The Role of ATP-Sensitive Potassium Channel Blockers in Ischemia-Reperfusion-Induced Renal Injury Versus Their Effects on Cardiac Ischemia Reperfusion in Rats

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European Review for Medical and Pharmacological Sciences 2009; 13: 81-93 The role of ATP-sensitive potassium channel blockers in ischemia-reperfusion-induced renal injury versus their effects on cardiac ischemia reperfusion in rats

MONA K. TAWFIKa, DINA M. ABO-ELMATTYb, AMAL A.M. AHMEDc aDepartment of Pharmacology, Faculty of Medicine, bDepartment of Biochemistry, Faculty of Pharmacy, cDepartment of Histology, Faculty of Veterinary Medicine, a,b,cSuez Canal University, Ismailia (Egypt)

Abstract. – In renal ischemia reperfusion Key Words: (I/R), opening of adenosine-triphosphate (ATP)- Renal ischemia reperfusion injury, Cardiac ischemia sensitive potassium (KATP) channels results in reperfusion injury, Neutrophil infiltration, Diazoxide, massive influx of neutrophils in both renal and Glibenclamide, Glimepiride. lung tissues. Our study was focused on the role of ATP-dependent potassium channel modula- tors, glimepiride and glibenclamide on I/R induced renal injury in rats. Additionally we evaluated their effects on normal heart and on ischemic reperfused heart subjected to ischemic Introduction preconditioning protection afforded by diazoxide. To test this hypothesis, we used renal I/R and cardiac I/R experiment. Renal ischemia Renal ischemia reperfusion (I/R) is an impor- reperfusion induced marked renal dysfunction tant cause of renal dysfunction in renal transplan- associated with significant increase in arterial tation and is associated with an increased rate of pressure, TNF-α level s, superoxide anion pro- 1,2 duction, and myeloperoxidase activity. Treat- acute and chronic rejection . In addition, it is ment with glibenclamide or glimepiride, demon- the most common cause of acute renal failure in 3 strated a significant improvement in the reperfu- shock, sepsis, and renal artery stenosis . sion-induced injury in both kidney and lung. The impairment of organ function derived Glimepiride has no effect on superoxide anion from ischemia/reperfusion injury is still an im- production. However glibenclamide induced a portant problem in solid organ transplantation. significant improvement in these measurements as compared to glimepiride group. Although the restoration of blood flow is the Before coronary artery ligation, neither dia- treatment of choice following acute ischemia of zoxide nor glimepiride pretreatment influenced vascular territory, restoration of oxygen delivery significantly the electrocardiographic parame- or reperfusion after ischemia can be accompa- ters in comparison with control group. Con- nied by significant injury to local and remote or- versely, glibenclamide supplementation in- gans, such as the lung, and systemic inflammato- duced a significant elevation in these parame- ry events that may limit the beneficial effects of ters. After left coronary artery ligation, reperfu- 4 sion of the ischemic hearts caused a signifi- blood flow restoration . The reperfusion-associ- cant elevation in the measured electrocardio- ated injury could be attributed to oxygen-derived +2 graphic parameters. These elevations were sig- free radicals and intracellular calcium (Ca ) nificantly ameliorated by the pretreatment with overload, which are associated with further in- diazoxide. In conclusion, the administration of jury, as well as, recruitment and activation of leu- glibenclamide significantly abolished the pro- cocytes and subsequent release of mediators of tective effects of diazoxide, while the pretreat- 3,5 ment with glimepiride didn't abolish it. So, the inflammatory process . glimepiride offers some promise for therapy of In the kidney, fall in intracellular ATP concen- renal I/R with minimizing the undesirable car- tration induces opening of adenosine-triphos- diac side effects. phate (ATP)-sensitive potassium (KATP) channels,

Corresponding Author: Mona K. Tawfik, MD; e-mail: [email protected] 81 Mona K. Tawfik, Dina M. Abo-ElMatty, Amal A.M. Ahmed that plays an important role in the massive influx to assess the role of ATP-dependent potassium of neutrophils early after reperfusion, exerting a channel modulation, comparing the protective ef- crucial role in the patho-physiology of post-is- fects of glimepiride and of glibenclamide on re- chemic renal failure, by the release of various in- nal I/R inflammatory injury and neutrophil ag- flammatory mediators as cytotoxic proteases and gregation. Moreover, we have observed whether oxygen-derived free radicals3,4,6. the inhibition of the inflammatory injury is ac- Renal I/R is a complex neutrophil-mediated companied by an improvement of renal dysfunc- syndrome, in which neutrophil and TNF-α are tion in rats. For comparison, we have also evalu- among the cell types and inflammatory media- ated the effects of the potassium channel opener tors, respectively, thought to be of major rele- diazoxide. Because the protective effect of KATP vance in the patho-physiology of I/R injury1. channel blockade in renal I/R stands in sharp Many of the pathological changes that follow I/R contrast to the harmful effects on the cardiac tis- injury in various organs are believed to be in- sues, our study was extended to evaluate the pos- duced by infiltrating activated neutrophils2. sible harmful effects of both sulfonylureas drugs Myeloperoxidase (MPO), an heme enzyme se- on normal heart and on ischemic reperfused heart creted by activated neutrophils, is an accepted in- subjected to ischemic preconditioning (IP) pro- dicator of neutrophil accumulation and oxidative tection afforded by diazoxide which is one of the 7 – activity . Superoxide anion (O2 ) generated by preconditioning mimetic agents. neutrophils, monocytes and macrophages is of a central importance in host defense mechanism and it is involved in augmenting neutrophils in- 8 Materials and Methods flux into the tissues . + ATP-sensitive K (KATP) channels belong to a family of inward rectifying potassium channels9, Animals: one hundred and fourteen (n=114) whose structure is believed to comprise a adult albino rats weighing 250 ± 10 g were used tetramer of four inwardly rectifying K+ channel in this study: seventy two of them were used for subunits, that are called the Kir subunits (form- the in renal I/R experiment and forty two rats for ing the ionic pore), coupled to four regulatory the in cardiac I/R experiment. They were pur- sulphonylurea receptors (SUR) subunits10,11. This chased from the Egyptian Organization for Bio- structure allowed to several subclasses of those logical Products and Vaccines (Egypt), and al- ionic channels12-14. The four regulatory SUR sub- lowed free access to food and water ad libitum. units contain the binding site for the anti-diabetic They were kept under constant conditions with sulfonylureas, and the pharmacological specifici- 12/12 h light/dark cycles and left for acclimatiza- ty of each sulfonylurea depends on the type of tion for one week before the start of the study. SUR protein present in each tissue11. Another The care and handling of the animals used in the protein called the Cystic Fibrosis Transmem- present study was in accordance with the guide- brane conductance Regulator (CFTR) is ex- lines of the National Institutes of Health (NIH). pressed abundantly in the kidney and associates Drugs: glibenclamide, glimepiride (Aventis with both ATP and sulfonylurea sensitivity10,15,16. Pharmaceutical Company, Egypt) and diazoxide The KATP is also abundant in the cardiac my- (Sigma Chemical Company, Egypt) were used. ocytes. Therefore, sulfonylurea may also bind the Glibenclamide, glimepiride and diazoxide were KATP channels of the cardiac myocytes with sub- given intraperitoneally at doses of 20 mg/kg, 5 sequent development of cardiac complications13. mg/kg and 30 mg/kg respectively4,19. All drugs Glimepiride is supposed to have several bene- were dissolved in dimethyl sulfoxide (DMSO)20. fits over conventional drugs such as glibenclamide. The benefits include rapid onset and 17 (I) Renal I/R Experiment longer duration of action , and more specificity than glibenclamide for the SUR1 receptor in the pancreatic beta cells18. Animal model of renal I/R: animals were giv- As treatment of renal I/R injury is still only en water but no food for 24 hours before the ex- supportive, the development of therapeutic inter- periment. Rats were anaesthetized with intra- ventions to prevent or reduce a renal tissue injury peritoneal injection of urethane in a dose of 1.25 after I/R remains the focus of research. In the mg/kg21. The renal I/R protocol was performed present study we have used the renal I/R model according to Pompermayer et al4. Left nephrecto-

82 Role of ATP-sensitive potassium channel blockers my was performed through a left flank incision. Group 5 – Renal I/R + glibenclamide group (I/R Renal ischemia was performed by right flank in- + Glibenclamide): rats were subject- cision and dissecting the right renal pedicle so as ed to I/R and were pretreated with to expose the renal vessels. A thick cotton thread glibenclamide. was passed over the renal vascular pedicle to in- Group 6 – Renal I/R + glimepiride group (I/R + duce ischemia. Both ends of the thread were ex- Glimepiride): rats were subjected to teriorized through the back of the rat and tied I/R and were pretreated with tightly above a length of latex tubing on the ex- glimepiride. ternal side of the skin. Ischemia was confirmed visually by blanching of the kidney. The renal Urine samples were collected using the meta- pedicle was released 45 minutes after occlusion bolic cadges. Rats from each group were anaes- by cutting the thread and pulling it out, and the thetized with (i.p.) urethane 1.25 mg/kg21. Arteri- wound was closed with 3-0 silk suture followed al pressure changes were measured through can- by 4 or 24 hours of reperfusion. Animals were al- nulation of the carotid artery. Blood samples lowed to recover from anesthesia without further were collected via the tail veins, then rats were interventions. sacrificed, kidney and lung tissues were also ob- Experimental Protocol tained and divided into two portions. The first one was immediately frozen at -80°C for the dif- All rats were subjected to left nephrectomy, ferent biochemical determinations, while the oth- then they were divided into six experimental er part was embedded in 10% neutral buffered groups (12 rats each). Each group was divided formalin and processed to perform histopatho- into two sub-groups according to the period of logical assay 4-6 µm thick paraffin sections were reperfusion at 4 hours (sub-group A, n=6) or 24 subjected to the hematoxyline and eosin stain. hours (sub-group B, n=6). Diazoxide was given Rats in sub-groups A were sacrificed 4 hours as a single intraperitoneal (i.p.) injection at 40 after the time of reperfusion, while rats in sub- minutes before reperfusion for both the 4 and 24 groups B were sacrificed 24 hours after the time hours reperfusion. of reperfusion. Glibenclamide and glimepiride were given as Measurement of Renal Function single dose 40 minutes before reperfusion for 4 hours reperfusion but for 24 hours reperfusion Renal dysfunction was evaluated by measur- they were given in two doses, one 40 minutes be- ing serum levels of blood urea nitrogen and crea- fore reperfusion and the other 8 hours after reper- tinine by standard urease assays and picric acid fusion4. reactions by colorimetric methods22, using Bio- clin kit (Santa Coloma, Spain). Group 1 – Sham operated control group (Sham): Determination of Blood Glucose Level rats were submitted to left nephrectomy and were used as control for the Serum glucose level was determined enzymat- reperfusion-induced injury. ically according to the principle of23, using Spin- Group 2 – Renal ischemia reperfusion group react Diagnostics Kits (Santa Coloma, Spain). (I/R): rats were submitted to 45 min- Measuring Systemic Arterial Blood Pressure utes of ischemia, and then the renal pedicles were released and followed Recording was done by using the PT400 blood by reperfusion period. This group was pressure transducer that was connected to the kept without treatment and served as FC137 strain gauge coupler which was fixed to a a positive control. channel of the oscillograph. A mid line longitudi- Group 3 – Renal I/R + solvent control group nal skin incision started just below the neck and (I/R + solvent): rats were subjected to extended to the sternum was done to expose the I/R and received DMSO i.p. and trachea and the common carotid artery. A tight served as a control group for the test- ligature of the artery at its distal end (cephalic ed drugs. end), and a loose ligature around its proximal Group 4 – Renal I/R + diazoxide group (I/R + end (thoracic end) were applied. A bulldog clamb Diazoxide): rats were subjected to was applied and a small snip across the artery I/R and were pretreated with dia- was opened by a small sharp scissors. A polyeth- zoxide. ylene arterial cannula filled with the heparinized

83 Mona K. Tawfik, Dina M. Abo-ElMatty, Amal A.M. Ahmed saline solution was inserted gently towards the was homogenized in phosphate buffer saline and heart, the ligature was tied around the cannula centrifuged at 16,000 × g for 15 minutes in cool- and the bulldog clamb was removed. About 10 ing centrifuge. The supernatant was added to 0.5 cm polyethylene tube with a clamp was connect- mM cytochrome C (Sigma Chemical Co, Egypt). ed to one side limb of the PT400 transducer, and This mixture was centrifuged again at 3000 × g the other limb of the transducer was connected for 10 minutes, the supernatant fractions were through polyethylene tube with a clamp to the ar- collected for subsequent spectrophotometrically terial cannula. Calibration was done by using the measurement at 550 nm. oscillograph at speed 0.25 mm/second24. Assay of Renal Myeloperoxidase (II) Cardiac I/R experiment (MPO) Activity Assaying of MPO activity was described Cardiac ischemia reperfusion model by left by4,25,26. Briefly, kidney or lung tissue (0.5 gm) coronary artery ligation was used for evaluating were homogenized in 10 ml of homogenization the side effects of both sulfonylureas (gliben- buffer pH (4.7) [0.1 mol/L NaCl, 0.02 mol/L clamide and glimepiride) on both normal and is- NaPO4 and 0.015 mol/L sodium ethylene di- chemic preconditioning rat’s hearts. amine tetracetic acid (EDTA)], centrifuge at 260 × Animal model of cardiac g for 10 minutes and the pellet underwent (dis- ischemia reperfusion solved) hypotonic lysis (0.2% NaCl) solution followed 1 minutes later by addition an equal vol- Rats were anaesthetized with intra-peritoneal ume of solution containing (1.6% NaCl and 5% injection of urethane in a dose of 1.25 mg/kg. glucose). After further centrifugation, the pellet The chest was opened in the fourth intercostal was then suspended in resuspension buffer pH space, and the heart was exposed and a loose 5.4 (0.05 mol/L NaPO4 containing 0.5% hexade- loop of silk was placed around the left main cyltrimethylammonium bromide) and rehomoge- coronary artery, approximately 2 mm from its nized. One milliliter aliquots of the suspension origin. Both ends of the ligature were led out of were freezed and thawed three cycles in liquid the thoracic cavity through flexible tubing. The nitrogen, then centrifuged for 15 minutes at 3000 heart was set back in its place and artificial respi- g. the pellet was discarded. MPO activity was as- ration was started. The standard electrocardio- sayed by measuring the change in optical density gram (lead II, ECG) was recorded using subcuta- at 450 nm using tetramethylbenzidine, as sub- neous needle electrodes. The animals were al- strate (1.5 mmol/L) and H2O2 (0.5 mmol/L). Re- lowed to stabilize for 10 minutes, then the loose sults were expressed as MPO relative units/100 loop of the coronary artery ligature was tightened mg tissue. One unit of MPO activity was defined and fixed by clamping on the silk and thus re- as the quantity of enzyme degrading one mmol gional myocardial ischemia was produced for 6 peroxide at 25°C. The activity of purified known minutes and then followed by reperfusion for 5 human neutrophil MPO was used as the standard minutes19. (Sigma Chemical Co, Egypt). Experimental Protocol Evaluation of TNF- in Lung and Kidney α Rats were divided into seven experimental Kidneys and lungs were homogenized using 0.1 groups (6 rats for each). Diazoxide, gliben- M phosphate buffer (pH 7.4) containing 0.05% clamide and glimepiride were given as a single (wt/vol) sodium azide at 4°C. Homogenates were i.p. dose 30 minutes before coronary artery liga- sonicated for 20 seconds and centrifuged (2000 g tion19. for 10 minutes at 4°C). The resulting supernatants were used for assaying TNF-α levels using Group 1 – Control group (cardiac I/R): rats ELISA (BioSource Europe S.A., Belgium)25. were submitted to six minutes of is- Assay of Superoxide Anion Production in chemia, and then the coronary liga- Kidney and Lung Tissues tion were released and followed by reperfusion period. This group was – Tissue O2 was determined according to the kept without treatment and served as modified method of Hassoun and Sthos27 follow- control group. ing the principle of Babior et al.28. Briefly, tissue Group 2 – Solvent treated group (cardiac I/R +

84 Role of ATP-sensitive potassium channel blockers solvent): rats were subjected to cardiac I/R and with a significant reduction in urine volume (P received DMSO i.p. 30 minutes before ligation <0.001) compared with sham-operated group, and served as a control group for the tested suggesting a significant degree of glomerular dys- drugs. function caused by renal I/R. These functional Group 3 – Diazoxide treated group (cardiac I/R + changes were prevented by treatment with Diazoxide): rats were subjected to car- sulphonylureas (glibenclamide & glimepiride) diac I/R and were pretreated 30 min- supplementation in comparison with I/R group. utes before ligation with diazoxide. However glibenclamide induced a significant im- Group 4 – Glibenclamide treated group (cardiac provement in renal function as compared to I/R + Glibenclamide): rats were sub- glimepiride group. Additionally renal I/R induced jected to cardiac I/R and were pre- a significant elevation in blood glucose level in treated 30 minutes before ligation comparison with the sham-operated group. While with glibenclamide. treatment with glibenclamide & glimepiride sig- Group 5 – Glimepiride treated group (cardiac I/R nificantly reduced this elevated glucose level as + Glimepiride): rats were subjected to compared to I/R group (Table I A, B). cardiac I/R and were pretreated 30 min- Table II A, B, Figure 1 showed that the renal utes before ligation with glimepiride. ischemic reperfusion model had a significant ele- Group 6 – Diazoxide + Glibenclamide treated vation in arterial blood pressure compared with group (cardiac I/R + Diazoxide + sham-operated group (P <0.001). These elevated Glibenclamide): rats were subjected measurements were improved by treatment with to cardiac I/R and were pretreated 30 sulphonylureas (glibenclamide & glimepiride) in minutes before ligation with both dia- comparison with I/R group (0.001). However zoxide and glibenclamide. glibenclamide induced a significant improvement Group 7 – Diazoxide + Glimepiride treated in these measurements as compared to glimepiri- group (cardiac I/R + Diazoxide + de group (P < 0.05). Glimepiride): rats were subjected to Table III A, B showed that reperfusion for (4 cardiac I/R and were pretreated 30 & 24 hours) I/R, leads to a significant elevation minutes before ligation with both dia- (P <0.001) in both renal and lung MPO activity .- zoxide and glimepiride. and O2 production in comparison with the Sham-operated group. Treatment with gliben- The anaesthetized rats of each group were sub- clamide significantly reduced both MPO activity .- jected to ECG monitoring using the two-channel and O2 production in renal and lung tissues oscillograph at speed of 50 mm/second before compared to the I/R. However, treatment with and after coronary ligation. The electrocardio- glimepiride significantly reduced MPO activity graph was adjusted so that each 20 mm height but, failed to affect the reperfusion-induced in- – represented 1 mv. The heart rate, T-wave voltage creased in the O2 production (P <0.001). and ST segment displacement were measured. Renal I/R induced a marked elevation in the concentration of TNF-α in both the renal and Statistical Analysis lung tissues. Treatment with glibenclamide or glimepiride suppressed the reperfusion-induced TNF-α (P <0.001) in the kidney and abolished SPSS program version 15 was used. The statis- its production in the lungs in both (4 & 24 hours tical significance was evaluated by using Student’s reperfusion with more potency of glibenclamide t test for comparison between the different groups. than glimepiride (Figure 2). P value <0.05 was considered significant. Diazoxide administration caused a significant increase in all studied measurements with signifi- Results cantly reduced urine volume in comparison with both glibenclamide &glimepiride (P <0.001) (I) Renal I/R Experiment (Tables I, III & Figure 2). As expected by its va- sodilatory effects, administration of diazoxide induced significant (P <0.001) reduction in the ele- Reperfusion of ischemic kidney induced signif- vated blood pressure as compared with I/R group icant elevation in serum levels of urea and creati- (II)(Table Cardiac II A, B, I/R Figure experiment 1). nine (in both 4 & 24 hours reperfusion periods)

85 Mona K. Tawfik, Dina M. Abo-ElMatty, Amal A.M. Ahmed

Table I. Effect of diazoxide (D) glibenclamide (Glib) and glimepiride (Glim) on serum glucose (mg/dl), creatinine (mg/dl), BUN (mg/dl) and urine volume (ml × 10-1/min) in rats exposed to renal I/R for 4 hours (a), for 24 hours (b). Groups A Sham I/R I/R + solvent I/R + D I/R + Glib I/R + Glim

Glucose 84.8 ± 10.8 160.0 ± 17.5† 162.5 ± 18.5† 165.6 ± 14.4† 115 ± 9.8# 108 ± 9.7# Creatinine 0.83 ± 0.27 2.18 ± 0.44† 2.06 ± 0.46† 2.26 ± 0.42† 1.06 ± 0.18# 1.5 ± 0.31#§ BUN 27.3 ± 4.08 65.0 ±7.8† 65.1 ± 7.1† 71.6 ± 5.8† 28.1 ± 4.9# 42.6 ± 5.9#§ Urine volume 1.26 ± 0.25 0.11 ± 0.034† .11 ± 0.028† 0.10 ± 0.044† 1.2 ± 0.22# 0.93 ± 0.18#§

Groups B Sham I/R I/R + solvent I/R + D I/R + Glib I/R + Glim

Glucose 94.5 ± 4.0 178 ± 13.3† 167.8 ± 13.5† 180.0 ± 10.7† 113 ± 10.0# 111 ± 10.8# Creatinine 0.91 ± 0.15 2.2 ± 0.28† 2.3 ± 0.2† 2.41 ± 0.47† 1.16 ± 0.18# 1.58 ± 0.14#§ BUN 28.6 ± 8.5 72.3 ± 5.3† 70.6 ± 7.5† 75.3 ± 7.8† 32.6 ± 5.8# 45.0 ± 6.4#§ Urine volume 1.5 ± 0.36 0.09 ± 0.016† 0.098 ± 0.017† 0.086 ± 0.013† 1.4 ± 0.31# 1.05 ± 0.13#§ n = 6 †Significantly different from sham group at p < 0.001; #Significantly different from I/R group at p < 0.001. §Significantly different from glibenclamide group at p < 0.05.

the protective effects of diazoxide, while pre- Before coronary artery ligation, neither dia- treatment with glimepiride didn’t abolish it. zoxide nor glimepiride pretreatment influenced Histological Findings significantly the electrocardiographic parameters (heart rates, T-waves voltages and ST segment As shown in Figure 3, histological examina- elevation) in comparison with the control group. tion of renal sections stained with H& E showed However, glibenclamide supplementation in- no histopathological changes in the kidney of duced a significant elevation (P <0.001) in all Sham-operated group in cortex and medulla (a) these parameters (Table IV & Figure 5). while kidney sections of I/R group showed im- After left coronary artery ligation, reperfusion munocompetent cell aggregation, tubular cell of the ischemic hearts caused a significant eleva- necrosis as well as shedding of the lining epithe- tion in the measured electrocardiographic para- lium and cytoplasmic eosinophilia (b). I/R pre- meters. These elevations were significantly (P treated with diazoxide was similar to I/R group <0.001) ameliorated by pretreatment with dia- in addition the renal tubules showed hyaline zoxide Table IV & Figure 5. Administration of casts (c). Treatment with glibenclamide was glibenclamide significantly (P <0.001) abolished nearly similar to Sham group except that there

Table II. Effect of diazoxide (D) glibenclamide (Glib) and glimepiride (Glim) on systolic and diastolic blood pressure (BP) (mm Hg) in rats exposed to renal I/R for 4 hours (A), for 24 hours (B). Groups A Sham I/R I/R + solvent I/R + D I/R + Glib I/R + Glim

Systolic B.P. 98.3 ± 8.2 170.8 ± 5.8† 169.1 ± 5.8† 112.5 ± 2.7# 122.2 ± 4.7# 131.6 ± 2.5#§ Diastolic B.P. 66.6 ± 6.0 136.6 ± 4.0† 133.3 ± 19.4† 81.6 ± 6.1# 87.0 ± 7.6# 101.6 ± 13.6#§

Groups B Sham I/R I/R + solvent I/R + D I/R + Glib I/R + Glim

Systolic B.P. 97.5 ± 5.2 172.5 ± 5.2† 171.6 ± 6.1† 113.3 ± 9.8# 119.2 ± 5.8# 130.8 ± 3.8#§ Diastolic B.P. 70.8 ± 7.3 132.5 ± 5.2† 133.3 ± 4.08† 83.3 ± 6.8# 86.6 ±7.5# 98.3 ± 5.1#§ n = 6 †Significantly different from sham group at p < 0.001; #Significantly different from I/R group at p < 0.001. §Significantly different from glibenclamide group at p < 0.05.

86 Role of ATP-sensitive potassium channel blockers

Sham-operated control group Renal ischemia-reperfusion group

A B

Renal I/R + Diazoxide group Renal I/R + Glibenclamide group

C D

Renal I/R + Glimepiride group

Figure 1. Effect of diazoxide, glibenclamide and glimepiride on systolic and diastolic blood pressure in rats exposed to renal ischemia and reperfusion (I/R).

Table III. Effect of diazoxide (D) glibenclamide (Glib) and glimepiride (Glim) on myeloperoxidase (MPO) activity (U/100 – -4 mg tissue) and O2 (nmol cytochrome C reduced /min/g tissue × 10 ) in the kidney and lung tissues of rats exposed to renal I/R for 4 hours (A), for 24 hours (B). Groups A Sham I/R I/R + solvent I/R + D I/R + Glib I/R + Glim

MPO Kidney 0.03 ± 0.01 0.45 ± 0.05† 0.43 ± 0.08† 0.47 ± 0.06† 0.05 ± 0.01# 0.103 ± 0.016#§ Lung 1.3 ± 36 12.1 ± 4.6† 11.46 ± 1.55† 12.8 ± 2.7† 2.5 ± 0.54# 5.9 ± 0.93#§ – O2 Kidney 1.05 ± 0.18 3.1 ± 0.5† 3.18 ± 0.37† 3.0 ± 0.53† 1.26 ± 0.24# 2.9 ± 0.19§ Lung 2.9 ± 0.66 6.3 ±1.0† 6.4 ± 0.63† 6.4 ± 0.86† 3.63 ± 0.5# 5.9 ± 1.3§

Groups B Sham I/R I/R + solvent I/R + D I/R + Glib I/R + Glim

MPO Kidney 0.048 ± 0.007 0.55 ± 0.12† 0.58 ± 0.15† 0.59 ± 0.08† 0.06 ± 0.017# 0.155 ± 0.03#§ Lung 2.0 ± 0.49 11.2 ± 1.83† 9.56 ± 1.55† 10.3 ± 1.7† 2.65 ± 0.7# 4.8 ± 0.7#§ – O2 Kidney 1.08 ± 0.19 3.2 ± 0.3† 3.3 ± 0.42† 3.16 ± 0.48† 1.25 ± 0.27# 3.0 ± 0.67§ Lung 2.2 ± 0.44 6.4 ± 0.9† 5.9 ± 0.66† 6.4 ± 0.86† 2.4 ± 0.47# 5.8 ± 0.66§ n = 6 †Significantly different from sham group at p < 0.001; #Significantly different from I/R group at p < 0.001; §Significantly different from glibenclamide group at p < 0.05.

87 Mona K. Tawfik, Dina M. Abo-ElMatty, Amal A.M. Ahmed (pg/ 100 mg tissue) (pg/ 100 mg tissue) TNF- α TNF- α A Groups Groups B

Figure 2. Effect of diazoxide (D), glibenclamide (Glib), and glimepiride (Glim) on TNF-α (pg/ 100 mg tissue) in the kidney (A) (B) * and lung tissues of rats exposed to renal I/R for 4 hr and 24 hr. n = 6. Significantly different from sham group at 4 and 24 h (p < 0.001); #Significantly different from I/R group at 4 and 24 h (p < 0.001); aSignificantly different from glibenclamide group at 4 and 24 h (p < 0.05).

A B

C D

E F

Figure 3. A, A photomicrograph renal sections stained with H & E showed no histopathological changes in kidney of sham B, C, operated group in cortex and medulla. While kidney sections of I/R group showed immunocompetent cell aggregation, D, tubular cell necrosis as well as shedding of the lining epithelium and cytoplasmic eosinophilia. I/R pretreated with diazoxide was similar to I/R group in addition to renal tubules showed hyaline casts. Treatment with glibenclamide was nearly similar (E) to sham group except that there was a light edema of tubular cells , however glimepiride had less improvement than gliben- (F) clamide . (H & E 400 ×).

88 Role of ATP-sensitive potassium channel blockers

Table IV. The effect of diazoxide (D) glibenclamide (Glib) and glimepiride (Glim) on heart rate (beats/min), T waves (mv) and ST segment elevation (mm) in rats before and after coronary artery ligation. Before ligation After ligation Groups Heart T-wave ST segment Heart T-wave ST segment rate voltage elevation rate voltage elevation

Cardiac I/R 278.3 ± 20.4 0.21 ± 0.04 1.7 ± 0.5 466.6 ± 37.2 0.65 ± 0.058 6.6 ± 0.58 Cardiac I/R + solvent 268.3 ± 19.4 0.18 ± 0.04 2.0 ± 0.66 460 ± 33.5 0.68 ± 0.04 6.3 ± 0.41 Cardiac I/R + D 270 ± 21.9 0.2 ± 0.037 1.9 ± 0.37 375 ± 23.2# 0.32 ± 0.051# 2.9 ± 0.58# Cardiac I/R + Glib 388.3 ± 22† 0.45 ± 0.058† 4.9 ± 0.73† 463.3 ± 51.25 0.68 ± 0.7 6.4 ± 0.58 Cardiac I/R + Glim 275 ± 23.5 0.24 ± 0.037 1.8 ± 0.25 453.3 ± 37.3 0.63 ± 0.068 6.2 ± 0.52 Cardiac I/R + D + Glib 395 ± 21.5† 0.46 ± 0.06† 5 ± 0.490† 463.3 ± 34.5 0.58 ± 0.06 5.9 ± 0.66 Cardiac I/R + D + Glim 266.6 ± 18.6 0.2 ± 0.052 1.8 ± 0.51 377.5 ± 22.1# 0.35 ± 0.04# 3.1 ± 0.52# n = 6 †Significantly different from cardiac I/R group before ligation at p < 0. 001; #Significantly different from cardiac I/R group after ligation at p < 0. 001.

A B

C D

Figure 4. A, A photomicrograph showed sections of the sham lung stained with H & E showed normal architecture with no B, C, histopathological changes. Sections of I/R and I/R pretreated with diazoxide showed marked increase in thickness of the inter-alveolar septa due to mononuclear cellular infiltration. This thickening of the inter-alveolar septa caused narrowing of the D, E, alveolar cavities. Treatment with glibenclamide and glimepiride preserved the normal histology of the lung. There was a significant decrease in the thickening of inter-alveolar septa. (H & E 400 ×, except a: 100 ×).

89 Mona K. Tawfik, Dina M. Abo-ElMatty, Amal A.M. Ahmed

Control group tion. So, renal ischemia followed by reperfusion results in severe injury that may contribute to renal damage29,30 and accompanies a lung inflam- A B mation, which complicates the reperfusion syn- 29,31 Diazoxide-treated group drome and contributes to the overall lethality . So, strategies that limit reperfusion-associated renal damage may be useful in these conditions. A B Our data demonstrated that, after 4 hours of renal reperfusion, there was a marked elevation Glibenclamide-treated group of the TNF-α level in kidney tissues, and these effects were extended and still present at 24 hours. This increase was due, in ischemic reper- A B fused injury, to complement activation and neu- Glimepiride-treated group trophil stimulation with accompanying oxygen radical-mediated injury29. Oxidants were released during the reperfusion of ischemic tissue; more- A B over, neutrophil activation stimulated transcrip- tion factors involved in TNF-α expression4,6,29. Diazoxide + Glibenclamide-treated group This notion was confirmed in our study by the – findings that renal tissue levels of O2 signifi- A B cantly were increased in association with the accumulated neutrophils and elevated TNF-α level Diazoxide + Glimepiride-treated group in 4 hours of reperfusion and extended also to 24 hours. These results agreed with those of Rah- 3 A B gozar et al. who emphasized that reperfusion of ischemic tissues imposes an oxidant burden in which the reduced form of molecular oxygen, Figure 5. The effect of diazoxide, glibenclamide and hydrogen peroxide, contributes to injury. Fur- (A) (B) glimepiride on ECG tracing in rats before and after thermore, the activation of oxidant-sensitive en- coronary artery ligation. zymes involved in TNF-α production represents an additional mechanism by which the oxidant stress induces a cellular damage. was a light edema of tubular cells (d). Glimepiri- Our study was extended by measuring the de induced less improvement than glibenclamide myeloperoxidase activity which is an accepted (e). indicator of neutrophil accumulation and oxida- Figure 4 shows sections of the Sham lung tive activity. An associated renal dysfunction ac- stained with H & E showing normal architecture companies the increase in the activity of MPO. with no histopathological changes (a). In con- Toybin et al.30 discovered that activated neu- trast, sections of I/R and I/R pretreated with dia- trophils produce superoxide, which can be dis- zoxide showed marked increase in the thickness mutated into hydrogen peroxide. Neutrophil of the inter-alveolar septa due to mononuclear MPO enzyme converts hydrogen peroxide to cellular infiltration. This thickening of the inter- hypochlorous acid that reacting with superoxide alveolar septa caused narrowing of the alveolar produces hydroxyl radicals. The elevated renal cavities (b), (c). Treatment with glibenclamide dysfunction also demonstrated in our study by an and glimepiride preserved the normal histology increase of both BUN and creatinine, raised of the lung. There was a significant decrease in blood pressure and decreased urinary volume can – the thickening of inter-alveolar septa (d & e). be due to the increase of TNF-α level, O2 production and MPO activity. Discussion As sulfonylureas are widely used as hypo- glycemic drugs, we measured the blood glucose levels at 4 and 24 hours of renal reperfusion and Reperfusion of an ischemic vascular bed is ac- we remarqued that glycemia was high in rats companied by local inflammatory injury that lim- submitted to renal I/R. These high levels of glu- its the potential benefits of blood flow restora- cose may be due to the surgical stress carried out.

90 Role of ATP-sensitive potassium channel blockers

However, treatment with sulfonylureas amelio- In studying the protective effects of both rated this elevated levels. glibenclamide and glimepiride on renal I/R, we In the current study, renal I/R-induced histo- noticed that the glibenclamide is more effective pathological changes included tubular necrosis, than the glimepiride in inhibiting the local and vascular congestion and neutrophil accumulation remote injury following I/R of the renal vascular in the outer medulla. These findings could be pedicle in rats. This inhibitory effect may be due considered the main site of inflammation where to the anti-oxidant effects of glibenclamide, that neutrophils are activated is the postcapillary was emphasized in this study by the inhibitory – venules feeding the proximal tubules in the kid- effects of glibenclamide on the O2 production, ney. In addition, the activated neutrophil-induced while glimepiride had no such effect. Another endothelial cell injury might lead to ischemia of explanation was afforded by Lee and Chou18 who the proximal tubules in the outer medulla. emphasized that the glimepiride was more spe- During renal I/R toxic products accumulate in- cific than the glibenclamide for the SUR1 recep- tracellular; after re-establishment or re-connection tor in the pancreatic beta cells. However, protein of the vasculature to the circulation, oxygen is re- making up the KATP channel in the pancreatic be- applied and repair mechanisms are set into place. ta cells and in the principal cells of the kidney 32 During that time, accumulated toxic metabolites were different . So, most probably the Kir1.1a/ are flushed into the system, which may affect other CFTR receptor that is expressed in the kidney organs and may negatively influence the process of was more efficiently inhibited by the gliben- regeneration in the ischemic organ. These events clamide more than by the glimepiride. Further re- occur secondary to the opening of ATP-sensitive searches are required before any definitive con- potassium channels after fall in intracellular ATP clusions can be made. concentration, causing massive influx of neu- As sulfonylureas have attracted a great deal of trophils in association with the release of various interest in experimental cardiology and the pro- inflammatory mediators and oxygen-derived free tective effect of KATP channel blockade in renal radicals. These effects called the “uremic lung”29,31. I/R stands in sharp contrast to the harmful effects Our data revealed the marked neutrophil and cy- on the cardiac tissues, our study was extended to tokines accumulation in the lung tissues that begin evaluate the possible harmful effects of both sul- as early as 4 hours and extended at 24 hours. fonylureas drugs on normal and ischemic reper- In this study we found that the treatment with fused heart. It is well established that KATP chan- both the glibenclamide and the glimepiride sig- nels are present in cardiac cells and also in vas- nificantly ameliorated the reperfusion-induced cular smooth muscle cells, and are implicated in neutrophil accumulation and elevation of TNF-α the regulation of myocardial and vascular func- level in both the kidneys and lungs. We also ob- tion. So, drugs which open or inhibit the opening served that the reperfusion-induced myeloperoxi- of these channels, might profoundly modify the dase activation in both the kidneys and lungs was cardiovascular functions both under physiologi- inhibited by using both sulfonylurea drugs. This cal and pathological conditions33. Questions are effect was secondary to their ability to improve now raised about the potential adverse effects of the production of TNF-α and neutrophil aggrega- these drugs on the cardiovascular functions. tion following renal I/R. Furthemore, we found The mechanism of protection against arrhyth- that sulfonylurea, because their anti-inflammato- mia associated with ischemia reperfusion is that ry effects, improved all the functional renal para- the myocardial sarcolemmal ATP-dependent meters as BUN, creatinine and the urinary vol- potassium channels, which are normally closed ume. In addition, sulfonylurea ameliorated the by high ATP concentration, open during ischemia level of blood pressure and the histo-pathological when the ATP generation decreases favoring changes associated with the renal I/R. In conclu- potassium efflux. This reduces the action poten- sion, the inhibition of the KATP channel due to the tial duration (APD) of the cardiomyocyte thus sulfonylurea is the mechanism by which these decreasing the time of calcium influx and over- drugs prevent the renal I/R injury in both the kid- load inducing arrhythmia34. Moreover, ischemic neys and lungs. The activation of ATP-sensitive preconditioning is considered to be one of the potassium channels during hypoxia plays an im- most potent mechanisms of protection against 10 portant role in the neutrophil accumulation and myocardial I/R injury . The mitochondrial KATP the development of inflammatory response that channel opening plays a central role in the acqui- accompany the renal I/R injury4. sition of this protection20.

91 Mona K. Tawfik, Dina M. Abo-ElMatty, Amal A.M. Ahmed

HAYAMA T, MATSUYAMA M, FUNAO K, TANAKA T, TSUCHI- Our data showed that, neither diazoxide nor 2) DA K, TAKEMOTO Y, KAWAHITO Y, SANO H, NAKATANI T, glimepiride pretreatment influenced significantly YOSHIMURA R the cardiac parameters before coronary artery lig- . Benefical effect of neutrophil elas- tase inhibitor on renal warm Ischemia-Reperfu- ation. However, glibenclamide significantly in- sion Injury in the rat. Transplant Proc 2006; 38: creased the heart rate associated with elevated T- 2201-2202. wave voltage and ST segment. These elevated RAHGOZAR M, WILLGOSS DA, GOBÉ GC, ENDRE ZH. 3) ATP- parameters increased more after transient coro- dependent K+ channels in renal ischemia reperfu- nary artery ligation followed by reperfusion. sion injury. Renal Failure 2003; 25: 885-896. Moreover, the harmful effects on the rat’s hearts POMPERMAYER K, SOUZA DG, LARA GG, SILVEIRA KD, CAS- 4) that occur secondary to transient coronary artery SALI GD, ANDRADE AA, BONJARDIM CA, PASSAGLIO KT, ASSREUY J, CUNHA FQ, VIEIRA MA, TEIXEIRA MM. ligation, were improved by a pretreatment with The diazoxide. Opening of the mitochondrial KATP ATP-sensitive potassium channel blocker gliben- channels and increasing the total outward potas- clamide prevents renal ischemia/reperfusion in- sium current shortens the action potential dura- jury in rats. Kidney Int 2005; 67: 1785-1796. VIÑAS JL, SOLA A, GENESCÀ M, ALFARO V, PÍ F, HOTTER tion, thereby decreases the calcium influx. Thus, 5) G causes an energy preservation and a mediation of . NO and NOS isoforms in the development of 10,20 apoptosis in renal ischemia/reperfusion. Free ischemic preconditioning . Radical Biol Med 2006; 40: 992-1003. We observed that pretreatment with gliben- DONNAHOO KK, MENG X, AYALA A, CAIN MP, HARKEN 6) clamide abolishes the cardio-protective effect of AH, MELDRUM DR . Early kidney TNF-alpha expres- diazoxide pretreatment presumably by inhibiting sion mediates neutrophil infiltration and injury af- the mitochondrial KATP channel opening in the ter renal ischemia-reperfusion. Am J Physiol 20 myocyte . These results agreed with Negroni et 1999; 277: R922-R929. 34 CARR AC, MCCALL MR, FREI B al. who referred that glibenclamide interferes 7) . Oxidation of LDL by with the beneficial action of KATP opening during myeloperoxidase and reactive nitrogen species: ischemia reperfusion events thus prolonging the reaction pathways and antioxidant protection. APD and calcium entry for a longer period of Arterioscler Thromb Vasc Biol 2000; 20: 1716- 1723. time, which is potentially harmful to the heart. KIRK EA, DINAUER MC, ROSEN H, CHIAT A, HEINECKE Conversely, the improvements that were associ- 8) JW ated with preconditioning afforded by diazoxide . Impaired superoxide production due to defi- ciency in phagocyte NADPH oxidase fails to in- were not significantly changed when the precon- hibit atherosclerosis in mice. Arterioscler Thromb ditioning protocol took place in the presence of Vasc Biol 2000; 20: 1529-1539. glimepiride. A possible explanation is that MATSUMOTO N, KOMIYAMA S, AKAIKE N 9) . Pre- and glimepiride, unlike glibenclamide, doesn’t block postsynaptic ATP-sensitive potassium channels the mitochondrial KATP channels known to play a during metabolic inhibition of rat hippocampal 13 crucial role in preconditioning protection . CA1 neurons. J Physiol 2002; 541: 511-520. YOKOSHIKI H, SUNAGAWA M, SEKI T, SPERELAKIS N Glimepiride appears to be significantly less 10) . ATP- harmful than glibenclamide to the normal and is- sensitive K+ channels in pancreatic, cardiac, and chemic reperfused hearts subjected to IP protec- vascular smooth muscle cells. Am J Physiol Cell tion afforded by diazoxide which is one of the Physiol 1998; 274: C25-C37. BATAILLE D preconditioning mimetic agents. 11) . Molecular mechanisms of insulin secre- In conclusion, glimepiride at equivalent pro- tion. Diabetes Metab 2002; 28(Suppl 6): 4S4-4S13. ASHCROFT FM, GRIBBLE FM. tective dose in renal I/R has less cardiovascular 12) Tissue-specific effects side effects than conventional sulfonylureas. of sulfonylureas: lessons from studies of cloned Therefore, it offers some promise for therapy of K(ATP) channels. J Diabetes Complications renal I/R minimizing the undesirable cardiac side 2000; 14: 192-196. CAULFIELD M, O’BRIEN K. effects. 13) Cardiovascular safety of oral antidiabetic agents: the insulin secretagogues. Clinical Diabetes 2002; 20: 81-84. References FELSCH H, LANGE U, HAMBROCK A, LÖFFLER-WALZ C, 14) RUSS U, CARROLL WA, GOPALAKRISHNAN M, QUAST U. ROUSCHOP KM, ROELOFS JJ, CLAESSEN N, DA COSTA 1) Interaction of a novel dihydropyridine K+ channel MARTINS P, ZWAGINGA JJ, PALS ST, WEENING JJ, opener, A-312110, with recombinant sulphony- FLORQUIN S . Protection against renal ischemia/ lurea receptors and KATP channels: comparison reperfusion injury by CD44 disruption. J Am Soc with the cyanoguanidine P1075. Br J Pharmacol Nephrol 2005; 16: 2034-2043. 2004; 141: 1098-1105.

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