Effect of U-74500A, A 21-Aminosteroid on Renal Ischemia-Reperfusion Injury in Rats

Abstract

Renal ischemia-reperfusion injury constitutes the most common pathogenic factor for acute renal failure and is the main contributor to renal dysfunction in allograft recipients and revascularization surgeries. Many studies have demonstrated that reactive oxygen species play an important role in ischemic acute renal failure. The aim of the present study was to investigate the effects of the synthetic antioxidant U-74500A, a 21-aminosteroid in a rat model of renal ischemia-reperfusion injury. Renal ischemia-reperfusion was induced by clamping unilateral renal artery for 45 min followed by 24 h of reperfusion. Two doses of U-74500A (4.0 mg/kg, i.v.) were administered 45 min prior to renal artery occlusion and then 15 min prior to reperfusion. Tissue lipid peroxidation was measured as thiobarbituric acid reacting substances (TBARS) in kidney homogenates. Renal function was assessed by estimating serum creatinine, blood urea nitrogen (BUN), creatinine and urea clearance. Renal morphological alterations were assessed by histopathological examination of hematoxylin-eosin stained sections of the kidneys. Ischemia-reperfusion produced elevated levels of TBARS and deteriorated the renal function as assessed by increased serum creatinine, BUN and decreased creatinine and urea clearance as compared to sham operated rats. The ischemic kidneys of rats showed severe hyaline casts, epithelial swelling, proteinaceous debris, tubular necrosis, medullary congestion and hemorrhage. U-74500A markedly attenuated elevated levels of TBARS as well as morphological changes, but did not improve renal dysfunction in rats subjected to renal ischemia-reperfusion. These results clearly demonstrate the in vivo antioxidant effect of U-74500A, a 21-aminosteroid in attenuating renal ischemia-reperfusion injury.

• Renal ischemia-reperfusion injury
• Oxidative stress
• U-74500A, 21-Aminosteroid

Introduction

Surgical procedures such as renal revascularization and renal transplantation are often associated with acute renal ischemia as a consequence of low perfusion states and shock. The renal damage resulting from ischemia-reperfusion is one of the leading causes of the early loss of transplanted organ.[[1]] Renal ischemia causes a series of cellular events, which occasionally lead to organ failure depending on the duration of the oxygen deprivation. It has been reported that reperfusion actually aggravates renal damage.[[2]] Several different mechanisms for this injury have been proposed, but the hypothesis about the role of oxygen-free radicals in generating ischemic-reperfusion injury seems the most reliable.

Reactive oxygen species (ROS) such as superoxide anion , hydrogen peroxide (H₂O₂) and hydroxyl radical () have been postulated to play a major role in this process.[[3]], [[4]] These species are generated in the ischemic kidney at the onset of reperfusion, when the oxygen supply is renewed.[[5]] Alternatively, it has been proposed that polymorphonuclear leukocytes that are activated during ischemia, presumably by cytokines, enter the kidney at the onset of reperfusion, causing tissue damage by releasing ROS.[[6]], [[7]] The oxygen free radicals generated during the proposed episodes of ischemia-reperfusion provoke severe deleterious changes at cellular level leading to cell death, because owing to their extreme reactivity, they attack essential cell constituents such as nucleic acids, proteins and lipids.[[8]] In particular, they induce lipid peroxidation of membrane lipids leading to the formation of toxic compounds, such as epoxides, aldehydes and new free radicals and disrupt membrane integrity and function and thus cause organ impairment and cell death.[[9]]

Tubular cell energy depletion seems to be a common phenomenon in all types of experimental renal failure. During ischemia, ATP is degraded to ADP, AMP and results in the formation of adenosine, inosine and hypoxanthine that leak out of the cell and thus further decrease the purine substrate pool.[[10]] Further, ischemic acute renal failure is associated with intracellular calcium (Ca²⁺) overload in vascular smooth muscle and endothelial cells[[11]], [[12]] that triggers the release of various vasoactive mediators.[[13]], [[14]] One of the main functional changes in ischemic renal failure is a decrease in glomerular filtration rate (GFR). Although numerous investigators have attempted, the pathophysiologic mechanisms for the initiation and maintenance of this filtration failure have not been clearly elucidated.

Irrespective of the origin of ROS, one would anticipate that treatment with antioxidants would minimize ischemia damage. However, treatment with antioxidants has produced only moderate success. Recently a new class of antioxidants, the 21-amiosteroids, named lazaroids have been developed and extensively investigated. The early animal studies suggested that lazaroids inhibit lipid membranes scavenging free radicals, a mechanism similar to that of vitamin E.[[15]] The lazaroids also have potent membrane stabilizing properties.[[16]] The protection action of the new 21-aminosteroid, U-74500A (pregna-1,4,9(11)-triene-3,20-dione, 21-(4-(5,6-bis(diethylamino)z-2-pyridinyl)-1-piperazinyl)-16-methyl, HCl (16 alpha)) has been shown in in vitro[[17]], [[18]] and in vivo[[19]], [[20]] studies in different animal models.

With this in background, the present study was designed to demonstrate the role of oxidative stress, renal dysfunction and to investigate whether the 21-aminosteroid, U-74500A provides protection against ischemia-reperfusion injury in rats.

Materials And Methods

Drugs

U-74500A as hydrochloride (Upjohn, USA) was dissolved in normal saline to give a final concentration of 4 mg/5mL. The drug solutions were made freshly at the beginning of each experiment.

Study Design

Wistar rats (150–200 g) of either sex bred in central animal house of Panjab University, Chandigarh, India were used. The animals were housed under standard conditions of light and dark cycle, with free access to food (Hindustan Lever Products, Kolkata, India) and water. All animals were given humane care in compliance with institutional guidelines. All the experimental procedures and protocols were approved by the Institutional Animal Ethical Committee, Panjab University, Chandigarh, India.

Animals were randomly divided into four groups, each comprising six animals. Group 1: A normal control group that was not subjected to any surgical procedure and drug treatment, group 2: Sham control group that was subjected to surgical procedure for renal artery identification but not occlusion, group 3: Ischemic control group that was subjected to renal artery occlusion for 45 min and reperfusion for 24 h, group 4: Drug-treated group that received a dose 4.0 mg/kg of U-74500A 45 min prior to renal artery occlusion, and the same dose of test substance was given intravenously 15 min before reperfusion of the kidney.

Surgical Procedure

The animals were anesthetized with 40 mg/kg thiopentone sodium and placed on a warm pad. The abdomen was opened by a midline incision and the left renal artery was identified. Renal ischemia was induced by occluding the left renal artery for 45 min and reperfused for 24 h. The abdomen was sutured and applied with topical antibiotic powder. The animals were allowed to recover from anesthesia and kept in individual metabolic cages to collect the urine for 24 h. Systolic blood pressure (SBP) was measured from the tail of the animals before the surgical procedure and 24 h after reperfusion using blood pressure recorder (UGO Basile, Italy). Animals were sacrificed at the end of reperfusion period. At this time, blood was collected in heparinized centrifuge tubes by the insertion of a cannula into the abdominal aorta. Both left and right kidneys were excised for the estimation of lipid peroxides and histopathological studies.

Assessment of Renal Function

Renal function was assessed by colorimetric estimation of serum and urine levels of creatinine and urea. Urea was measured as blood urea nitrogen (BUN). Creatinine and urea clearances as an index of GFR were calculated. Urinary protein was estimated by Folin–Lowry's method.

Estimation of Lipid Peroxides

Malondialdehyde (MDA), an indirect index of lipid peroxidation, was assayed in the form of thiobarbituric acid reacting substances (TBARS).[[21]] The left kidney was isolated immediately after sacrificing the rat and placed in ice-cold saline. It was weighed and properly minced. To 0.2 mL of 10% (w/v) tissue homogenate prepared in 1.15% potassium chloride, 0.2 mL of 8.1% sodium lauryl sulfate, 1.5 mL of 20% acetic acid solution adjusted to pH 3.5 with sodium hydroxide and 1.5 mL of 0.8% of aqueous solution of thiobarbituric acid were added. The mixture was made up to 4.0 mL with distilled water, and heated at 95°C for 60 min. After cooling with tap water, 1.0 mL of distilled water and 5.0 mL of the mixture of n-butanol and pyridine (15 : 1, v/v) was added and centrifuged. The organic layer was taken out and its absorbance was measured at 532 nm. The TBARS were quantified using an extinction factor of 1.56 × 10⁵ M⁻¹ cm⁻¹ and expressed as nmol of TBARS per mg protein. The tissue protein was estimated using Folin–Lowry method of protein assay.

Renal Histology

The right kidney was isolated immediately after sacrificing the animal and washed with ice cold saline. Then it was fixed in 10% neutral buffered formalin solution, embedded in paraffin and was used for histopathological examination. Five micro meter thick sections were cut, deparaffinized, hydrated and stained with hemotoxylin-eosin. The renal sections were examined by a qualified pathologist in blind fashion for hyaline casts, epithelial swelling, proteinaceous debris, tubular necrosis, medullary congestion and hemorrhage in all the groups. A minimum of 20 fields from each kidney slide were examined and assigned for severity of changes using scores on a scale of: − none, + mild, ++ moderate, +++ severe damage.

Statistical Analysis

The data were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett's test. The data were expressed as mean ± SEM and p < 0.05 was considered statistically significant.

Results

Effect of U-74500A on Systolic Blood Pressure

Renal artery occlusion followed by reperfusion significantly increased SBP in rats. U-74500A had no effect on elevated SBP as compared to ischemic group (Table 1).

Table 1. Effect of U-74500A (4.0 mg/kg, i.v.) on systolic blood pressure in rats subjected to renal ischemia-reperfusion injury

Treatment	Normal control	Sham control	Ischemic control	U-74500A group
% change in SBP	1.04 ± 1.11	1.29 ± 0.18	18.45 ± 2.67a	12.21 ± 4.56a

^ap<0.05 as compared to control group.

Effect of U-74500A on Ischemia-Reperfusion Induced Lipid Peroxidation

Forty five minutes of ischemia followed by 24 h of reperfusion produced a significant increase in renal TBARS as compared to sham control animals. Animals treated with U-74500A had shown significant decrease in TBARS as compared to ischemia-reperfused rats (Fig. 1).

Figure 1. Effect of U-74500A on ischemia-reperfusion induced lipid peroxidation in kidneys. Values expressed as mean ± SEM. *p<0.05 as compared to sham control group, **p<0.05 as compared to ischemic control group (One-way ANOVA followed by Dunnett's test).

Effect of U-74500A on Ischemia-Reperfusion Induced Renal Dysfunction

Renal ischemia-reperfusion significantly increased the serum creatinine and BUN in rats, while pretreatment with U-74500A had not prevented elevated creatinine level (Fig. 2) but decreased urea levels significantly (Fig. 3). There was a significant decrease in creatinine and urea clearance in rats subjected to renal ischemia-reperfusion injury, however, concomitant treatment with U-74500A had not improved renal dysfunction (Figs. 4 and 5). In addition, there was an increased level of proteins in urine in ischemic controls, whereas this effect was not observed with U-74500A treatment (Fig. 6).

Figure 2. Effect of U-74500A on serum creatinine in rats subjected to ischaemia-reperfusion. Values expressed as mean ± SEM. *p<0.05 as compared to sham control group, **p<0.05 as compared to ischemic control group (One-way ANOVA followed by Dunnett's test).

Figure 3. Effect of U-74500A on blood urea nitrogen (BUN) in rats subjected to ischaemia-reperfusion. Values expressed as mean ± SEM. *p<0.05 as compared to sham control group, **p<0.05 as compared to ischemic control group (One-way ANOVA followed by Dunnett's test).

Figure 4. Effect of U-74500A on creatinine clearance in rats subjected to ischemia-reperfusion. Values expressed as mean ± SEM. *p<0.05 as compared to sham control group, **p<0.05 as compared to ischemic control group. (One-way ANOVA followed by Dunnett's test).

Figure 5. Effect of U-74500A on urea clearance in rats subjected to ischemia-reperfusion. *p<0.05 as compared to sham control group, **p<0.05 as compared to ischemic control group (One-way ANOVA followed by Dunnett's test).

Figure 6. Effect of U-74500A on proteinuria in rats subjected to ischemia-reperfusion. Values expressed as mean ± SEM. *p<0.05 as compared to control group, **p<0.05 as compared to ischaemic control group (One-way ANOVA followed by Dunnett's test).

Figure 7. Light microscopic observations of kidney sections of different treatment groups: (A) renal cortex of a sham control rat (HE, X280); (B) severe hyaline casts, tubular necrosis and medullary congestion in kidneys of rats subjected to ischaemia-reperfusion. (HE, X280); (C) mild hyaline casts and medullary congestion in kidneys of ischaemia-reperfused rats pretreated with U-74500A 4.0 mg/kg (HE, X280).

Effect of U-74500A on Renal Morphological Changes

The histopathological changes were graded and summarized in Table 2. The sham control group did not show any morphological changes (Fig. 1). By contrast, the kidneys of ischemia-reperfused rats showed marked histological changes in the cortex and outer medulla. The sections of ischemic-reperfused kidneys showed severe hyaline casts, epithelial swelling, proteinaceous debris, tubular necrosis, medullary congestion and hemorrhage (Fig. 2). The renal sections of rats pre-treated with U-74500A demonstrated normal morphology except for the presence of mild hyaline casts and medullary congestion (Fig. 3).

Table 2. Effect of U-74500A (4.0 mg/kg, i.v.) administration on morphological changes as assessed by histopathological examination of kidneys of rats subjected to renal ischemia-reperfusion injury

Group	Hyaline casts	Epithelal swelling	Proteinaceous debris	Tubular necrosis	Medullary congestion	Hemorrhage
Normal control	—	—	—	—	—	—
Sham control	—	—	—	—	—	—
Ischemic control	+++	+++	+++	+++	+++	+++
U-74500A 4.0 mg/kg, i.v.	+	—	—	—	+	—

− none, + Mild, +moderate, +++ Severe.

Discussion

Intrinsic acute renal failure is characterized by deterioration of the renal function over a period of hours to days, and results in the failure of the kidney to excrete nitrogenous waste products and to maintain fluid and electrolyte homeostasis.[[22]] Despite the progress in animal research concerning the pathophysiology and the progress in clinical practice regarding the methods of therapy, the incidence and mortality due to ischemic renal failure remain exceptionally high. Renal ischemia-reperfusion constitutes the major contributor to renal dysfunction in allograft recipients and associated poor graft function and survival.[[1]]

The severity of ischemia-induced damage depends on the duration of ischemic insult. An ischemia of less than 25 min duration causes mild renal injury whereas 40–60 min ischemia causes severe though reversible renal damage.[[23]] However, an ischemic insult of more than one hour produces irreversible renal injury.[[24]] Furthermore, impairment of renal function after ischemia is documented to reach its peak after 24 h of reperfusion.[[2]] Therefore in an attempt to produce significant renal damage, unilateral renal artery occlusion in rats for 45 min followed by reperfusion for 24 h was employed in the present study.

Ischemic-reperfusion injury is a complex interrelated sequence of events that classically involves the vascular endothelium and activated leukocytes. During ischemic phase, the endothelium is primed both to produce free radicals and to secrete chemoattractants. ROS have been demonstrated to play a major role in post ischemic renal injury.[[3]], [[4]] MDA, a stable lipid hydroperoxide, provides an index of the peroxidation of lipids in biological tissues.[[21]] In the present study, ischemic-reperfusion injury caused a marked production of MDA measured as TBARS in the kidney indicating significant oxygen free radical generation. The role of ROS in human kidney transplantation seems to correlate with results obtained in animal studies. In human transplantation studies, increase in myeloperoxidase and lipid peroxide products has been demonstrated in the blood of transplant recipients.[[25]]

In the present study, pretreatment with U-74500A prevented elevated TBARS levels in rats subjected to renal ischemia-reperfusion injury. Findings from in vitro and in vivo studies have demonstrated that U-74500A has an ability to inhibit lipid peroxidation and also scavenge free radicals similar to that of vitamin E.[[17]], [[18]], [[19]], [[20]] It also preserves mitochondrial function and energy metabolism in ischemic cells.[[26]], [[27]] The lazaroids also have potent membrane stabilizing effects. They have a high affinity for the lipid bilayer.[[28]] The positively charged piperazine nitrogen in U-74500A interacts with negatively charged phosphate-containing head groups of the membrane lipids.[[16]] The localization of bulky 21-aminosteroid moiety towards the surface compresses the membrane phospholipids head groups. This membrane stabilizing action restricts the movement of lipid peroxy radicals within the membrane so that their interaction with neighboring fatty acids is reduced, thus inhibiting lipid peroxidation. This compound has also been shown to protect cellular integrity by scavenging the ROS, inhibiting free radicals formation, and subsequent lipid peroxidation after ischemic insult of the kidney.[[18]], [[19]] Further, this in vivo study supports U-74500A as a potent antioxidant, and suggests that through anti-lipoperoxidative and free radical scavenging effects, it might contribute to the decreased lipid peroxides in ischemic-reperfused kidneys.

We found a significant correlation between lipid peroxidation and renal dysfunction. Various studies have demonstrated that ROS mediated lipid peroxidation is accompanied by loss of renal function and subcellular damage.[[29]], [[30]], [[31]] In our study, ischemia-reperfusion induced lipid peroxidation was associated with impaired renal function as evidenced by increased serum creatinine and urea, and decreased creatinine and urea clearance. It is clear that decreased GFR and RBF due to renal vasoconstriction and hypoperfusion of renal microcirculation caused by renal ischemia might contribute to these alterations. In addition, proximal tubular obstruction, reduction in renal blood flow (RBF) and tubular back leak of filtrate have been implicated as the important factors in the decrease of GFR in ischemic-reperfusion injury.[[15]], [[32]] Furthermore, increased intracellular Ca²⁺ as well as membrane damage can promote the formation of a variety of vasoactive mediators that can affect renal function directly by causing renal vasoconstriction or decreasing the glomerular capillary ultrafiltration coefficient, and thus reduce glomerular filtration rate.[[33]]

One would expect that the scavenging effects of U-74500A on free radicals, inhibition of lipid peroxidation and subsequent decrease in the production of vasoconstrictive mediators should play a role in improving renal dysfunction in ischemic-reperfused kidneys. However, our data showed that U-74500A had not improved renal dysfunction caused by ischemia-reperfusion injury. One possibility is that ischemia causes the increase in intracellular calcium results in the release of vasoconstrictors such as angiotensin II,[[13]] platelet activating factor,[[14]] thromboxane,[[34]], [[35]] and endothelin.[[36]], [[37]] The antagonists of these mediators have shown improved renal function and prevented morphological alterations[[34]], [[35]], [[36]], [[37]] suggesting the pivotal role of vasoactive mediators also besides oxidative stress in ischemia-reperfusion injury. The failure of U-compound to reverse renal dysfunction may be related to the dose used. On the other hand, the dosage of U-compound used in the present study (4 mg/kg) has been demonstrated to exert protective effects in various experimental models.[[19]], [[20]] Unlike other studies, animals were administered with two doses of U-compound (4 mg/kg). So, the observed effect in this study due to low dose can be ruled out. Thus the dosage does not seem to account for the observed failure in reversal of renal dysfunction in the present study. our results are in agreement with the other animal studies where U-compounds have not demonstrated complete organ protection.[[38]], [[39]], [[40]]

Reactive oxygen species mediated peroxidation of lipid structures of the tissue results in extensive subcellular damage.[[41]] In our study, the kidneys of rats subjected to ischemia-reperfusion have shown characteristic morphological findings such as hyaline casts, epithelial swelling, proteinaceous debris, tubular necrosis, medullary congestion and hemorrhage. The marked histological changes were prominent in epithelial cells, especially in the proximal tubules of the kidney. Due to limited oxygen availability, these structures are particularly vulnerable to ischemia. Alteration of the cytoskeleton, disruption of normal cell matrix interaction and shedding of the apical microvilli into tubular lumen leads to the inability of the renal epithelium to maintain polarity and normal renal function.[[41]] Furthermore, the vasoconstriction induced by ischemia produces local hypoxic environment, which leads to a number of cellular changes, such as deterioration in membrane integrity. The marked morphological protection by U-74500A may be related to its significant antioxidant effect.

We concluded that U-74500A attenuated post-ischemic renal morphological changes and oxidative stress in this rat model without altering renal dysfunction.

Acknowledgment

The authors would like to thank Ms. Saraswati Gupta for technical assistance.