Apart from the Other Members of PDE Inhibitors' Family, Enoximone Does Not Enhance Renal Ischemic Reperfusion Injury: The Effects of Enoximone on Renal Ischemia Reperfusion

Abstract

Many pharmacological agents were investigated for the prevention of renal ischemic reperfusion (IR) injury as well as the phosphodiesterase (PDE) inhibitors. The aim of the study was to examine the possible renoprotective effect of enoximone as a member of this family on IR injury. Thirty-six Wistar-Albino rats were allocated to six groups. Sham (S) and control groups (E1, E2) only received 0.09% NaCl, 5 mg/kg and 10 mg/kg enoximone via caudal caval vein, respectively. In ischemia (I) and treatment groups (IE1, IE2), the rats were subjected to bilateral renal artery occlusion and were given 0.09% NaCl, 5 mg/kg and 10 mg/kg enoximone in the same route, respectively. Bilateral kidneys were removed at the sixth hour of laparotomy for histopathological and biochemical analysis, such as superoxide dismutase, myeloperoxidase, malonyldialdehyde, and nitric oxide end products. Blood samples were taken in order to evaluate renal function tests. The data were analyzed by using one-way analysis of variance, and p < .05 was considered to be statistically significant. The worst results were achieved in ischemia group (p < .05). Treatments groups showed nearly similar findings with this group (p < .05). There was no significant difference between control and sham groups. In this study, we found that apart from the other members of the PDE inhibitors' family, enoximone did not contribute to the attenuation of IR injury of kidney.

Keywords:

• renal ischemia reperfusion
• enoximone
• PDE III inhibitor

INTRODUCTION

As a consequence of hemorrhage, shock, cardiac arrest, renal artery surgery, or renal transplantation, ischemic reperfusion (IR) injury remains a major clinical problem. Preventing the renal tissue from IR injury is the main aim of the intense researches about this ischemic phenomenon.

Phosphodiesterase (PDE) inhibitors are the compounds that inhibit or antagonize the biosynthesis or actions of PDEs. The effects of many members in this family have been studied especially on IR injury.^[1–5] The general acceptation is that these vasodilator and inotropic agents attenuate the renal injury related to IR. This study aimed to evaluate the “possible” renoprotective effects of enoximone, a selective PDE III inhibitor, on IR injury.

MATERIALS AND METHODS

The study protocol was approved by the Animal Ethics Committee and performed according to the guidelines of the Research Committee of Faculty of Medicine at Gazi University. The study comprised 36 male Wistar-Albino rats weighing 300–350 gr. All animals were kept under controlled temperature (21 ± 2°C) and humidity (55.5%) with a 14-hour light and 10-hour dark cycle. They were fed standard rat chow and had free access to water. There were no water and light restrictions throughout the experiment. All animals received human care in compliance with Principles of Laboratory Animal Care formulated by National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources published by the National Institutes of Health. All surgical procedures were performed by the same two surgeons in the sterile conditions. Every surgical intervention was performed under 80 mg.kg⁻¹ ketamine hydrochloride (Ketalar, Eczacibasi, Turkey) and 10 mg.kg⁻¹ xylazine hydrochloride (Alfazyne, Ege Vet, Turkey) anesthesia.

Thirty-six male Wistar-Albino rats were divided into six groups, six animals in each group. The animals in sham (S) and control (E1, E2) groups were not subjected to ischemia and administered 0.09% NaCl and 5 and 10 mg/kg enoximone via the caudal caval vein, respectively. The rest of the animals were allocated to three groups in order to constitute the ischemia (I) and treatment (IE1, IE2) groups. The laparotomy was done through midline incision. After taking the bowel out of the abdominal cavity, bilateral renal vessels were visualized. Only a dissection was performed around the renal hilus in the S, E1, and E2 groups. Renal ischemia was created by clamping of bilateral renal hilus for 45 minutes in I, IE1, and IE2 groups, giving, just before the reperfusion, 0.09% NaCl, 5 mg/kg enoximone, and 10 mg/kg enoximone using the same route, respectively. After removal of the clamps, abdominal cavity was closed. At the 6^th hour of reperfusion, the animals were re-entered and kidneys were removed. Blood samples were taken for serum blood urea nitrogen (BUN) and creatinine before scarification of the subjects. Half of the either side nephrectomy material was wrapped in aluminium foils and placed in liquid nitrogen to keep at −80°C for biochemical analysis. Other halves of the materials were placed in 10% formaldehyde for histopathogic evaluation. All specimens were coded and analyzed by the same individuals who were blinded to the group assignments.

Serum BUN and creatinine levels were determined with an Abbott-Aeroset autoanalyzer (Chicago, Illinois, USA) using the original kits.

Myeloperoxidase (MPO) activity was assessed by measuring the H₂O₂-dependent oxidation of O-dianisidine. Brown colored oxidized dianisidine was measured spectrophotometrically, and the results were given as U/mg-protein. One unit of MPO activity was defined as the amount of enzyme caused absorbance change in 1 minute.

Malondialdehyde (MDA, the lipid peroxidation end product) levels were determined using the thiobarbituric acid (TBA) test based on the calorimetric measurement of the concentration obtained from the pink colored end product of the reaction between lipid peroxides and TBA. Renal tissues were weighed and homogenized in KCl at a ratio of 1/9 (w/v). Phosphoric acid and TBA were added to the homogenate. After the addition of n-butanol, the solution was centrifuged. The absorbent of the supernatant was measured spectrophotometrically. 1,1,3,3-tetraetoxypropane was measured as standard. The difference between two absorbance values was accepted as the amount of MDA in nmoles/g tissue.

Nitric oxide end products (NOx), nitrite, and nitrate are the primary oxidation products of nitric oxide subsequent to the reaction with oxygen; therefore, the nitrite/nitrate concentration in the renal homogenate was used. Quantification of nitrite and nitrate was based on the Griess reaction, in which a chromophore with a strong absorbance is formed by reaction of nitrite with a mixture of naphthylethylenediamine and sulfanilamide. Nitrate was reduced to nitrite by 30 minutes' incubation with nitrate reductase in the presence of nicotinamide adenine dinucleotide 3-phosphate and flavinadenine dinucleotide. Then, the Griess reagent was added to the solution, and this reagent converted the nitrite into a deep purple azo compound. The total nitrite/nitrate concentration was calculated by using the standard of sodium nitrate, and the results were expressed as micromoles/liter.

For superoxide dismutase (SOD) measurement, the tissues were homogenized at room temperature in nine volumes of 50 mmol/L potassium phosphate buffer, pH 7.4, and containing 10⁻⁴ mmol/L edetic acid (EDTA) for SOD assay. Homogenates were then centrifuged at 4°C for 10 minutes at 700g in a Damon IEC B-20A refrigerated centrifuge. Protein concentration was measured in tissue homogenate by the method of Lowry using bovine serum albumin standard. The SOD levels were expressed as U/mg tissue.

For histopathological evaluation, tissue samples were fixed in a 4% paraformaldehyde solution and embedded in paraffin, and 5 μm sections were stained with hematoxylin and eosin. Ten glomeruli and tubules in either side of each kidney were randomly selected, the damaged glomeruli and tubules were counted, and the mean number of damaged tissues was determined. The epithelial and interstitial injuries were also noted, and mean epithelial and interstitial injury scores were found.

Statistical Analysis

All statistical analyses were made by using the statistical package SPSS for Windows, version 10 (SPSS, Chicago, Illinois, USA). All values are expressed as the groups mean ± standard deviation (SD) for the results found for all rats in each group. Biochemical distributions of the groups were calculated by using one-way analysis of variance (ANOVA) followed by a Newman-Keuls test. Histological damage among the groups was compared using the Kruskal-Wallis test. The value of p < .05 was considered statistically significant.

RESULTS

The results of the groups are shown in Table 1. The mean serum BUN and creatinine levels in the I, IE1, and IE2 groups were found significantly higher than for the sham and control groups (p < .05).

Table 1  The serum levels of BUN and creatinine and the tissue levels of MPO, MDA, NOx, and SOD

Groups	BUN (mg/dl)	Creatinine(mg/dl)	MPO (U/mg-protein)	MDA (nmoles/g tissue)	NOx(micromoles/liter )	SOD (U/mg tissue)
S	16.5	0.39	0.71	10	2.1	2.5
E1	17.4	0.32	1.2	14	3.3	3.6
E2	16.2	0.34	1.25	17	3	3.7
I	39.4	0.73	1.1	23	4	4.1
IE1	37.3	0.69	1.4	15	5	4.1
IE2	36.9	0.67	1.6	16	3.7	4.3

*p < .05 when compared to groups S, E1, and E2.

Abbreviations: BUN = blood urea nitrogen, MPO = myeloperoxidase, MDA = malondialdehyde, NOx = nitric oxide end products, SOD = superoxide dismutase.

The highest MPO levels were found in enoximone treatment groups (IE1 and IE2) compared to the others (p < .05). There was no significant difference between the other groups.

The mean tissue MDA level was the highest in group I compared to the others (p < .05), though there existed no significant difference between the other groups.

According to the results, NOx levels in the I, IE1, and IE2 groups were significantly higher than in the sham and control groups (p < .05). Similarly, SOD levels were significantly higher in the I, IE1, and IE2 groups than the others (p < .05). There was no statistical difference between the sham and control groups in terms of SOD and NOx levels.

Histopathological evaluation revealed no significant difference between the treatment groups and ischemia group.

DISCUSSION

The effects of the members of selective and nonselective PDE inhibitors on the renal IR injury have been comprehensively studied in the literature.^[1–5] The accepted modality is that of their enhancements on the IR injury. As a member of this family, the effect of enoximone was investigated in this study and found to be distinct from the other PDE inhibitors as having no beneficial effect on IR injury of kidney.

Williams et al. demonstrated that two hours' reperfusion following a 45-minute ischemia caused impairment in the renal function tests that worsened through 24 hours. A minimum duration of four hours of renal reperfusion has proven to be adequate for IR injury in terms of the changes took place in renal function, renal histopathology, and MPO levels.^[6] In this study, six-hour reperfusion following 45 minutes ischemia was created in order to evaluate the IR injury. Serum blood urea nitrogen and creatinine levels have been chosen to evaluate the altered glomerular hemodynamics, which appears as a result of renal ischemia and its treatment with the agent. Both of the functions were found impaired after ischemia, and the treatment with enoximone did not alter them.

IR injury is characterized by the generation of free radicals and neutrophil infiltration. The determination of the tissue levels of MPO, MDA, and NOx is regarded as a reliable way of documenting free radical production and neutrophil infiltration in an IR injury. In this study, the levels of MDA, NOx, and SOD were found to be elevated at the sixth hour of reperfusion in the ischemia groups compared to sham and control groups as an indicator of tissue damage (p < .05). MDA, NOx, and SOD levels of the treatment groups were found identical to the ischemia group, suggesting an ongoing injury despite enoximone treatment (p < .05). However, MPO levels increased only in the treatment groups, suggesting more neutrophil infiltration.

Similarly, the histopathological findings of the treatment groups showed no improvements when compared to the ischemia group.

Cyclic nucleotide PDEs are the enzymes that regulate the cellular levels of second messengers, cAMP and cGMP. Eleven types of different PDE families and their isoforms in each family were defined in the literature. Every type with its own properties modulates distinct regulatory pathways in the cell; thus, targeting specific PDE offers a fine way to treat a disease. The type III PDEs are shown to be present in the heart, vascular smooth muscle, kidney, adipocytes, hepatocytes, oocyte, developing sperms, platelets, T lymphocytes, and macrophages.^[7]

In one study, Jackson et al. demonstrated that the type IV PDE is the predominant isoenzyme in the rat kidney vasculature.^[8] They have shown that type IV PDE blockage significantly altered cAMP metabolism not only in the perfused kidney but also in the cultured preglomerular vascular smooth muscle cells, and considered the type IV PDE inhibitors' clinical value in treating renal diseases in which elevated renal vascular cAMP levels are desired. Even though the predominant PDE is proven to be type IV PDE in kidney, the proportions of the other isoenzymes are not known.

The type III PDE inhibitors' family is characterized by its high affinity for cAMP and competitive inhibition by cGMP. Enoximone is an imidazole derivative that selectively inhibits the type III PDE enzyme in cardiac and vascular smooth muscle. The inhibition of the enzyme results in an accumulation of intracellular cAMP and leads the enhanced activity of cAMP-dependent protein kinases. These enzymes activate other proteins, which cause a decrease in intracellular Ca⁺² levels and the suppression of the sensitivity of contractile elements to Ca⁺². These intracellular changes are the reasons behind the positive inotropism and vascular relaxation effects of the drug.^[9]

The findings of this study showed that as a therapeutic agent, enoximone has no effect on IR injury of kidney. Karabulut et al. showed the protective effects of amrinone (the prototype of type III PDE inhibitors) on ischemic reperfusion injury of kidney.^[2] Anas et al. reported the beneficial effects of olprinone (the other member of type III PDE inhibitors) in the renal ischemia reperfusion in terms of increased flow due to vasodilatation, maintained endothelium and tubular epithelium integrity, and preserved anti-inflammatory effects.^[5] Setoyama et al. pointed out that milrinone (the other member of type III PDE inhibitors) has increased the renal blood flow under hypoxic conditions.^[10] In their study on healthy dogs with different inotropic agents, Tobato et al. found that the renal blood flow was altered with the use of amrinone and milrinone, though these alterations were not statistically significant.^[11] However, all members of the type III PDE inhibitors have similar clinical effects, as the former three drugs, being biypridine derivatives, are chemically diverse from enoximone, an imidazole derivative. This chemical diversity can be speculated as the reason for different effects on the renal tissue. Moreover, the proportion of the isoenzymes in the renal vasculature can be blamed as the reason behind unlike effects of the same group drugs. However, amrinone is the prototype of the type III PDE inhibitors, as it inhibits type I and II PDE isoenzymes to some extent.^[12] As amrinone inhibits type I, II, and III isoenzymes and enoximone inhibits type III isoenzyme, and as the effects of amrinone are found to be superior to enoximone in renal IR injury, we can speculate about the proportion of other isoenzymes in the renal vasculature. In kidney vasculature, where predominance of type IV is present, the proportion of type I and II is much more than type III. Moreover under special conditions like hypoxia, it is probable that the expression of the PDE isoenzymes might change in the renal tissue. It is obvious that further studies are needed to prove these hypotheses.

Because of its positive inotropic and vasodilatory effects, enoximone is commonly used in the clinic practice. However, as the drug is known to improve hemodynamic status, the clinic effects of enoximone on renal function are controversial. In an earlier study, Leier et al. stated that either 1 or 2 mg/kg doses of enoximone led to an increase in cardiac output and a decrease in systemic vascular resistance in a placebo-controlled crossover trial of 12 patients with severe chronic heart failure.^[13] However, neither dose caused a change in the renal blood flow or in the renal vascular resistance. Clifton et al. found significant acute reductions in glomerular filtration rates (18%) and effective renal plasma flow (20%) after intravenous administration of the drug.^[14] They also reported that these effects are not to be seen after an oral route. On the contrary, Berti et al. reported beneficial effects in the renal hemodynamics after 24 hours infusion of the drug in terms of improved renal plasma flow.^[15] This effect was found to last 48 hours after the discontinuation of the drug. However, they interpreted this to be a less marked but a more lasting effect on renal hemodynamics. In a study on 42 patients, Boldt et al. evaluated the effects of prophylactic usage of a beta-blocker (esmolol) and type III PDE inhibitor (enoximone) in elder cardiac surgery patients.^[16] They examined the serum creatinine clearance and urine N-acetyl-beta-D-glucosaminidase levels in order to determine the renal functions. They found that the kidney functions were preserved from the negative effects of cardiopulmonary bypass with the use of this combination. They also attributed this effect to be related to the effects of the type III PDE inhibitor on (micro-)perfusion.

The type III PDE inhibitors are known to attenuate the mediator response in systemic inflammation. These agents act as antiinflammatory agents and cause a reduction in the formation and secretion of pro-inflammatory chemokines. In the case of ischemic renal reperfusion injury, most of their favorable consequences were attributed to their anti-inflammatory effects.^[1,5] In a study by Loick et al., endotoxin levels were found to be minimized with enoximone use in the patients undergoing cardiopulmonary bypass.^[17] In another study, Santarpino et al. demonstrated the anti-inflammatory response of the drug to cardiopulmonary bypass in patients undergoing myocardial revascularization.^[18] The unchanged levels of MPO in the treated groups in our study suggested a distinct inflammatory effect of the drug that may be attributed to proinflammatory cytokines in the damaged area and proinflammatory/anti-inflammatory leukotrienes ratio.

The diverse findings of our study can be explained with a few more mechanisms. The first mechanism is connected with metal-mediated free radical attacks. Oxidative damage generally leads metal ions to release from metalloproteins within the cell, and these ions tend to bind to macromolecules serving as centers for repeated production of free radicals.^[19] These metal ions may be reduced into their lower oxidative states in the damaged tissues. These ions in their new forms in return further promote free radical injury. That is how the administration of a “reducing” antioxidant agent could worsen the damage on an ischemic basis. Another explanation is that enoximone cannot be an appropriate antioxidant agent in the prevention of metal-mediated damage like vitamin E or melatonin. Moreover, the differences in redox state and susceptibility to oxidant-induced changes in cell functions among different cell types can be blamed as the reason behind these conflicting results.

In conclusion, enoximone was found to be ineffective in renal IR injury. These controversial findings of renal functions in the clinic studies and the findings of our experimental study revealed that enoximone, apart from the other PDE inhibitors, have supported some conflicting effects on kidney. We believe that the drug neither enhances ischemic reperfusion injury nor has absolute beneficial effects on renal functions. It should be a piece of wise advice to postpone using this agent for the cardiac patients with a known renal impairment. We think that this drug deserves further investigations in order to be used safely in the cardiac patients with renal impairment.

ACKNOWLEDGMENTS

The authors declare no conflicts of interest.