial and interdependent, involving hyp- oxia, free radical damage and inflammatory responses

To whom correspondence should be addressed: Patricia D. Wil- liams, Ph.D., BioChem Therapeutic Inc., 275 Armand-Frappier Blvd., Lava], QuCbec H7V 4A7 Canada.

Revised and accepted October 15, 1996.

Journal of Pharmacological and Toxicological Methods 37, 1-7 (1997) 0 1997 Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010

(Paller, 1994). Thus, agents proposed to be useful in the clinical setting of renal ischemia/reperfusion damage include free-radical scavengers (Dillon et al., 1993; Sabbatini et al., 1994) and inflammatory cell adhesion inhibitors (Kelly et al., 1994; Rabb et al., 1994). The development and use of animal models to evaluate these investigational agents is critical to the discovery and advancement of new drug therapies. Thus, this study was designed to examine an animal model to mimic isch- emia/reperfusion injury observed clinically. Specifically, the time course of damage to renal tissue following reperfusion injury was of interest 1) in order to design a protocol that could efficiently evaluate new chemical entities and 2) anticipate when therapies might prove to

1056-8719/97/$17.00 PII 1056s8719(96)00141-4

2 JPM Vol. 37, No. 1 February 1997:1-7

Table 1. BUN, Creatinine, and Renal MPO Data”

Group

Normal Sham Tohb Sham T,,b Sham Tbhb Sham TShb Ischemia T,,’ Ischemia T,,,,’ Ischemia T,,C Ischemia T,,’ Ischemia T,,’ Ischemla TshC Ischemia T,,’ Ischemla TzhhC Ischemia T1,kc

BUN (mg/dl) Creatine (mg/dl) (Mean ? SEM) (Mean ? SEM)

16.60 2 0.98 (5) 0.84 2 0.02 (5) 15.67 + 0.67 (3) 0.77 ? 0.03 (3) 14.25 ? 1.31 (4) 0.73 2 0.03 (4)* 14.25 f. 0.48 (4) 0.73 k 0.03 (4)’ 13.67 ? 1.67 (3) 0.70 2 0.00 (3) 18.00 2 2.07 (5) 0.94 -c 0.02 (5)t 27.20 2 1.66 (5)* 1.00 2 0.00 (5) 26.60 k 1.57 (5)* 1.08 ? 0.05 (5)* 31.80 k 0.58 (5)*t 1.24 + 0.02 (S)*t 39.20 2 1.56 (5)*t 1.38 5 0.06 (5)*t 48.20 2 3.35 (5)*t 1.40 2 0.10 (5)* 41.25 2 6.69 (4)* 1.20 k 0.20 (4)* 60.60 ? 13.85 (5)* 1.56 2 0.24 (5)* 20.20 2 4.95 (5) 0.84 ir 0.07 (5)

MPO (mOD/min) (Mean r+_ SEM)

0.62 2 0.31 (4) 0.27 2 0.09 (4) 0.60 L 0.30 (4) 0.92 2 0.63 (4) 1.62 t 0.40 (4)* 9.99 t 6.81 (4)*t 1.84 t 0.84 (5)* 2.02 t 0.73 (5)* 6.91 t 7.45 (5)*t 8.07 t 9.10 (5)*t

41.85 t 19.51 (5)*t 28.86 t 12.58 (4)* 8.68 t 7.06 (5)* 4.77 ? 5.32 (5)*

“Shown are mean values 2 SEM; () = number of animals per group. bAnimals in these groups received sham operations without ischemia, and blood and kidney samples were taken immediately after the operation

(TO,,) or 2, 4, or 6 h after the operation (T,,, T,,, or T,,, respectively). “Animals in these groups were subjected to 45 min renal ischemia, and blood and kidney samples were taken immediately after ischemia (TO,,)

or 30 min, lh, 2h, 4h, 6h, 9h, 24h, or 1 week after ischemia (T,,,, T,,, T,,, T,,, T,,, T9,,, TZ4,,, and T,,, respectively). *p < 0.05; two-tailed Mann-Whitney test, relative to normal values. tp < 0.05; two-tailed Mann-Whitney test, relative to sham values of the same time point.

be the most effective. Renal injury was assessed by serum BUN and creatinine concentrations, renal his- topathologic analyses, and myeloperoxidase (MPO) ac- tivity. MPO activity is indicative of neutrophil infiltra- tion, an event hypothesized to play a key role in subsequent renal injury following reperfusion (Kelly et al., 1994). In a previous study by Kelly and coworkers (Kelly et al., 1994), an antibody to intracellular adhesion molecule 1 (ICAM-l), inhibited neutrophil adhesion to endothelial cells, consequently protecting against renal ischemia-reperfusion. Thus, the ICAM- antibody (ICAM-Ab) was also tested in the present study as an agent reportedly able to limit renal ischemia/reperfusion injury.

Materials and Animals

Male Sprague-Dawley rats

Methods

weighing approximately 140-160 g (Charles River, Hollister, CA) were accli- mated for at least 3 days prior to surgery. Rats were housed in groups of two and had free access to food (Simonsen Rodent Maintenance Diet) and water, and they were selected for study based upon good general physical condition and body weight (-150 g).

IschemialReperfusion Injury

Rodent anesthesia mixture [5.0 mL acepromazine maleate injection (10 mg/mL Fermenta Animal Health Company, Kansas City, MO] were combined with 1.0 ml

xylazine sterile solution 100 mg/ml (Lloyd Laboratories, Shenandoah, IA), 25.0 ml ketamine HCl injection, USP 100 mg/ml (Fort Dodge Laboratories, Inc., Fort Dodge, IA), and 15.0 ml water for injection (Ampro Pharma- ceutical, Arcadia, CA) in a 50-ml conical tube. The solution was vortexed and stored at room temperature before use. At the time of use, the solution was vortexed and drawn into a syringe for injection. Isoflurane USP (SOLVAY Animal Health, Inc., Mendota Heights, MN) was used for additional anesthesia as needed.

The procedure was a modification of the design described by Kelly et al. (1994). The rats were anesthe- tized with the rodent anesthesia mixture i.m. at a dose of 3 ml/kg, and a midline laparotomy was performed. The animals were then subjected to 45 min of bilateral renal occlusion using atramatic vascular clamps (Fine Science Tools, Foster City, CA) followed by reestablishment of renal perfusion. At reestablishment of blood flow, the animals were randomly grouped. At TO,-,, T1,2h, Tlh, TZh, Tdh, TGh, Tgh, TZdh, and Tlwk blood samples (-1.5 ml) were collected under anesthesia via the dorsal aorta, and standard hematology and serum chemistry parameters, including BUN and creatinine, were measured (Consol- idated Veterinary Diagnostics, Inc., West Sacramento, CA). Immediately following blood collections, animals were euthanized (100% ETOH @l ml intracardiac), and kidneys were excised and bisected for histopathology and MPO activity. Five untreated rats had blood samples and kidneys taken to determine normal values. Other rats were given sham operations without bilateral renal occlusion; at T,, T,, T,, and T, blood and urine samples

P.D. WILLIAMS ET AL. RENAL ISCHEMIA-REPERFUSION INJURY

3

80

U Nor&

. . . . . . . 0 . . . . . . Sham

--- __-. -0 Ischernia

Time after Treatment

Figure 1. BUN time course following &hernia. Shown are mean values 2 SEM. Details of the procedure are found in the Methods section.

were collected, and kidneys were excised and bisected for histopathology and MPO activity.

In addition, an ICAM- antibody was tested in this model to confirm the renal protection previously re- ported by Kelly et al. (1994). An anti-rat CD54 (ICAM-1) antibody (ICAM-Ab), Lot No. 8443-04, was obtained from Pharmingen (San Diego, CA) in a 1-mg/ml solution in saline. Immediately after reestab- lishment of renal blood flow (within 5 min), the animals received an intravenous bolus injection of ICAM-Ab at 1 mg/animal. Twenty-four hours later, blood samples were taken from all animals for analysis of BUN and creatinine levels (indicators of renal insufficiency). Both kidneys were harvested for histopathology and MPO analyses 24 h after reestablishment of blood flow.

Myeloperoxidase (MPO) Assays

MPO activity was measured in kidneys excised at various time periods after reperfusion in a procedure

similar to that documented by Hillegass et al. (1990). Kidney samples were homogenized in 50-mM potassium phosphate buffer (PB, pH 6.0), and centrifuged at 41,400 g (10 min); pellets were suspended in 50-mM PB con- taining 0.5% cetyltrimethylammonium bromide (CTAB). After three freeze and thaw cycles with soni- cation between cycles, the samples were centrifuged at 41,400g for 10 min. Aliquots (0.05 ml), in pure form and in two-, four-, and eightfold dilutions, were added to 0.1 ml of reaction mixture containing 50 mM PB, O-diani- sidine, and 30% H,O, solution. Absorbance was mea- sured at 450 nm for 5 min; MPO activity was expressed as mOD/min.

Renal Histopathology

Histopathologic examination of left and right kidneys from untreated (normal and sham) animals, as well as animals subjected to ischemia and sacrificed at the various time points was performed. The samples were

JPM Vol. 37, No. 1 February 1997:1-7

2

1.75

1.5 =i %I g 0 1.25

18 8 3 1

0.75

0.5

U Normal

. . . . . . . 0 . . . . . . Sham

m-w Q - - - Ischemia

Figure 2. Serum creatinine time course following ischemia. Shown are mean values ? SEM. Details of the procedure are found in the Methods section.

evaluated for specific lesions-cytoplasmic degenera- tion, mitosis, necrosis, inflammation, basophilia, calcifi- cation, fibrosis, and tubular dilatation, atrophy, and casts (Hottendorf 1984).

Statistical Analysis

Statistical analysis was carried out using GraphPad Instat 1.1 (GraphPad Software, San Diego, CA). Results are expressed as calculated means with standard devia- tion of the mean (SEM), and statistical significance was calculated using a nonparametric Mann-Whitney test reporting a two-tailed p value; p values less than 0.05 were considered significant.

Results Clinical Chemistry Parameters

As illustrated in Table 1 and Figures 1 and 2, animals that underwent renal ischemia exhibited significant in- creases, in serum BUN and creatinine compared to normal and sham animals. Serum BUN and creatinine

levels exhibited a steady increase at the T,,, T,,, and T,, time points leading to a peak at the Tz4,, time point.

The remaining clinical chemistry and hematology parameters (alkaline phosphatase, bilirubin, globulin albumin, total protein, WBC, monocytes) revealed few deviations from normal or sham animal values with the exception of CPK (isoenzyme not related to cardiac pathology) which was elevated at the 2-h time point.

Myeloperoxidase (MPO) Levels

As illustrated in Table 1 and Figure 3, MPO activity was increased in animals that underwent ischemia com- pared to normal and sham animals. MPO activity showed a slight increase during the T,, and T,, time points, with a peak elevations occurring at the T,, and T,, time point.

Renal Histopathology

Observations regarding lesions and progress of tissue and tubular damage in relation to time are shown in

P.D. WILLIAMS ET AL. RENAL ISCHEMIA-REPERFUSION INJURY

60

U Normal

. . . . . . V . . . . . . Sham

Time after Treatment

Figure 3. Renal MPO time course following ischemia. Shown are mean values -t SEM. Details of the procedure are found in the Methods section.

(Table 2). Renal injury was evident at the early time points following &hernia with peak necrosis of the proximal tubular segments observed at the 24-h time point. Renal changes proceeded from early cytoplasmic degeneration indicated at To through the actual necrosis of proximal convoluted tubule cells which peaked at 24 h to the regeneration (basophilia) of the epithelium re- vealed at 24 h and 1 week after ischemia. Renal injury was still evident 1 week following renal injury. In addi- tion, quantitative scores reflecting the extent of the sample affected by these lesions (1 = less than 10%; 2 = lo-50%; 3 = 50-90%; 4 = >90%) were assigned to kidneys from the 24-h and l-week periods. The individ- ual scores were then added to yield a final score per kidney. The histopathology scores for kidneys of these animals are summarized in Figure 4. The progression from the acute necrotic phase to the regeneration of proximal convoluted tubules is illustrated in Figure 4. The severity of damage that was produced acutely by the

ischemia is indicated by the significant number of tu- bules that did not recover after 1 week. The tubular basophilia observed is indicative of regeneration or repair; however, atrophic tubules and focal fibrosis represent permanent tubular loss despite the return of the BUN and creatinine to normal levels at 1 week. No evidence of inflammatory cell infiltration was observed at any of the time points following ischemia.

ICAM-Ab Treatment

The results of treatment with ICAM-Ab are shown in Table 3. Serum BUN and creatinine levels exhibited a decrease in the ICAM-Ab-treated animals compared to the placebo group, but these decreases were not statis- tically significant. Renal histopathologic evaluation re- vealed a significant decrease in histopathology scores in the ICAM-Ab-treated animals. MPO analysis revealed no significant changes.

6 JPM Vol. 37, No. 1 February 1997:1-7

Table 2. Renal Histopathology Following 45-Minute Bilateral Ischemia

Time Pointa Observations

Controls

TOh

T 0.5 h

T 1.0 h

T 2.0 h

T 4.0 h

T 6.0 h

T 9.0 h

T 24 h

T 1 wk

Relatively clean kidneys; few small focal areas of inflammatiombasophilic tubules

Corticomedullaxy congestion, some subcapsular cytoplasmic degeneration of proximal convoluted tubules

<Congestion, <subcapsular degeneration of PCT, bdegeneration of PCI in corticomedullary junction (CMJ)

>Eosinophilic casts and subcortical granular degeneration (lysosomes), >CMJ cytoplasmic degeneration, clear cut PCT necrosis especially at CMJ

>Tubular dilatation/degeneration/eosinophilic casts at CMJ

<Tubular dilatation, >eosinophilic casts, >necrosis at CMJ

>Necrosis of PCT at CMJ, %ytoplasmic granular degeneration throughout cortex

>Necrosis of PCT at CMJ especially, some subcortical basophilia of PCT (regeneration)

Massive amounts of necrosis of PCT (including calcification of cellular debris in 4/5 animals) at CMJ, tubular basophilia more obvious; least amount of change in animal no. 1 (BUN 30), severe changes in animal no. 13 (BUN 26)

PCT basophilia, tubular dilatation and atrophy, focal fibrosis, necrosis still present to some degree

“post-ischemia

Discussion The results of this study suggest that renal injury may

be initiated as early as 4 h following 45min ischemia in

rats as judged by increases in BUN and serum creati- nine, renal MPO levels, and histopathologic evaluations. Thus, therapeutic interventions within 6 h following ischemia/reperfusion may be the most effective in limit- ing renal injury. Peak renal injury occurred 24 h follow- ing ischemia, and this time point may be useful in monitoring the protective effects of new investigational agents.

It is noteworthy in the characterization of this model that permanent renal damage was evident in histopatho- logic examinations performed 1 week following isch- emia. Damage was evident despite the return to normal clinical pathologic parameters of BUN and serum cre- atinine. Renal function will remain within the normal range unless more than 50% of the nephrons are destroyed (Baum et al., 1975). Thus, the lack of corre- lation between the morphologic assessment and renal function tests (BUN and creatinine) after 1 week reflects the greater sensitivity of the morphologic assessment which scores the tubular basophilia equally with tubular necrosis (Hottendorf 1982; Hottendorf and Williams, 1986). While clinicians rely primarily on renal function for assessing renal status, the morphologic findings described here underscore the need to recognize that subclinical renal injury may be present in the face of normal function.

Inflammation and the migration of any type of in- flammatory cells into the renal parenchyma was not a significant part of the histopathology of renal ischemia/ reperfusion injury induced in this model. Thus, the elevations in renal MPO levels were not correlated with

c Control

q 24 hr

0 lwk

Figure 4. Quantitative renal histopathology on kidneys following 45 minutes of ischemia.

P.D. WILLIAMS ET AL. RENAL ISCHEMIA-REPERFUSION INJURY

Table 3. BUN, Creatinine, Renal Histopathology, and MPO Activity Following ICAM-Ab Treatment=

Parameter Normalt’

BUN (mg/dl) 15.46 ? 0.22 (13) Creatinine (mg/dl) 0.84 + 0.09 (13) Histopathology score per kidney 0.82 + 0.2 (5) MPO (mOD/min) 0.62 2 0.31 (4)

“Shown are mean values + SEM. Number of animals is in parentheses. bNormal data obtained from animals without ischemia treatment.

Placebo’

93.90 2 14.67 (lo)* 2.41 2 0.36 (lo)* 8.65 _t 0.58 (20) 3.14 2 0.78 (10)

ICAM-Abd

65.89 i 11.26 (9)* 1.66 2 0.26 (9)* 7.45 2 0.32 (20)t 4.80 2 1.27 (9)

‘Animals in the placebo group were administered saline via 200 PI intravenous bolus injection, immediately after reestablishment of blood flow following 45 min renal ischemia.

dAnimaIs in the ICAM-Ab treatment group were administered 1 mg of ICAM- antibody via intravenous bolus injection, immediately after reestablishment of blood flow following 45 min renal ischemia.

*p < 0.05; two-tailed Mann-Whitney test, relative to normal values. tp < 0.05; two-tailed Mann-Whitney test, relative to placebo values.

observations of intact inflammatory the kidney.

cell infiltration in References

Lastly, our results have confirmed the protective activity of the cellular adhesion antibody, ICAM-Ab, as reported by Kelly et al. (1994). However, in our hands, the magnitude of the protection by ICAM-Ab was not as dramatic as previously reported, nor were renal MPO levels significantly attenuated. Our results are also in contrast to those reported by Laight et al. (1994), who measured a reduction in renal MPO levels in rats following ischemia and treatment with antibodies, in- cluding ICAM-1. However, they did utilize a different MPO assay method than that employed in the present study and also reported that the reduction in ischemia- induced MPO levels was attenuated nonspecifically by other IgG antibodies as well. Thus, while elevations in renal MPOs appear to occur following renal ischemia/ reperfusion, their obligatory role in the pathogenesis of renal injury remains to be established.

In summary, renal injury following bilateral ischemia in rats was demonstrated as early as 4 h post-ischemia as judged by changes in renal function, MPO levels, and renal histopathology. Peak injury was observed 24 h post-ischemia with evidence of renal damage still present 1 week following injury. The results indicate that early intervention in the course of ischemia-reperfusion injury may be most effective therapeutic strategy. Fur- thermore, assessment of protection should include time points representative of peak injury as well as follow-up to insure full recovery.

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